The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 1449 -1457
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Iwamoto, S.
Right arrow Articles by Miyazaki, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Iwamoto, S.
Right arrow Articles by Miyazaki, A.

TNF-{alpha} Drives Human CD14+ Monocytes to Differentiate into CD70+ Dendritic Cells Evoking Th1 and Th17 Responses

Sanju Iwamoto1,*, Shin-ichi Iwai{dagger}, Kazuko Tsujiyama{dagger}, Chika Kurahashi{dagger}, Kumiko Takeshita{ddagger}, Michio Naoe{ddagger}, Atsuko Masunaga§, Yoshio Ogawa{ddagger}, Katsuji Oguchi{dagger} and Akira Miyazaki*

* Department of Biochemistry, {dagger} Department of Pharmacology, and {ddagger} Department of Urology, School of Medicine, Showa University, Tokyo, Japan; and § Department of Surgical Pathology, Showa University Fujigaoka Hospital, Yokohama, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Many mechanisms involving TNF-{alpha}, Th1 responses, and Th17 responses are implicated in chronic inflammatory autoimmune disease. Recently, the clinical impact of anti-TNF therapy on disease progression has resulted in re-evaluation of the central role of this cytokine and engendered novel concept of TNF-dependent immunity. However, the overall relationship of TNF-{alpha} to pathogenesis is unclear. Here, we demonstrate a TNF-dependent differentiation pathway of dendritic cells (DC) evoking Th1 and Th17 responses. CD14+ monocytes cultured in the presence of TNF-{alpha} and GM-CSF converted to CD14+ CD1alow adherent cells with little capacity to stimulate T cells. On stimulation by LPS, however, they produced high levels of TNF-{alpha}, matrix metalloproteinase (MMP)-9, and IL-23 and differentiated either into mature DC or activated macrophages (M{phi}). The mature DC (CD83+ CD70+ HLA-DR high CD14low) expressed high levels of mRNA for IL-6, IL-15, and IL-23, induced naive CD4 T cells to produce IFN-{gamma} and TNF-{alpha}, and stimulated resting CD4 T cells to secret IL-17. Intriguingly, TNF-{alpha} added to the monocyte culture medium determined the magnitude of LPS-induced maturation and the functions of the derived DC. In contrast, the M{phi} (CD14highCD70+CD83–HLA-DR–) produced large amounts of MMP-9 and TNF-{alpha} without exogenous TNF stimulation. These results suggest that the TNF priming of monocytes controls Th1 and Th17 responses induced by mature DC, but not inflammation induced by activated M{phi}. Therefore, additional stimulation of monocytes with TNF-{alpha} may facilitate TNF-dependent adaptive immunity together with GM-CSF-stimulated M{phi}-mediated innate immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Not only does TNF-{alpha} have multiple physiological functions (1) but it also has been implicated in a variety of human diseases including sepsis, cerebral malaria, and certain autoimmune diseases such as rheumatoid arthritis (RA),2 psoriasis, and Crohn’s disease, as well as cancer (2). Over the past decade, anti-TNF therapy for autoimmune diseases has become widespread, efficiently preventing tissue injury. Moreover, studies in patients and experimental models have confirmed the central role of TNF-{alpha} in the etiology of several autoimmune diseases (3, 4, 5) and have advanced TNF biology. Thereby, questions as to the initial rationale supporting the use of anti-TNF (3, 4), and the mode of action of this therapy have arisen (6, 7, 8, 9, 10). In particular, although important for the etiology of disease, data on the impact of anti-TNF therapy on IL-23 production by dendritic cells (DC; Refs. 6 and 7), Th1 responses (6, 9, 10) and IL-23/IL-17 axis inflammation (11, 12, 13, 14, 15) are essentially absent. Recently, Banchereau et al. (16) proposed the existence of two different types of autoimmunity; one IFN dependent and the other TNF dependent. These cross-regulate one another (17) and involve induction of differentiation of DC, which further induce cytokine-dependent immunity (16). However, the precise differentiation pathways of these DC are unclear.

DC orchestrate a variety of immune responses by stimulating the differentiation of naive CD4 T cells into helper T effectors such as Th1, Th2, Th3, and Th17 cells (18, 19, 20, 21). The cytokine environment influencing DC differentiation may be an important factor in determining which type of Th cell is induced (22). To date, although a variety of in vitro-cultured DC has been studied (23, 24, 25, 26, 27), DC-driven TNF-dependent immunity has not been elucidated. Here, we have addressed such TNF-dependent differentiation of human CD14+ monocytes into mature DC. Uniquely, IL-4 (generally used for monocyte-derived DC differentiation) was not used here because of its anti-TNF/inflammatory effects (28, 29). Instead, a combination of TNF-{alpha} and GM-CSF was used for DC preparation, because of their effect on circulating monocytes in the sera of patients with autoimmune disease (4, 30). Although human monocytes cultured with this cytokine combination failed to generate functional DC (31), we did find that. LPS from Gram-negative bacteria induces not only maturation of DC but also production of IL-23 by these DC via TLR-4 ligation (32). The present study took advantage of human CD70+ DC, as previously described (33). These DC can persistently induce Th1 responses and the expression of IL-23 mRNA, whereas CD70– DC manifest exhaustion after transient activation (34). Such functions may be favorable for DC evocation of persistent Th1 and IL-23/IL-17 axis inflammation. The type of DC described here is another form of CD70+ DC; we analyzed the TNF-dependent differentiation pathway from CD14+ monocytes to CD70+ DC. Additionally, differentiation of these cells may also result in CD70+ macrophages (M{phi}) producing large amounts of TNF-{alpha} and matrix metalloproteinase (MMP)-9, which is also important for their mechanism of action in autoimmune disease (35, 36, 37). Here, we propose a novel model for the TNF-dependent differentiation of a monocyte-DC/M{phi} lineage and discuss where TNF-{alpha} orchestrates Th1 responses, and IL-23/IL-17 axis inflammation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Human rTNF-{alpha}, rGM-CSF, rIL-4, agonistic mAb to human CD3 (B-B11), and CD28 (B-T3) were from Diaclone Research. Human soluble type I TNF receptor (sTNFRI) was from R&D Systems. Culture grade LPS from Escherichia coli (L4516) was from Sigma-Aldrich. Keyhole limpet hemocyanin (KLH) was from Wako Pure Chemical.

Cell culture

T cells, monocytes, and DC were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS (Life Technologies-BRL) plus 100 U/ml penicillin G and 100 µg/ml streptomycin.

Preparation of DC and M{phi}

Samples from healthy volunteers were obtained with informed consent approved by the ethics committee of Showa University (Tokyo, Japan). PMBC were isolated by Histopaque (Sigma-Aldrich) density gradient (1.077) centrifugation in a Leuco Sep (Greiner Bio-One) that can separate granulocytes from CD14+ monocytes. After depletion of platelets, monocytes were isolated using CD14 Ab-conjugated magnetic microbeads (Miltenyi Biotech) according to the manufacturer’s instructions. The isolated CD14+ monocytes (5 x 105/ml) were cultured for 7 days in the presence of GM-CSF (10 ng/ml) and/or TNF-{alpha} (10 ng/ml). Subsequently, these cells were washed three times with warmed 10% FBS containing RPMI 1640 and cultured in the presence of LPS (10 µg/ml) and KLH (10 µg/ml). Floating cells were collected by gentle pipetting at indicated times after stimulation. Adherent cells in the cultures were collected by vigorous pipetting after incubation in 2 mM EDTA-PBS solution at 37°C for 5 min.

Preparation of CD4 T cells

CD4 T cells were isolated from PBMC-depleted CD14+ cells. Cells were treated with anti-CD45RO Ab-conjugated magnetic microbeads (Miltenyi Biotech). After absorbing CD45RO+ cells to a negative selection column (MACS 25 LD; Miltenyi Biotec), CD45RO– cells in the passthrough fractions were treated with anti-CD4 Ab-conjugated magnetic microbeads (Miltenyi Biotec), and the resulting CD4+CD45RO– cells were collected by positive selection. Finally, activated T cells and APCs including DC or B cells were depleted by M-450 pan human HLA class II and M-450 CD19 Dynabeads (Dynal Biotech), respectively. The naive CD4 T cell fraction (CD4+CD45RA+ cells, >98%) was then used for these experiments. Occasionally, purified CD4 T cells (>98%) without depletion of CD45RO+ cells were used. The CD4 T cell fractions containing memory type T cells were isolated using the pan-T isolation kit (Miltenyi Biotec) and anti-CD4 Ab-conjugated magnetic microbeads.

FACS

Cells fixed with 4% paraformaldehyde were adjusted to a concentration of 1 x 106 cells/ml and incubated at 4°C for 30 min with appropriate Abs. After two washings with ice-cold PBS containing 0.3% BSA, cells were analyzed by FACS with CellQuest software (BD Pharmingen). Ab used for flow cytometry were as follows: FITC-conjugated mAb to HLA-ABC (w6/32; Diaclone); CD14 (M5E2; BioLegend); CD70 (Ki-24; BD Pharmingen); CD80 (B-L2; Diaclone); and PE-labeled mAb to HLA-DR (B-F1), and CD40 (B-B20) from Diaclone. mAbs to CD86 (IT2.2), and CD83 (HB15e) were from BD Pharmingen. Staining for intracellular cytokines in T cells was described in a previous report (33). Ab used for the latter experiments were rabbit anti-hIL-17 polyclonal Ab or isotype control Ab (MBL), FITC-labeled goat anti-rabbit IgG-Fab (PARIS), FITC-labeled monoclonal anti-IFN-{gamma}-Ab (4S.B3; BioLegend), PE-labeled mAb to IL-4 (MP4-25D2; BioLegend); and TNF-{alpha} (Mab11, BioLegend).

Cytokine measurement

The concentrations of cytokines in DC culture medium were determined using ELISA kits for IFN-{gamma}, TNF-{alpha}, IL-12/IL-23p40, IL-12p70, and IL-23p19/p40 (eBioscience). Data were acquired by a VERSAmax microplate reader (Molecular Devices) and analyzed by SOFTmax PRO (Molecular Devices).

T cell proliferation assay

T cell proliferation was determined by incorporation of [3H]thymidine as described in a previous report (33). T cells (2 x 105 cells) were cultured alone or cocultured with DC for 3 days. [3H]Thymidine (0.4 µCi; Amersham Pharmacia) was added to each well, and nuclear incorporation was stopped by the addition of cold thymidine after a further 18 h of culture. Isotope incorporation was determined by liquid scintillation counting in a TracorAnalytic Mark III machine, after three washings with PBS containing 0.3% BSA.

Quantitative real-time RT-PCR

Total RNA and synthesis of first-strand cDNA was from 5 x 105 cells/assay. mRNA for several different cytokines were quantified by real-time RT-PCR using a Syber Green Realtime PCR Master Mix (Toyobo) in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems Japan). Amplification was conducted in a total volume of 50 µl for 40 cycles of 15 s at 94°C, 20 s at 60°C and 1 min at 72°C. mRNA levels were determined after normalization of RNA concentration against beta-actin, and values were expressed as fold increase of expression above the DC control. All samples were run in triplicate. The primers used in the experiments were as follows: beta-actin: forward, 5'-GCACCACACCTTCTACATGAGC; reverse, 5'-GCACAGCTTTCTCCTTAATGTCACGC. TNF-{alpha}: forward, 5'-GAGTGACAAGCCTGTAGCCCATGTTGTAGC; reverse, 5'- GCAATGATCCCAAAGTAGCCTGCCCAGAC. IL-6: forward, 5'-GA AAGCACAAAGAGGCACT; reverse, 5'-GCGCAGAATGAGATGAGATTG; IL-15: forward, 5'-ATGAGAATTTCGAAACCACATTG; reverse, 5'-CCATTAGAAGACAAACTGTTCTTTGC. Primers for IL-12p35 (33), IL-12/23p40 (33), and IL-23p19 (33) were as described in previous reports. MMP-9 primers (HA040500) were purchased from Takara Bio.

Zymography

MMP-2 and MMP-9 were measured by gelatin zymography on 10% polyacrylamide gels containing 1 mg/ml gelatin in a polymerization mixture of SDS as described in previous report (37). Samples containing equal protein concentrations were mixed with sample buffer. After SDS-PAGE, SDS was washed out of the gels with 2.5% Triton X-100 for 30 min at room temperature before they were incubated in a solution of 50 mM CaCl2 and 0.02% sodium azide (pH 7.5) at 37°C for 1 day. Gels were then stained with 0.1% Coomassie blue and destained. Clear area indicated proteolytic activity. The 72-kDa purified proenzyme MMP-2 and the 92-kDa purified proenzyme MMP-9 were used as positive control and standard for the gels (Oncogene Research Products). The zymography bands were digitized using the Gel Doc/Chemic Doc Imaging System, and the data were analyzed using the Quantity One analysis software program (Bio-Rad). Detected activities of positive controls were set at 100%.

Statistical analysis

The unpaired two-tailed Student t test was used to compare differences. A p value of ≤0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Combination of TNF-{alpha} and GM-CSF induces differentiation of CD14+ monocytes into CD14+ DC/M{phi} precursors

CD14+ monocytes (>98% purity) isolated from PMBC were cultured in the presence of 10 ng/ml GM-CSF plus 10 ng/ml TNF-{alpha} or 10 ng/ml IL-4 for 7 days. Most of the cells cultured with GM-CSF plus TNF-{alpha} were converted into round or spindle-shaped adherent M{phi}-like cells (Fig. 1A). These cells expressed high levels of CD14, a marker of monocyte/M{phi}, and substantial levels of CD86 (Fig. 1B) and are termed CD14+ pre-DC/M{phi} hereafter. They also expressed HLA-DR, CD1a, CD40, and CD80, but at lower levels than immature DC cultured with GM-CSF and IL-4 for 7 days (IL-4 DC). Furthermore, these CD14+ pre-DC/M{phi} stimulated much less proliferation of allogeneic naive CD4 T cells than immature IL-4 DC (data not shown).


Figure 1
View larger version (40K):
[in this window]
[in a new window]

  		FIGURE 1. Comparison of CD14+ pre-DC/M{phi} and immature IL-4 DC. A, Morphological features in light microscopic micrographs of cells cultured with GM-CSF plus TNF-{alpha}. Original magnification, x100. B, Phenotypic differences of these cells. Surface Ags were analyzed by FACS after staining with FITC- or PE-labeled murine mAb. Histograms show expression of surface Ags (solid) compared with the IgG isotype control (line). Numbers indicate the percentage of positive cells. Data are representative of three similar experiments.

 
The production of TNF-{alpha}, IL-12/IL-23p40 (homodimer of p40 subunits), IL-12p70 (heterodimer of p35 and p40 subunits), and IL-23p19/p40 (heterodimer of p19 and p40 subunits) in response to 10 µg/ml LPS is shown in Fig. 2. CD14+ pre-DC/M{phi} produced extremely large amounts of TNF (>150 ng/ml at peak), as well as IL-12/IL-23p40 and IL-23p19/p40, but very little IL-12p70. They maintained high levels of IL-12/IL-23p40 and IL-23p19/p40 production for up to at least 5 days after LPS stimulation. In contrast, IL-4 DC produced much smaller amounts of TNF, IL-12/IL-23p40, and IL-23p19/p40, whereas IL-12p70 was produced in far greater amounts. Conclusively, CD14+preDC/M{phi} have M{phi}-like rather than DC-like characteristics and retain the high responsiveness to LPS reflected in production of TNF-{alpha} and IL-23 rather than IL-12.


Figure 2
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 2. Qualitative differences in cytokine production of monocyte-derived cells on LPS stimulation. CD14+preDC/M{phi} ({square}) or immature IL-4 DC ({blacksquare}), 5 x 105 cells/ml, were cultured in the presence of LPS (10 µg/ml). Culture-conditioned medium were collected at the indicated times after stimulation. Concentrations of TNF-{alpha}, IL-12/23p40, IL-12p70, and IL-23p19/p40 in the supernatant were determined by ELISA. Values are means ± SD (n = 3). Data are representative of three similar experiments. ND, Not detectable.

 
LPS induces dual differentiation of CD14+ pre-DC/M{phi} into mature DC or activated M{phi}-like cells

Differentiation of CD14+ pre-DC/M{phi} after LPS stimulation was analyzed. Some cells began to convert to DC-like floating cells with extended dendrites (Fig. 3A) 2 days after LPS stimulation, and the number of floating cells increased to up to one-third of the total at 6 days (Fig. 3B). Phenotyping of the floating cells showed that these were mature DC, because substantial levels of HLA-DR, CD40, CD70, CD80, CD86, and CD83 were expressed, but only little CD14 (Fig. 3C). In contrast, adherent cells maintained high levels of CD14 and CD86, but only low-level HLA-DR and CD83; CD70 and CD86 expression was also lower than in DC-like cells (Fig. 3C). TNF-DC (The floating cells are termed TNF-DC hereafter.) stimulated naive CD4 T cells to the same extent as mature IL-4 DC induced by LPS (Fig. 4A). Furthermore, TNF-DC retained IFN-{gamma}-inducing capacity much better than mature IL-4 DC (data not shown). In contrast, the adherent cells had typical activated M{phi} functions (i.e., excellent phagocytic activity and production of TNF-{alpha}) but stimulated proliferation only poorly (data not shown). These results indicate that LPS stimulation induces dual differentiation of CD14+ pre-DC/M{phi} to mature DC or activated M{phi}.


Figure 3
View larger version (48K):
[in this window]
[in a new window]

  		FIGURE 3. LPS induces dual differentiation of CD14+ pre-DC/M{phi} either into mature DC or activated M{phi}-like cells. CD14+ pre-DC/M{phi} were stimulated with 10 µg/ml LPS, and floating cells and adherent cells were collected. A, Floating cells possessed morphological characteristics of DC. Lanes a and b, micrographs of floating cells. Lane a, Light microscopic image; original magnification, x100; lane b, May-Grünwald staining; original magnification, x400; lane c, micrographs of adherent cells 4 days after LPS stimulation. B, Percentages of floating cells per total number of cells. Cells were counted by trypan blue exclusion. Values are means ± SD (n = 3). C, Phenotypic differences. Floating and adherent cells were collected 4 days after stimulation, and surface Ags were stained with FITC- or PE-labeled murine mAbs. Histograms show expression of surface Ags (solid lines) compared with IgG isotype control (broken lines). Numbers not in parentheses and those in parentheses indicate the percentage of positive cells and mean fluorescence intensity, respectively.

 

Figure 4
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 4. Comparison between TNF-DC and IL-4 DC in naive T cell-stimulatory capacity. CD14+ pre-DC/M{phi} and immature IL-4-DC were pulsed with KLH (10 µg/ml) and stimulated by LPS (10 µg/ml) for 3 days and then cocultured with autologous naive CD4+ T cells. A, Incorporation of [3H]thymidine. After 3 days, DC and T cell cocultured cells were pulsed with [3H]thymidine overnight. Values are means ± SD (n = 4). B, Profiles of cytokines in T effectors generated by IL-4-DC and TNF-DC after stimulation by LPS. Naive CD4+ T cells were cocultured with DC for 12 days. Negative controls were T cells cultured in the presence of 10 µg/ml anti-IL-12 Ab and 10 ng/ml TGF-beta. Intracellular cytokines were stained using FITC-labeled anti-IFN-{gamma} Ab plus PE-labeled Ab to IL-4, or TNF, and analyzed by FACS. Numbers indicate the percentage of positive cells.

 
Comparison of T effectors induced by TNF-DC or IL-4 DC

Cytokine profiles of CD4 T cell stimulated by TNF-DC or mature IL-4 DC were compared (Fig. 4B). Autologous naive CD4 T cells were added to DC pulsed with KLH and treated with LPS for 3 days. Intracellular cytokines were analyzed by FACS 12 days after starting the DC-T cultures. TNF-DC promoted more Th1-dominant polarization than mature IL-4 DC. T effectors stimulated by TNF-DC contained many cells producing both of IFN-{gamma} and TNF-{alpha}, which was not the case for T cells cocultured with mature IL-4 DC. According to the current paradigm for the functions of activated DC (34), the T cell induction capacity of mature IL-4 DC shifts from Th1 to Th2 polarization shortly after LPS stimulation, but the TNF-DC investigated here did not show this increase in Th2 polarization. Even much later after LPS stimulation, Th2 polarization was very limited in TNF-DC (data not shown).

TNF-{alpha} stimulation of monocytes determines maturation and Th1-inducing activity of TNF-DC

The effects on subsequent LPS-induced DC maturation and function of TNF-{alpha} added into monocyte cultures were analyzed. Because low levels of TNF-{alpha} were detected in the cultures of monocytes cultured for 7 days with GM-CSF alone (~50 pg/ml), we tested monocytes cultured with GM-CSF plus anti-TNF reagents (50 ng/ml sTNFRI) or titrated concentrations of TNF-{alpha} (0–5 ng/ml). Cells in these cultures were also converted to floating DC or adherent M{phi} after LPS stimulation. Differences between phenotypes of cells cultured in the presence or absence of TNF-{alpha} were not apparent. However, after LPS stimulation, expression of DC maturation markers (HLA-DR, CD70, and CD86) were markedly different (Fig. 5A). Activity was increased in a dose-dependent manner by adding TNF-{alpha} into the monocyte culture medium. Likewise, the Th1-inducing capacity of these cells was analyzed. Quantitative analysis of IFN-{gamma} production in combination cultures with naive CD4 T cells also revealed that the Th1-inducing capacity rose in parallel with the dose of TNF-{alpha} in the monocyte cultures (Fig. 5B). FACS also revealed that the addition of TNF-{alpha} enhanced Th1-dominant responses induced by DC (data not shown). Supporting these data, expression of mRNA for IL-12 family members (IL-12p35, IL-12/IL-23p40, and IL-23p19) and proinflammatory cytokines (TNF-{alpha}, IL-6, and IL-15) 2 days after stimulation was also amplified in the same manner (Fig. 5C). These results indicate that CD14+ monocytes are primed with TNF-{alpha} before LPS-induced maturation into TNF-DC, and this controls the magnitude of Th1 inducing capacity of CD70+ DC. Additionally, TNF-{alpha} stimulation of monocytes had to be from the beginning of the culture, having no effect 3 days later.


Figure 5
View larger version (38K):
[in this window]
[in a new window]

  		FIGURE 5. TNF priming determines magnitude of maturation and Th1-inducing capacity of TNF-DC. Monocytes were cultured with GM-CSF plus different TNF-{alpha} concentrations (0–10 ng/ml) or sTNFRI, and then stimulated by LPS (10 µg/ml). Floating DC were collected 2–4 days after LPS stimulation. A, FACS of surface Ags. Cells were stained with mAb to HLA-DR (PE), CD70 (FITC), and CD86 (PE) at 4 days after LPS stimulation. Numbers not in parentheses and those in in parentheses indicate the percentage of positive cells and mean fluorescence intensity, respectively. B, Effects of TNF-{alpha} exposure of CD14+ monocytes on subsequent Th1 induction. Floating DC were collected 3 days after the addition of LPS and KLH and cocultured with autologous naive CD4+ T cells. IFN-{gamma} production. Supernatants of 3-day DC-T cocultures were collected, and IFN-{gamma} was determined by ELISA. Values are mean ± SD (n = 3). C, Quantitative analysis of mRNA by real-time RT-PCR. Cells were collected 2 days after stimulation, and mRNA for beta-actin, IL-12p35, IL-12/23p40, IL-23p19, IL-6, and IL-15 was determined. mRNA levels are expressed as fold increased expression above control mRNA (beta-actin). Values are means ± SD (n = 3). Results are representative of at least three independent experiments.

 
TNF priming facilitates capacity of DC to induce IL-17 production from resting CD4 T cells

The capacity of TNF-DC to induce IL-17 production by purified CD4 T cells was tested. T cells were extensively depleted of APC or activated T cells because crude T cell fractions contained cells spontaneously producing IL-17. CD4 T cells were cultured alone or with LPS-induced DC pretreated with GM-CSF alone (GM-CSF DC), or GM-CSF plus sTNFRI (anti-TNF DC), or 10 ng/ml TNF (TNF-DC) for 6 days. T cells alone contained only low levels of IL-17-producing cells. T cells stimulated with 1 µg/ml agonistic anti-CD3 Ab and anti-CD28 Ab in the absence of DC did not yield increased ratios of Th17 cells. IFN-{gamma}- and/or IL-17-positive cells cocultured with LPS-stimulated DC are presented in Fig. 6A. TNF-DC induced three types of T effectors (IL-17+IFN-{gamma}–, IL-17+IFN-{gamma}+, and IL-17–IFN-{gamma}+). In experiments using isotypic control IgG instead of rabbit anti-IL17 Ab, IL-17+ cells were significantly reduced (Fig. 6A). The increase in all these cell types was facilitated by TNF priming. Similar results were obtained when data on total IL-17-producing cells (IL-17+IFN-{gamma}– and IL-17+IFN-{gamma}+ cells) from four independent experiments were considered (Fig. 6B). In experiments on Th17 induction from naive T cells (CD4+CD45RA+ >98%), TNF-DC could induce small but significantly increased numbers of these cells (negative control; 0.04 ± 0.03%, n = 8, vs TNF-DC; 0.24 ± 0.05%, n = 8, p < 0.005). These were almost all IL-17+IFN-{gamma}+ (data not shown). Therefore, these data indicate that the TNF-DC had the capacity to induce Th17 responses and that the priming of monocyte with TNF-{alpha} facilitated this activity.


Figure 6
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 6. Induction of Th17 responses by TNF-DC. Monocytes were cultured with GM-CSF plus sTNFRI (anti-TNF DC), or TNF-{alpha} (TNF-DC) or with GM-CSF alone (GM-CSF DC) for 7 days. They were then pulsed with 10 µg/ml LPS and KLH. Floating DC were collected 3 days after stimulation, and autologous resting CD4+ T cells (>98%) were cocultured alone, or with these LPS-stimulated DC for 6 days. Cells were fixed and analyzed by FACS. A, FACS of IFN-{gamma}- and IL-17-producing T cells induced by DC. Numbers in gated areas are percentages of positive cells in the total counted cells. Isotype IgG control was used rabbit polyclonal Ab instead of specific Ab to anti-human IL-17. B, Total IL-17-producing cells induced by DC. Values are mean ± SD of four independent experiments.

 
Production of MMPs and TNF-{alpha} by DC and M{phi}

Production of MMP-9 by LPS-stimulated DC or M{phi} was analyzed. Conditioned medium from cultures of DC 3 days before and after LPS stimulation were tested in gelatin zymography. In Fig. 7A, MMP-9 activity is shown to be facilitated in cultures of anti-TNF-DC, GM-CSF DC, and TNF-DC, but not IL-4-DC, after LPS stimulation. In contrast, MMP-2 activity was consistently detected in all samples. Semi quantitative analysis using the Gel Doc/Chemic Doc Imaging System showed that the MMP-9 activity of TNF-DC cultures was ~5 times of that of IL-4 DC. However, MMP-9 activity of anti-TNF-DC, GM-CSF, and TNF-DC was significantly high. Analysis by real-time RT-PCR revealed that expression of MMP-9 mRNA was high in M{phi}, but very low in DC, especially IL-4 DC (Fig. 7B). These data suggest that large amounts of MMP-9 in cultures of anti-TNF-DC, GM-CSF-DC, and TNF-DC were mainly produced by M{phi}. Likewise, TNF-{alpha} mRNA expression by M{phi} in TNF-DC culture (1640 ± 531, n = 4) was much higher than DC (Fig. 5B). Mean values were not significantly different from those of other M{phi} (data not shown). These data suggest that LPS-stimulated M{phi} produced large amounts of MMP-9 and TNF-{alpha} in a manner independent of TNF priming.


Figure 7
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 7. Production and expression of MMP-9 by DC and M{phi}. Monocytes (5 x 105 cells/ml) were cultured in the presence of 10 ng/ml GM-CSF plus sTNFRI (lane a), no cytokine (lane b), TNF-{alpha} (lane c), or IL-4 (lane d) for 7 days. Subsequently, cells were stimulated by 10 µg/ml LPS for 3 days. A, Gelatin zymograpy analysis of MMP-9 and MMP-2 activity in conditioned medium of monocyte cultures before (top) and 3 days after (bottom) LPS stimulation. Active bands at 92 and 72 kDa indicate MMP-9 and MMP-2, respectively. Similar results were obtained in four experiments. B, Analysis of expression of MMP-9 by real-time RT-PCR. RNA samples were obtained from M{phi} and DC 3 days after LPS stimulation. The relative expression of each MMP-9 normalized to that of beta-actin was plotted. Values are means ± SD (n = 3). Results are representative of at least three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this report, we document TNF-dependent differentiation of CD14+ monocytes into mature DC that evoke Th1 and Th17 responses. This takes place in a two-step stimulation model. First, monocytes are stimulated with TNF-{alpha}; second, mature DC are induced by LPS stimulation. The first step determined the maturation and functions of the derived DC. The mode of differentiation is different from that of conventional IL-4-DC. This represents a unique role of TNF-{alpha} in stimulating monocytes for DC differentiation, in addition to its accepted maturational effect on immature DC (38). The former effect of TNF-{alpha} is more important than the latter, as supported by several studies using clinical samples. First, sera of RA patients stimulate monocytes from healthy donors to differentiate into active DC, which is reduced by anti-TNF therapy (30); second, monocyte-DC from patients are more active than those from healthy donors (39); third, a combination of TNF-{alpha} and GM-CSF not only enhances DC differentiation (40, 41) but also facilitates autologous MLR (42); Fourth, serum TNF/ sTNFR (endogenous agonists/inhibitors) ratios correlate with the severity of autoimmune disease (43); fifth, injection of anti-TNF reagents into the blood rather than joint space is effective (4). Taken together, TNF-{alpha} elevated in patients’ sera may render monocytes susceptible to the TNF priming described here for TNF-DC differentiation. Intriguingly, CD14 positivity of DC precursors in RA synovial fluid (42) is similar to pre-TNF-DC/M{phi}, but not IL-4-DC. Further in vivo studies remain necessary to clarify the roles of TNF priming for the differentiation of DC that evoke abnormal inflammatory responses.

Knowledge of how to maintain TNF priming could be important for persistence in Th1 and Th17 responses. This could be provided by large amounts of TNF produced by pre-TNF-DC/M{phi} stimulated with LPS. Such phenomena were noted in previous experimental animal models where hemorrhagic necrosis was induced (44). Alternatively, T effectors producing TNF-{alpha} or IL-17 induced by TNF-DC could stimulate circulating monocytes and M{phi} (45). Such routes for positive feedback stimulation could maintain persistent inflammation (46). Thus, anti-TNF therapy could reduce Th1 and Th17 responses by blocking TNF-dependent differentiation of DC and thence the TNF-dependent positive feedback system. Hence, we propose a model for the differentiation pathway of DC and M{phi} as shown in Fig. 8. In this model, inflammation would be terminated once pathogens are cleared in the case of a bacterial infection. Unlike infections, endogenous agonists for TLR-4 could evoke unremitting inflammation (47). Endogenous TLR-4 agonists such as hyaluronan, fibrinogen, fibronectin, high mobility group box 1 protein, and heat shock proteins may be involved in this process (48). However, this model cannot apply to every autoimmune disease such as multiple sclerosis and systemic lupus erythematosus that involve in IFN-dependent immunity (17).


Figure 8
View larger version (25K):
[in this window]
[in a new window]

  		FIGURE 8. Model for TNF-dependent differentiation of DC. Circulating monocytes stimulated with GM-CSF convert to CD14+ pre-DC/M{phi}, and are then recruited to inflamed tissues. Subsequently, cells would be stimulated with exogenous or endogenous TLR-4 ligands and activate to produce large amounts of TNF-{alpha}. In this case, innate immunity, but not adaptive immunity, could be evoked because mature DC could not be generated (these pathways shown in white). The TNF-{alpha} secreted by the GM-CSF M{phi} resulted in elevated serum levels, which could prime circulating monocytes to differentiate into functional DC (following processes shown by the black arrows). After being recruited into inflamed tissues CD14+ pre-DC/M{phi} primed with TNF-{alpha} could be stimulated through TLR-4 signaling and differentiate into TNF-DC that could then promote Th1 and Th17 responses to mediate adaptive autoimmunity. Inflammatory cytokines including TNF-{alpha}, IFN-{gamma}, and IL-17 secreted by T effectors could prime circulating monocytes and encourage M{phi} to be more active. These would in turn prime DC differentiation in a positive loop circuit and exacerbate inflammation by excess production of MMP-9 and TNF-{alpha}.

 
Although induction of human Th-17 by DC has not been reported thus far, TNF-DC induced IL-17 production by highly purified resting CD4 T cells, which did not respond to stimulation with anti-CD3/CD28 Ab. The persistent IL-23 production in response to LPS (Fig. 2) is a feature that would evoke IL-23/IL-17 axis inflammation in TNF-DC differentiation, but not IL-4-DC. Furthermore, the TNF priming enhanced both of IL-23 mRNA expression (Fig. 5C) and induction of IL-17-producing cells by TNF-DC (Fig. 6). Thus, TNF-{alpha} may drive IL-23/IL-17 axis inflammation due to promoting monocytes to differentiate into TNF-DC.

To date, some differences between human and murine system have been found concerning the induction of Th17 cells. Th1-type IL-17 producing cells (IFN-{gamma}+IL-17+ cells) as shown in Fig. 6 can be found in patients with autoimmune disease (49), but not in mice, because IFN-{gamma} inhibits IL-17 production (18, 20). In addition, IL-17 production from human naive CD4 T cells cannot be induced by stimuli as can be done in mice (50). Accordingly, it is suggested that IL-17 induction may be more complex than in mice, and conditions for inducing substantial Ag-specific Th17 cells in humans are largely unknown. Although TNF-DC had potential ability to promote naive CD4 T cells to Th17 cells, the activity was rather low. Because precise definition of these conditions is ultimately crucial for understanding the pathogenesis of autoimmune disease, further studies will be required to delineate specific condition in vitro. In this study, we also investigated monocyte-derived M{phi} differentiation by stimulation with LPS after culture with GM-CSF alone or GM-CSF plus anti-TNF or TNF-{alpha}. These findings suggested that such differentiated M{phi} could evoke abnormal inflammation because they produced large amounts of TNF-{alpha} and MMP-9, even, without TNF-priming. However, GM-CSF is known to induce M{phi}, termed proinflammatory type 1 M{phi} (51), which have important roles in defense against infection (52), but a limited role in adaptive immunity (53). These data are consistent with our results. Namely, GM-CSF plus TNF stimulation of monocytes may form a bridge to adaptive immunity from the innate immunity mediated through TLR-4 signaling. In contrast, TLR-7 or -9 signaling, which leads to IFN-{alpha} production by plasmacytoid DC initiates IFN-dependent immunity by generating DC derived form monocytes cultured with GM-CSF plus IFN-{alpha} (16). Concerning infectious immunity based on GM-CSF-dependent differentiation to type 1 M{phi}, the additional cytokine stimulation of monocytes could modify the mode of differentiation of DC/M{phi} appropriate to adaptive immunity against bacterial or viral infection.

CD70 positivity of the TNF-DC and M{phi} presented in this report is unusual. CD70+ DC and M{phi} are found in lymph nodes in experimental infectious models in mice (54) and in atherosclerotic plaques in humans (55), respectively. Interestingly, CD70+ DC augment CD4 T cell-independent cellular immunity (32, 56) through CD27/CD70 alternative pathway interactions. These CD70+ cells may share functions in infectious immunity or pathogenesis of chronic inflammatory diseases. Further studies are required to clarify the precise details.

In conclusion, this differentiation of TNF-DC has provided a new rationale to explain the mechanism of action of TNF-dependent Th1 and Th17 responses. Hence, the model presented here is useful to better understand TNF immunity and the pathology of disease. In future, support for this model needs to be acquired in animals, but some studies, in particular, regarding IL-17-dependent inflammation must use human materials.


   Acknowledgments
 
We thank Professors Yoichiro Iwakura, Kouji Matsushima, Seigi Shioda, and Ken Takeda for helpful suggestions and discussion.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 Address correspondence and reprint requests to Dr. Sanju Iwamoto, Department of Biochemistry, School of Medicine, Showa University, Hatanodai 1-5-8, Shinagawa-ku, Tokyo, Japan. E-mail address: iwasanju{at}med.showa-u.ac.jp Back

2 Abbreviations used in this paper: RA, rheumatoid arthritis; DC, dendritic cell; M{phi}, macrophage; MMP, matrix metalloproteinase; sTNFRI, soluble type I TNF receptor; KLH, keyhole limpet hemocyanin. Back

Received for publication January 24, 2007. Accepted for publication May 25, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Bonavida, B.. 1992. TNF as immunomodulatory agent. B. B. Aggarwal, and J. Vilcek, eds. Tumor Necrosis Factors 315-340. Marcel Dekker, New York.
  2. Ruuls, S. R., J. D. Sedgwick. 1999. Unkilling tumor necrosis factor biology from the major histocompatibility complex. Am. J. Hum. Genet. 65: 294-301. [Medline]
  3. Feldmann, M., M. J. Elliott, J. N. Woody, R. N. Maini. 1997. Anti-tumor necrosis factor: a therapy of rheumatoid arthritis. Adv. Immunol. 64: 283-350. [Medline]
  4. Feldmann, M., F. M. Brennan, E. Paleolog, A. Cope, P. Taylor, R. Williams, J. Woody, R. N. Maini. 2004. Anti-TNF{alpha} therapy of rheumatoid arthritis. Novartis Found. Symp. 256: 53-69. [Medline]
  5. Suryaprasad, A. G., T. Prindiville. 2003. The biology of TNF blockade. Autoimmun. Rev. 2: 346-357. [Medline]
  6. Gottilieb, A. B., F. Chamain, S. Masud, I. Cardinale, M. V. Abello, M. A. Lowes, F. Chen, M. Magliocco, J. G. Krueger. 2005. TNF inhibition rapidly down-regulates multiple proinflammatory pathways in Psoriasis plaques. J. Immunol. 175: 2721-2729. [Abstract/Free Full Text]
  7. Kirman, I., R. L. Whelan, O. H. Nielsen. 2004. Infleximab: mechanism of action beyond TNF-{alpha} neutralization in inflammatory bowel disease. Eur. J. Gastroenterol. Hepatol. 16: 639-641. [Medline]
  8. Danese, S., M. Sans, F. Scaldaferri, A. Sgambato, S. Rutella, A. Cittadini, J. M. Pique, J. Panes, J. A. Katz, A. Gasbarrini, C. Fiocchi. 2006. TNF-{alpha} blockade down-regulates the CD40/CD40L pathway in the mucosal microcirculation. J. Immunol. 176: 2617-2624. [Abstract/Free Full Text]
  9. Agnholt, J., K. Kaltoft. 2001. Infleximab downregulates interferon-{gamma} production in activated gut T-lymphocytes from patients with Crohn’s disease. Cytokine 15: 212-222. [Medline]
  10. Ross, S. E., R. O. Williams, L. J. Mason, C. Mauri, L. Marinova-Mutufchieva, A.-M. Malfait, R. N. Maini, M. Feldmann. 1997. Suppression of TNF-{alpha} expression, inhibition of Th1 activity and amelioration of collagen-induced arthritis by rolipram. J. Immunol. 159: 6253-6259. [Abstract]
  11. Palucka, A. K., J.-P. Blanck, L. Bennett, V. Pascual, J. Banchereau. 2005. , Cross-regulation of TNF and IFN-{alpha} in autoimmune diseases. Proc. Natl. Acad. Sci. USA 102: 3372-3377. [Abstract/Free Full Text]
  12. Kolls, J. K., A. Linden. 2004. Interleukin-17 family members and inflammation. Immunity 21: 467-476. [Medline]
  13. Lubberts, E., M. I. Koenders, W. B. van den Berg. 2005. The role of T cell interleukin-17 in conducting distractive arthritis. Arthritis Res. Ther. 7: 29-37. [Medline]
  14. Nakae, S., Y. Komiyama, A. Nambu, K. Sudo, M. Iwase, I. Homma, K. Sekikawa, M. Asano, Y. Iwakura. 2002. Antigen-specific T cell sensitization is impaired in IL-17-deficient mice, causing suppression of allergic cellular and humoral responses. Immunity 17: 375-387. [Medline]
  15. Chen, Y., C. L. Langrish, B. Mckenzie, B. Joyce-Shaikh, J. S. Stumhofer, T. McClanahan, W. Blumenschein, T. Churakovasa, J. Low, L. Presta, et al 2006. Anti-IL-23 therapy inhibits multiple inflammatory pathways and ameliorates autoimmune encephalomyelitis. J. Clin. Invest. 116: 1317-1326. [Medline]
  16. Banchereau, J., V. Pascual. 2006. Type I interferon in systemic lupus erythematosus and other autoimmune diseases. Immunity 25: 383-392. [Medline]
  17. Langrish, C. L., Y. Chen, W. M. Blumenschein, J. Mattson, B. Basham, J. D. Sedgwich, T. McClanahan, R. A. Kastelein, D. J. Cau. 2005. IL-23 drives a pathogenic T cell pollution that induces autoimmune inflammation. J. Exp. Med. 201: 233-240. [Abstract/Free Full Text]
  18. Park, H., Z. Li, X. O. Yang, S. H. Chang, R. Nurieva, Y.-H. Wang, Y. Wang, L. Hood, Z. Zhu, Q. Tian, C. Dong. 2005. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat. Immunol. 6: 1133-1141. [Medline]
  19. Wynn, T. A.. 2005. Th-17: a giant step from Th1 and Th2. Nat. Immunol. 6: 1069-1070. [Medline]
  20. Harrington, L. E., R. D. Hatton, P. R. Mangan, H. Turner, T. L. Murphy, K. M. Murphy, C. T. Weaver. 2005. Interleukin 17 producing CD4+ effector T cells develop via a lineage distinct from the helper type 1 and 2 lineage. Nat. Immunol. 6: 1123-1132. [Medline]
  21. Tato, C. M., J. J. O’Shea. 2006. What dose it mean to be just 17?. Nature 1441: 166-168.
  22. Vieiva, P. C., E. C. de Jong, E. A. Wierenga, M. L. Kapsenberg, P. Kalinski. 2000. Development of Th1-inducing capacity in myeloid dendritic cells requires environmental instruction. J. Immunol. 164: 4507-4512. [Abstract/Free Full Text]
  23. Zou, G. M., Y. K. Tam. 2002. Cytokines in the generation and maturation of dendritic cells. Eur. Cytokine Network 13: 186-199. [Medline]
  24. Caux, C., C. Massacrier, B. Vandervliet, B. Dubois, I. Durand, M. Cella, A. Lanzavecchia, J. Banchereau. 1997. CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to granulocyte-macrophage colony-stimulating factor plus tumor necrosis factor {alpha}. Blood 90: 1458-1470. [Abstract/Free Full Text]
  25. Lane, P. J. L., F. M. McConnell. 2001. Dendritic cell subsets and costimulation for effector T-cell responses. Curr. Opin. Pharmacol. 1: 409-416. [Medline]
  26. Sato, K., N. Yamashita, M. Baba, T. Matsuyama. 2003. Modified myeloid dendritic cells act as regulatory dendritic cells to induce anergic and regulatory T cells. Blood 101: 3581-3589. [Abstract/Free Full Text]
  27. Colonna, M., G. Trinchieri, Y.-J. Liu. 2004. Plasmacytoid dendritic cells in immunity. Nat. Immunol. 5: 1219-1226. [Medline]
  28. Hart, P. H., G. F. Vitti, D. R. Brugess, G. A. Whitty, D. S. Piccoli, J. H. Hamilton. 1989. Potential anti-inflammatory effects of interleukin-4. Proc. Natl. Acad. Sci. USA 86: 3803-3807. [Abstract/Free Full Text]
  29. Levings, M. K., J. W. Schrader. 1999. IL-4 inhibits the production of TNF-{alpha} and IL-12 by STAT6-dependent and -independent mechanisms. J. Immunol. 162: 5224-5229. [Abstract/Free Full Text]
  30. Tokayer, A., S. E. Carsons, B. Chokski, F. Santiago-Schwarz. 2002. High levels of interleukin 13 in rheumatoid arthritis sera are modulated by tumor necrosis factor antagonist therapy. J. Rheumatol. 29: 454-461. [Abstract/Free Full Text]
  31. Sallusto, F., A. Lanzavecchia. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor a. J. Exp. Med. 179: 1109-1118. [Abstract/Free Full Text]
  32. Happel, K., P. J. Dubin, M. Zheng, N. Ghilardi, C. Lockhart, L. J. Quinton, A. R. Odden, J. E. Shellito, G. J. Bagby, S. Nelson, J. K. Kolls. 2005. Divergent roles of IL-23 and IL-12 in host defense against Klebsiella pneumoniae. J. Exp. Med. 202: 761-769. [Abstract/Free Full Text]
  33. Iwamoto, S., M. Ishida, K. Takahashi, K. Takeda, A. Miyazaki. 2005. Lipopolysaccharide stimulation converts vigorously washed dendritic cells (DC) to non-exhausted DC expressing CD70 and evoking long-lasting type 1 T cell responses. J. Leukocyte Biol. 78: 383-392. [Abstract/Free Full Text]
  34. Langenkamp, A., M. Messi, A. Lanzavecchia, F. Sallusto. 2000. Kinetics of dendritic cell activation. Nat. Immunol. 4: 311-316.
  35. Agrawal, S., P. Anderson, M. Durbeej, N. van Rooijen, F. Ivars, G. Opdenakker, L. M. Sorokin. 2006. Dystroglycan is selectively cleaved at parenchymal basement membrain at site of leukocyte extravasation in experimental autoimmune encephalomyelitis. J. Exp. Med. 203: 1007-1019. [Abstract/Free Full Text]
  36. McClellan, S. A., X. Huang, R. P. Barrett, S. Lighvani, Y. Ahang, D. Richiert, L. D. Hazlett. 2006. Matrix metalloproteinase-9 amplifies the immune responses to pseudomonous aeruginosa corneal infection. Invest. Opthalmol. Visual Sci. 47: 256-264. [Abstract/Free Full Text]
  37. Iwai, S., A. Aljada, A. Higa, T. Nakanishi-Ueda, S. Fukuda, M. Kamegawa, S. Iwabuchi, T. Ueda, S. Caballero, R. Browne, et al 2006. Activation of AP-1 and increased synthesis of MMP-9 in the rabbit retina induced by lipid hydroperoxide. Curr. Eye Res. 31: 337-346. [Medline]
  38. Rieser, C., G. Bock, H. Klocker, G. Bartsch, M. Thurnher. 1997. Prostaglandin E2 and tumor necrosis factor {alpha} cooperate to activate human dendritic cells: synergistic activation of interleukin 12 production. J. Exp. Med. 186: 1603-1608. [Abstract/Free Full Text]
  39. Radstake, T. R., P. L. van Lent, G. J. Pesman, A. B. Blom, F. G. Sweep, J. Ronnelid, G. J. Adema, P. Barrera, W. B. van den Berg. 2004. High production of proinflammatory and Th1 cytokines by dendritic cells from patients with rheumatoid arthritis, and down regulation upon FcCR triggering. Ann. Rheum. Dis. 63: 696-702. [Abstract/Free Full Text]
  40. Santiago-Schwarz, F., E. Blilos, B. Diamond, S. E. Carsons. 1992. TNF in combination with GM-CSF enhances the differentiation of neonatal cord blood stem cells into dendritic cells and macrophages. J. Leukocyte Biol. 52: 274-281. [Abstract]
  41. Chomarart, P., C. Dantin, L. Bennett, J. Banchereau, A. K. Palucka. 2003. TNF skews monocyte differentiation from macrophages to dendritic cells. J. Immunol. 171: 2262-2269. [Abstract/Free Full Text]
  42. Thomas, R., L. S. Davis, P. Lipsky. 1994. Rheumatoid synovium is enriched in mature antigen-presenting dendritic cells. J. Immunol. 152: 2613-2623. [Abstract]
  43. Rooney, M., H. Varsani, P. R. Lombard, J.-M. Dayer, P. Woo. 2000. Tumor necrosis factor {alpha} and its soluble receptors in juvenile chronic arthritis. Rheumatology 39: 432-438. [Abstract/Free Full Text]
  44. Satoh, M., H. Inagawa, H. Minagawa, T. Kajikawa, H. Oshima, S. Abe, M. Yamazaki, D. Mizuno. 1985. Endogenous production of TNF in mice with immune complex as primer. J. Biol. Response Modif. 5: 140-147.
  45. Burger, D.. 2000. Cell contact interactions in rheumatology. Arthritis Res. 2: 472-476. [Medline]
  46. Veldhoen, M., R. J. Hocking, R. A. Flavell, B. Stockinger. 2006. Signals mediated by transforming growth factor-beta initiate autoimmune encephalomyelitis but chronic inflammation is needed to sustain disease. Nat. Immunol. 7: 1151-1156. [Medline]
  47. Jiang, D., J. Liang, J. Fan, S. Yu, S. Chen, Y. Luo, G. D. Prestwich, M. M. Mascarenhas, H. G. Garg, D. A. Quinn, et al 2005. Regulation of lung injury and repair by Toll-like receptors and hyaluronan. Nat. Med. 11: 1173-1179. [Medline]
  48. Brentano, F., D. Kyburz, O. Schorr, R. Gay, S. Gay. 2005. The role of Toll-like receptor signaling in the pathogenesis of arthritis. Cell. Immunol. 233: 90-96. [Medline]
  49. Page, G., A. Sattler, S. Kersten, A. Thiel, A. Radbruch, P. Miossec. 2004. Plasma cell like morphology of Th1-cytokine-producing cells associated with the loss of CD3 expression. Am. J. Pathol. 164: 409-417. [Abstract/Free Full Text]
  50. Schnurr, M., T. Toy, A. Shin, M. Wanger, J. Cebon, E. Maraskovsky. 2005. Extracellular nucleotide signaling by P2 receptors inhibits IL-12 and enhances IL-23 expression in human dendritic cells. Blood 105: 1582-1589. [Abstract/Free Full Text]
  51. Verreck, F. A. W., T. de Boer, D. M. L. Langenberg, M. A. Hoeve, M. Kramer, E. Vaisberg, R. Kastelein, A. Kolk, R. de Wall-Malefyt, T. H. M. Ottenhoff. 2004. Human IL-23-producing type 1 macrophages promote but IL-10-producing type 2 macrophages subert immunity to (myco)bacteria. Proc. Natl. Acad. Sci. USA 101: 4560-4565. [Abstract/Free Full Text]
  52. Komuro, I., Y. Yokota, S. Yasuda, A. Iwamoto, K. S. Kagawa. 2003. CSF-induced and HIV-1-mediated distinct regulation of Hck and C/EBPbeta represent a heterogenous susceptibility of monocyte-derived macrophages to M-tropic HIV-1 infection. J. Exp. Med. 198: 443-453. [Abstract/Free Full Text]
  53. Xu, J., R. Lucas, M. Schuchmann, S. Kuhnle, T. Meergans, A. P. Barreiros, A. W. Lohse, G. Otto, A. Wendel. 2003. GM-CSF restores innante, but not adaptive, immune responses in glucocorticoid-immunosuppressed human blood in vitro. J. Immunol. 171: 938-947. [Abstract/Free Full Text]
  54. Akiba, H., Y. Miyahira, M. Atsuta, K. Takeda, C. Nohara, T. Futagawa, H. Matsuda, T. Aoki, H. Yagita, K. Okumura. 2000. Critical contribution of OX40 ligand to T helper cell type 2 differentiation in experimental leishmaniasis. J. Exp. Med. 191: 375-380. [Abstract/Free Full Text]
  55. de Boer, O. J., F. Hirsch, A. C. van der Wal, C. M. van der Loos, P. K. Das, A. E. Becke. 1997. Costimulatory molecules in human atheroschlerotic plaques. Atherosclerosis 133: 227-234. [Medline]
  56. Bullock, T. J., H. Yagita. 2005. Induction of CD70 on dendritic cells through CD40 or TRL stimulation contributes to the development of CD8+ T cell responses in the absence of CD4 T cells. J. Immunol. 174: 710-717. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 The Journal of RheumatologyHome page
S.-Y. KAWASHIRI, A. KAWAKAMI, N. IWAMOTO, K. FUJIKAWA, T. ARAMAKI, M. TAMAI, K. ARIMA, M. KAMACHI, S. YAMASAKI, H. NAKAMURA, et al.
Proinflammatory Cytokines Synergistically Enhance the Production of Chemokine Ligand 20 (CCL20) from Rheumatoid Fibroblast-like Synovial Cells in vitro and Serum CCL20 Is Reduced in vivo by Biologic Disease-modifying Antirheumatic Drugs
J Rheumatol, November 1, 2009; 36(11): 2397 - 2402.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Noris, P. Cassis, N. Azzollini, R. Cavinato, D. Cugini, F. Casiraghi, S. Aiello, S. Solini, L. Cassis, M. Mister, et al.
The Toll-IL-1R Member Tir8/SIGIRR Negatively Regulates Adaptive Immunity against Kidney Grafts
J. Immunol., October 1, 2009; 183(7): 4249 - 4260.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
M. Hashimoto-Okada, T. Kitawaki, N. Kadowaki, S. Iwata, C. Morimoto, T. Hori, and T. Uchiyama
The CD70-CD27 interaction during the stimulation with dendritic cells promotes naive CD4+ T cells to develop into T cells producing a broad array of immunostimulatory cytokines in humans
Int. Immunol., August 1, 2009; 21(8): 891 - 904.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
A. M. Keller, Y. Xiao, V. Peperzak, S. H. Naik, and J. Borst
Costimulatory ligand CD70 allows induction of CD8+ T-cell immunity by immature dendritic cells in a vaccination setting
Blood, May 21, 2009; 113(21): 5167 - 5175.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
S. A. Gujar and T. I. Michalak
Primary Occult Hepadnavirus Infection Induces Virus-Specific T-Cell and Aberrant Cytokine Responses in the Absence of Antiviral Antibody Reactivity in the Woodchuck Model of Hepatitis B Virus Infection
J. Virol., April 15, 2009; 83(8): 3861 - 3876.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
C. A. Notley, J. J. Inglis, S. Alzabin, F. E. McCann, K. E. McNamee, and R. O. Williams
Blockade of tumor necrosis factor in collagen-induced arthritis reveals a novel immunoregulatory pathway for Th1 and Th17 cells
J. Exp. Med., October 27, 2008; 205(11): 2491 - 2497.
[Abstract] [Full Text] [PDF]

Home page
 GutHome page
M. Shale and S. Ghosh
Beyond TNF, Th1 and Th2 in inflammatory bowel disease
Gut, October 1, 2008; 57(10): 1349 - 1351.
[Full Text] [PDF]

Home page
 BloodHome page
K. M. Dhodapkar, S. Barbuto, P. Matthews, A. Kukreja, A. Mazumder, D. Vesole, S. Jagannath, and M. V. Dhodapkar
Dendritic cells mediate the induction of polyfunctional human IL17-producing cells (Th17-1 cells) enriched in the bone marrow of patients with myeloma
Blood, October 1, 2008; 112(7): 2878 - 2885.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
S. Mariotti, V. Sargentini, C. Marcantonio, E. Todero, R. Teloni, M. C. Gagliardi, A. R. Ciccaglione, and R. Nisini
T-cell-mediated and antigen-dependent differentiation of human monocyte into different dendritic cell subsets: a feedback control of Th1/Th2 responses
FASEB J, September 1, 2008; 22(9): 3370 - 3379.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
S. A. Gujar, A. K. Jenkins, C. S. Guy, J. Wang, and T. I. Michalak
Aberrant Lymphocyte Activation Precedes Delayed Virus-Specific T-Cell Response after both Primary Infection and Secondary Exposure to Hepadnavirus in the Woodchuck Model of Hepatitis B Virus Infection
J. Virol., July 15, 2008; 82(14): 6992 - 7008.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Iwamoto, S.
Right arrow Articles by Miyazaki, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Iwamoto, S.
Right arrow Articles by Miyazaki, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS