Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

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
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mizuno, K.
Right arrow Articles by Horio, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mizuno, K.
Right arrow Articles by Horio, T.
(Journal of Leukocyte Biology. 2001;70:386-394.)
© 2001 by Society for Leukocyte Biology

Muramyl dipeptide and mononuclear cell supernatant induce Langhans-type cells from human monocytes

Kana Mizuno, Hiroyuki Okamoto and Takeshi Horio

Department of Dermatology, Kansai Medical University, Moriguchi, Osaka 570-8507, Japan

Correspondence: Hiroyuki Okamoto, MD, Department of Dermatology, Kansai Medical University, 10-15 Fumizono, Moriguchi, Osaka 570-8507, Japan. E-mail: hokamoto{at}takii.kmu.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Muramyl dipeptide (MDP) in bacterial cell walls reportedly evokes epithelioid cell granulomas. We examined its effects on multinucleated-giant-cell (MGC) formation from monocytes. Supernatant of concanavalin A-stimulated peripheral blood mononuclear cells (conditioned medium) generated MGCs from monocytes. MDP significantly increased the fusion index of Langhans-type MGCs (LGCs) but did not affect total MGCs. N-Acetylmuramyl-L-alanyl-L-isoglutamine, an MDP analogue, had no effect on MGC formation. MGCs were produced by conditioned medium from CD14++/CD16- monocytes. MDP enhanced the LGC fusion index from CD14++/CD16- monocytes. MGCs were not produced from CD14+/CD16+ monocytes or immature dendritic cells induced by granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL) 4 and only weakly produced from macrophage (M)-CSF- or GM-CSF-induced macrophages. Added MDP did not generate MGCs from CD14+/CD16+ monocytes or dendritic cells but enhanced LGC formation from macrophages. Because IFN-{gamma}, IL-3, and GM-CSF reportedly are important in LGC induction, we added anti-IFN-{gamma}, anti-IL-3, or anti-GM-CSF monoclonal antibody (mAb) concomitantly to the monocyte culture treated with conditioned medium alone or plus MDP. Anti-IFN-{gamma} mAb completely abrogated MGC generation, whereas anti-GM-CSF and anti-IL-3 mAbs significantly inhibited LGCs. These findings suggest that CD14++/CD16- monocytes are fused to form LGCs by MDP derived from granulomatous-disease-causing pathogens with inflammatory mediators such as IFN-{gamma}, IL-3, and GM-CSF.

Key Words: CD14++/CD16- monocytes • granuloma • macrophages • dendritic cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The presence of multinucleated giant cells (MGCs) in the tuberculous granuloma was first described by Langhans in 1868 [1 ]. MGCs are also seen in other types of granulomas and considered to originate from the fusion of monocyte-macrophage lineage cells. Langhans-type giant cells (LGCs) are seen in many infectious granulomatous disorders such as tuberculosis and leprosy or in unknown pathological inflammatory granulomatous disorders such as sarcoidosis, whereas foreign-body-type giant cells (FGCs) are characteristic in foreign-body granulomas. They are also induced in vitro from human blood monocytes by the use of supernatants of lectin-stimulated mononuclear cells or cytokines [2 3 4 5 6 7 8 9 10 11 12 13 14 ]. Among the cytokines, interferon (IFN) {gamma} was reported to be one of the essential factors promoting monocyte fusion [2 , 4 ]. Morphologically, MGCs are classified into LGCs and FGCs. LGCs show a circular peripheral arrangement of nuclei, and FGCs have the nuclei scattered in an irregular fashion throughout the cell. McNally and Anderson [9 ] reported that differential regulation of morphological variants of MGC formation by interleukin (IL) 4 and IFN-{gamma}. IL-4 is closely related to FGC formation, and IFN-{gamma} together with either IL-3 or granulocyte macrophage (GM)-colony stimulating factor (CSF) initiates the formation of morphologically distinguishable LGCs. However, which subpopulation of monocyte-macrophage lineage cells is the precursor of MGCs in granulomatous diseases has not been determined.

The monocyte populations can be defined in human peripheral blood based on the expression of the CD14 and CD16 antigens (Ags). The major population of strongly CD14-positive monocytes (CD14++/CD16- monocytes) and the minor population of CD14-positive and CD16-positive monocytes (CD14+/CD16+ monocytes) can be recognized [15 ]. Expression of class II Ag on CD14+/CD16+ monocytes was higher than that on CD14++/CD16- monocytes. By contrast, the ability to perform Fc-receptor-mediated phagocytosis and adherence to plastic surfaces was increased in the CD14++/CD16- monocyte subset as compared with CD14+/CD16+ monocytes [16 ]. Recently, it has been reported that CD14+/CD16+ monocytes are increased in various diseases including sepsis [17 ], AIDS [18 19 20 21 ], tuberculosis [22 ], and Kawasaki disease [23 ]. The production of proinflammatory cytokines such as tumor necrosis factor (TNF), IL-1, and IL-6 by CD14++/CD16- monocytes was much higher than that by CD14+/CD16+ monocytes [24 ]. On the other hand, the anti-inflammatory cytokine IL-10 was produced by CD14++/CD16- monocytes but not by CD14+/CD16+ monocytes when they were stimulated by lipopolysaccharide [25 ]. However, it is unclear what the pathophysiological role of a CD14++/CD16- or CD14+/CD16+ monocyte population might be in inflammatory diseases, particularly in granulomatous disorders.

Muramyl dipeptide (MDP) is a peptidoglycan portion of the common structure of bacterial cell walls. MDP was originally found as a minimal essential structure for adjuvant activity of bacterial cell walls in studies using different fractions obtained from enzymatic digestion of cell walls of several bacteria [26 , 27 ]. It has been reported that MDP causes macrophage activation [28 29 30 31 ] and produces massive epithelioid granulomas in guinea pig [32 ] and rat [33 ] tissues that are indistinguishable from those produced by tubercle bacilli when injected as Freund-type water in oil emulsion [32 ]. In vitro examination shows that MDP augments the expression of adhesion molecules on monocytes [34 ]. From these findings, we hypothesize that MDP plays a role in the formation of MGC and in the development of granulomas by directly affecting monocyte-macrophage lineage cells.

In this study, we examined the effects of MDP on MGCs induced from various types of human monocyte-macrophage lineage cells by conditioned medium.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
RPMI 1640 medium (Nikken Biochem, Kyoto, Japan) was supplemented with 100µg/mL of streptomycin and 100 U/mL of penicillin (Gibco BRL, Grand Island, NY). MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine) and N-acetylmuramyl-L-alanyl-L-isoglutamine (L-MDP) were purchased from Sigma (St. Louis, MO). Recombinant human (rh) GM-CSF was kindly provided by Schering-Plough Japan (Osaka, Japan). rhIL-4 and rh macrophage-CSF (rhM-CSF) were obtained from Genzyme Corp. (Cambridge, MA). Mouse immunoglobulin G1 and mouse anti-human IL-3 monoclonal antibody (mAb) were obtained from R&D Systems (Minneapolis, MN). Mouse anti-human IFN-{gamma} mAb and mouse anti-human GM-CSF mAb were purchased from Genzyme-Techne (Cambridge, MA).

Isolation of monocytes
Isolation of human monocytes from peripheral blood mononuclear cells (PBMCs) was performed by depletion of nonmonocytes with an indirect-magnetic-labeling system monocyte isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). PBMCs were isolated from the heparinized blood of healthy adult volunteers by density gradient centrifugation with LymphoprepTM (Nycomed, Oslo, Norway) and washed twice in phosphate-buffered saline (PBS). For depletion of T cells, natural killer (NK) cells, B cells, dendritic cells, and basophils from PBMCs, the cells were indirectly magnetically labeled using a cocktail of hapten-conjugated CD3, CD7, CD19, CD45RA, CD56, and anti-IgE antibodies (Abs) and MACS MicroBeads coupled to an antihapten mAb. The magnetically labeled cells were depleted by retaining them on a MACS column in the magnetic field of the MidiMACS (Miltenyi Biotec). The isolated cells contained at least 90% monocytes as shown by their morphology and phenotype analyzed by cytofluorography on a FACScan (Becton Dickinson Immunocytometry System; Becton Dickinson, Mountain View, CA). These monocytes were used in our studies.

Isolation of CD14++/CD16- or CD14+/CD16+ monocytes
Magnetically sorted monocytes were stained for 15 min at room temperature with phycoerythrin (PE)-conjugated anti-CD14 mAb (PharMingen, San Diego, CA) and fluorescein isothiocyanate (FITC)-conjugated anti-CD16 mAb (PharMingen). The monocyte fractions were purified CD14++/CD16- monocytes and CD14+/CD16+ monocytes by EPICS ALTRATM (Beckman Coulter, Hialeah, FL).

Preparation of monocyte-derived immature dendritic cells or macrophages
To prepare immature dendritic cells or macrophages, a modified protocol described by Brand et al. [35 ] and Becker et al. [36 ] was used. Immature dendritic cells were differentiated from monocytes by culturing with rhGM-CSF (1,000 U/mL) + rhIL-4 (1,000 U/mL) for 4 days. Macrophages were induced from monocytes by culturing with rhM-CSF (1,000 U/mL) or rhGM-CSF (500 U/mL) for 4 days. These dendritic cells or macrophages were evaluated for morphology, phagocytic ability of latex particles, and positivity of CD1a, CD14, and CD68.

Generation of MGCs
PBMCs were cultured in RPMI 1640 supplemented with 10% fetal calf serum (Gibco BRL) and 16 µg/mL of concanavalin A (ConA; Sigma) at a density of 2 x 106 cells/mL for 72 h. The cell-free supernatant was used as conditioned medium and stored at -40°C before use. By the method of Möst et al. [13 ], MGCs were induced by culturing monocytes in RPMI 1640 with a final concentration of 50% conditioned medium. Monocytes were added to 48-well (Falcon 3078; Becton Dickinson, Lincoln Park, NJ) or 96-well (Falcon 3072; Becton Dickinson) tissue culture plates or eight-chamber slides for tissue culture (Lab-Tek; Nunc Inc., Naperville, IL) at a density of 2 x 105 cells/mL. To investigate the possible influence of concentration of MDP and L-MDP on MGC formation, monocytes were cultured with MDP (1, 10, 100, and 1,000µg/mL) or L-MDP (1, 10, and 100µg/mL). Three days later, the medium was removed, and cells were stained with Giemsa (Merck, Tokyo, Japan) in the plate. To disclose any possible influence of culture periods on MGC formation, monocytes were cultured with 10µg/mL of MDP for different periods. The medium was renewed every 3 days. At 1, 3, 5, 7, 10, 12, and 14 days after culturing, the medium was removed, and cells were stained with Giemsa in the plate. MGCs were defined as cells with more than three nuclei per cell. As reported previously [37 ], MGCs show the morphological features of LGCs when cytoskeletal components (microfilaments and microtubules) are maintained at equilibrium. On the other hand, disruption of these components by in vitro incubation with cytochalasin B and/or colchicine induce more typical FGCs. Therefore, LGCs have been defined as MGCs with a nuclear arrangement that is circular only. When MGCs had randomly arranged nuclei or both circularly arranged and unarranged nuclei, they were judged as FGCs, because the architecture of their cytoskeleton seemed already to be disrupted.

Determination of the fusion index
The fusion rate of monocytes was determined by examining the stained plate under a microscope using a x20 or x60 objective lens with x10 eyepieces and counting the number of nuclei within MGCs (more than three nuclei per cell) in a given area and the total number of nuclei in the same area. The fusion index (FI) was calculated according to the following formula: FI (%) = (number of nuclei within MGCs)/(total number of nuclei counted) x 100. Between 300 and 500 nuclei from selected representative fields were counted for each experiment.

Phenotyping with mAbs and expression of angiotensin-converting enzyme
The expression of cell surface markers, cytoplasmic antigens, and angiotensin-converting enzyme (ACE) of freshly isolated monocytes and MGCs was assessed using the following mAbs: anti-CD1a, anti-CD11a [lymphocyte function-associated antigen (LFA)-1], anti-CD54 [intercellular adhesion molecule (ICAM)-1], anti-CD68 (Serotec Ltd., Oxford, England), anti-CD11b (Ancell, Bayport, MN), anti-CD14, anti-CD16 (PharMingen), and anti-ACE (Chemicon, Temecula, CA). Monocytes and MGCs in an eight-well chamber slide were washed twice with PBS and fixed with 2% paraformaldehyde solution for 20 min at room temperature. After rinsing twice with PBS, the slide was incubated with propidium iodide (PI; Sigma) for 30 min at 4°C to stain the nuclei. After three rinses with PBS, the slide was incubated with each mAb for 60 min at 37°C. After three rinses with PBS, the slide was treated with FITC-conjugated rabbit anti-mouse IgG Ab (Caltag, Burlingame, CA) for 30 min at 37°C. The positive cells with fluorescence were observed with a personal confocal microscope system (FLUOVIEW; Olympus, Tokyo, Japan). To determine the extent of adhesion molecule expression, single-cell suspensions of cultured monocytes for 24 h were stained with anti-CD11a (LFA-1) and anti-CD54 (ICAM-1) mAbs. Monocytes were placed in microcentrifuge tubes and fixed with 1% paraformaldehyde. Cells were stained with 1:50 dilutions of the mAbs described above, by incubation on ice for 60 min. The cells were washed and then incubated with a 1:50 dilution of FITC-conjugated rabbit anti-mouse IgG Ab. They were then washed again and analyzed by cytofluorography on a FACScan immunocytometry system (Becton Dickinson).

Enzyme-linked immunosorbent assay for cytokine determination
Protein levels of IFN-{gamma}, GM-CSF, and IL-3 in the culture supernatants were measured by a commercially available enzyme immunoassay kit (Genzyme Corp.) according to the manufacturer’s instructions. The supernatants were obtained from cultures of monocytes in 50% conditioned medium with or without MDP and stored at -20°C. Each sample was examined in duplicate.

Neutralization with anti-IFN-{gamma} , anti-GM-CSF, and anti-IL-3 mAbs
To investigate the possible influence of IFN-{gamma}, GM-CSF, and IL-3 on MGC formation, MGCs were induced in the presence of anti-IFN-{gamma} mAb , anti-GM-CSF mAb, or anti-IL-3 mAb at concentrations of 50 µg/mL. The mAbs were added at the beginning of the culture and continued to present throughout the culture periods. Three days later, the medium was removed, and cells were stained with Giemsa in the plates.

Statistical analysis
Statistical significance of differences was determined by Student’s t-test, and a P value of <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of MDP or L-MDP on MGC formation from monocytes cultured with conditioned medium
Human monocytes were cultured in conditioned medium with or without MDP (1, 10, 100, or 1,000 µg/mL) or L-MDP (1, 10, or 100 µg/mL) for 3 days. The doses of these agents did not affect the viability of monocytes assessed by trypan blue exclusion. LGCs and FGCs were generated by conditioned medium (Fig. 1 ), and LGCs were preferentially induced by addition of MDP. FIs of LGCs, FGCs, and total MGCs reached 76.1 ± 7.5%, 16.5 ± 9.6%, and 92.6 ± 2.3% in the presence of MDP (10 µg/mL), whereas these FIs were 33.3 ± 12.9%, 45.8 ± 13.8%, and 81.0 ± 4.4% in conditioned medium only, respectively (Fig. 2 ). On the other hand, when monocytes were cultured in 16 µg /mL of ConA without conditioned medium, FIs of LGCs, FGCs, and total MGCs were 2.2 ± 0.1%, 4.9 ± 0.9%, and 7.1 ± 0.8%, respectively. L-MDP had no effect on MGCs induced by conditioned medium; the FIs of LGCs, FGCs, and total MGCs reached 21.1 ± 3.1%, 62.8 ± 9.7%, and 83.9 ± 4.6% in the presence of 100 µg/mL of L-MDP.



View larger version (101K):
[in this window]
[in a new window]
  		Figure 1. Photomicrographs of Giemsa-stained MGCs in 48-well tissue culture plates. (A) MGCs obtained from peripheral blood monocytes cultured with conditioned medium and 10 µg/mL of MDP for 3 days. (B) LGC with an annular or semilunar nuclear array. (C) FGC with randomly arranged nuclei. Original magnifications: A, x200; B and C, x600._art>

 


View larger version (49K):
[in this window]
[in a new window]
  		Figure 2. Influence of MDP on FI after culture for 3 days with conditioned medium. The FI in LGCs was increased by any concentration of MDP. *, P < 0.05 compared with the FI of each MGC from monocytes cultured without MDP. Results are expressed as the mean ± SD of three separate experiments.

 
Time Course of MGC formation from monocytes cultured with conditioned medium and MDP
Monocytes were cultured in conditioned medium with 10 µg/mL of MDP for 1 to 14 days (Fig. 3 ). After 1 day of culture, both types of MGCs were similarly induced, and the FIs of LGCs and FGCs were almost the same (LGCs, 12.7 ± 1.5%; FGCs, 15.7 ± 2.7%). The maximal FIs of LGCs and FGCs were obtained at 3 days of culture (LGCs, 66.5 ± 2.8%; FGC, 24.5 ± 2.6%). After that, they decreased with advancing duration of the culture to day 14.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 3. Time course of the FI in culture with conditioned medium and 10 µg/mL of MDP. The maximal FIs in LGCs and FGCs were reached at 3 days and decreased thereafter to day 14 of culture. Results are expressed as the mean ± SD of three separate experiments.

 
Cell Surface antigen expression and ACE expression
Expression of cell surface markers and ACE of freshly isolated monocytes and monocyte-derived MGCs were examined. The majority of monocytes expressed CD11a and CD14, whereas no monocytes expressed CD1a. CD11b, CD16, CD54, and CD68 were expressed on some monocytes. Both types of MGCs expressed CD14, CD11a, CD54, and CD68 in the cytoplasmic area (Fig. 4 ). CD14 and CD54 were also expressed on the cell surface of some MGCs. However, MGCs did not express CD1a, CD11b, or CD16. There was no difference in the extent and distribution pattern of the expression of cell surface markers between LGCs and FGCs. ACE is released from epithelioid cells from the granulomatous disorders, and serum levels are reported to be correlated with total granuloma mass [38 ]. ACE was detected in the cytoplasmic area of most MGCs (Fig. 5 ) but not in monocytes. There were no differences in the extent and distribution pattern of ACE expression between LGCs and FGCs. To determine whether the expression of adhesion molecules on monocytes is enhanced by MDP before fusion, single-cell suspensions of cultured monocytes for 24 h were stained with anti-CD11a (LFA-1) and anti-CD54 (ICAM-1) mAbs and analyzed by cytofluorography on a FACScan. Figure 6 shows that the expression of CD54 on monocytes was enhanced, whereas that of CD11a was not changed. However, there were no differences in the extent of CD54 expression between monocytes cultured in conditioned medium and those in conditioned medium with MDP.



View larger version (41K):
[in this window]
[in a new window]
  		Figure 4. Expression of surface markers for monocyte-macrophage lineage cells and adhesion molecules in MGCs. MGCs were stained with mAb recognizing FITC-rabbit anti-mouse IgG Ab (green), and the nuclei were stained with PI (red). Both types of MGCs expressed CD11a, CD14, CD54, and CD68 in the cytoplasmic area. CD14 and CD54 were also expressed on the cell surface of some MGCs. However, MGCs did not express CD1a, CD11b, or CD16. There was no difference in the extent and distribution pattern of the expression of cell surface markers between LGCs and FGCs._art>

 


View larger version (25K):
[in this window]
[in a new window]
  		Figure 5. Expression of ACE in MGCs. MGCs were stained with anti-ACE Ab recognizing FITC-rabbit anti-mouse IgG Ab (green), and the nuclei were stained with PI (red)._art>

 


View larger version (27K):
[in this window]
[in a new window]
  		Figure 6. Expression of CD54 on monocytes cultured with conditioned medium with or without 10 µg/mL of MDP for 24 h. The expression of CD54 on monocytes was enhanced, whereas that of CD11a was not changed. There were no differences in the extent of CD54 expression between monocytes cultured in conditioned medium and those in conditioned medium with 10 µg/mL of MDP.

 
Generation of MGCs from subpopulations of monocytes, dendritic cells, or macrophages
To distinguish the precursor cells of MGCs, we examined MGC formation by sorted cells in CD14 and CD16 (Fig. 7 ) or differentiated cells from monocytes stimulated with cytokines (Table 1 ). CD14++ /CD16- monocytes, the major population of monocytes, exhibited the same FIs of LGCs, FGCs, and total MGCs as those in whole monocytes. On the other hand, MGCs were not produced from CD14+/CD16+ monocytes by conditioned medium with or without MDP. Four days were necessary to differentiate monocytes into dendritic cells (group 4) or macrophages (groups 5 and 6). These cells were then stimulated by conditioned medium with or without MDP. When monocytes were cultured in RPMI 1640 medium for 4 days and then stimulated by conditioned medium plus MDP for 3 days, the FI of MGCs was 4.6 ± 0.7% (group 1). However, when freshly isolated monocytes were cultured in conditioned medium with MDP for 7 days, the FI of MGCs was 73.7 ± 3.5% [group 3 (positive control)]. MGCs were not generated from dendritic cells induced by GM-CSF+Il-4 when cultured with conditioned medium for 3 days. Addition of MDP did not induce MGCs. On the other hand, only a small number of MGCs were obtained from macrophages induced by M-CSF or GM-CSF when cultured with conditioned medium (FIs: M-CSF, 3.6 ± 1.1%; GM-CSF, 2.3 ± 1.2%) for 3 days. Addition of MDP slightly enhanced formation of MGCs from M-CSF-induced macrophages (FI = 8.3 ± 1.2%), most of which were LGCs.



View larger version (30K):
[in this window]
[in a new window]
  		Figure 7. MGC formation from CD14++/CD16- or CD14+/CD16+ monocytes cultured in conditioned medium. (A) Two-color immunofluorescence analysis of monocyte subpopulation. Magnetically sorted monocytes was stained with PE-conjugated anti-CD14 Ab and FITC-conjugated anti-CD16 Ab. (B) FI in MGCs from each monocyte subpopulation. CD14++/CD16- monocytes exhibited the same FI of LGCs, FGCs, and total MGCs as those in whole monocytes. CD14+/CD16+ monocytes did not lead to MGCs by conditioned medium with or without MDP. *, P < 0.05 compared with the FI of each MGC from monocytes cultured without MDP. Results are expressed as the mean ± SD of three separate experiments. ND, not detected.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Fusion Index of MGCs from Immature Dendritic Cells or Macrophages

 
Determination of IFN-{gamma}, GM-CSF, and IL-3 in supernatants
The supernatants were obtained from 3-day cultures of monocytes in 50% conditioned medium with or without MDP. Before exposure to monocytes, protein levels of IFN-{gamma}, GM-CSF, and IL-3 in conditioned medium were 32.1 ± 7.5 IU/mL, 650.6 ± 19.6 pg/mL, and 580.0 ± 22.3 pg/mL, respectively. These cytokine levels in culture supernatants without MDP harvested after 3-day culturing were 65.1 ± 1.6 IU/mL, 1,308.3 ± 13.2 pg/mL, and 513.0 ± 15.3 pg/mL, respectively. On the other hand, when cultured with MDP, the levels of IFN-{gamma}, GM-CSF, and IL-3 in the supernatants were 66.8 ± 2.9 IU/mL, 1,322.6 ± 17.3 pg/mL, and 511.0 ± 18.9 pg/mL, respectively. The results indicated that MDP did not affect the cytokine production from monocytes cultured with conditioned medium.

Effects of anti-IFN-{gamma}, anti-GM-CSF, and anti-IL-3 mAbs on LGC generation
To further understand the association of IFN-{gamma}, GM-CSF, and IL-3 with LGC formation, the effect of mAbs against these cytokines on LGC formation was examined. When anti-IFN-{gamma} mAb was added concomitantly to the culture of monocytes treated with conditioned medium alone or conditioned medium and MDP, MGC generation was completely abrogated. On the other hand, when anti-GM-CSF or anti-IL-3 mAb was added, only LGC generation was significantly inhibited (Fig. 8 ). However, in the presence of MDP, 33.0 ± 4.5% or 25.2 ± 5.2% of the FI of LGCs was still observed in the anti-GM-CSF mAb- or anti-IL-3 mAb-treated group, respectively.



View larger version (33K):
[in this window]
[in a new window]
  		Figure 8. Effect of anti-IFN-{gamma}, anti-GM-CSF, and anti-IL-3 mAbs on MGC generation (A) in conditioned medium- or (B) conditioned medium and 10µg/mL MDP-induced MGCs. Normal mouse IgG, anti-IFN-{gamma} mAb, anti-GM-CSF mAb, or anti-IL-3 mAb was added at the beginning of the culture. Each antibody was used at the final concentration of 50µg/mL. Anti-IFN-{gamma} mAb completely inhibited MGC generation, whereas anti-GM-CSF and IL-3 mAb partially inhibited this process, predominantly for LGCs. *, P < 0.05 compared with the FI of each MGC without Abs. Results are expressed as means ± SD of three separate experiments. ND, not detected.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
MDP is a minimal common structure that is contained generally in bacterial cell walls. It is known to have biologic properties such as pyrogenicity and resistance against bacterial infections [39 ]. Kuroe et al. [40 ] showed that MDP induced granulomatous enterocolitis in rabbits by injection together with Freund’s incomplete adjuvant. Massive epithelioid granulomas in the skin were also produced by injecting MDP into rat footpads [33 ] or MDP and Freund’s incomplete adjuvant into guinea pig footpads [32 ]. MDP can induce granulomas more effectively than tubercle bacillus [32 ]. Also, MDP has elicited uveitis characterized by monocyte infiltrate [41 ]. Thus, MDP induces monocyte-associated inflammatory disorders. Furthermore, there is growing evidence that MDP enhances the function of monocyte-macrophage lineage cells, including phagocytosis, migration activity of monocytes [42 ], macrophage spreading, and adherence and synthesis of the collagenase enzyme [30 ]. The current study shows that MDP but not an analogue of MDP without adjuvant activity preferentially induced LGCs, which are characteristic cells in tuberculosis and sarcoidosis, from human monocytes stimulated together with conditioned medium.

The mechanisms by which MDP modulates MGC formation in vitro are unknown. The involvement of cell adhesion molecules has been documented in the process of cell fusion. Most et al. [4 ] reported that the expression of LFA-1 (CD11a) is required for monocyte fusion. To support the importance of adhesion molecules in granulomas, Saha et al. [43 ] observed that the expression of CD54 molecules increased by 108% on Mycobacterium tuberculosis-infected murine macrophages. CD11a/CD54 interaction has also been associated with the formation of MGCs from HIV-infected lymphocytes [7 , 44 45 46 ]. MDP has been reported to increase the expression of CD11a, b, and c; CD18; and CD54 on monocytes [34 , 47 ]. Our fluorescein-activated cell sorter analysis also demonstrated that the expression of CD54 on monocytes was enhanced when monocytes were cultured with conditioned medium and MDP for 24 h. However, there were no differences in the extent of CD54 expression between monocytes cultured in conditioned medium and those in conditioned medium with MDP. CD54 has been localized on the membrane where the cell-to-cell contact occurs on clustering of monocytes, but it stains in the center of the giant cell in newly formed MGCs [8 ]. This result was also seen in our studies. Therefore, it is likely that the expression of cell adhesion molecules of monocytes is important for MGC formation but not for predominant induction of LGCs.

McNally and Anderson [9 ] described the differential regulation of macrophage fusion by various cytokines. IL-4 leads to formation of FGCs, whereas IFN-{gamma} induced LGCs together with IL-3 or GM-CSF. We measured the content of IFN-{gamma}, IL-3, and GM-CSF in the supernatants of our culture system. These cytokines were detected at almost the same levels regardless of the presence or absence of MDP. Therefore, MDP augmented the formation of LGCs independently on these cytokines. On the other hand, when we added anti-IFN-{gamma}, anti-IL-3, or anti-GM-CSF mAb concomitantly to the culture of monocytes treated with conditioned medium alone or conditioned medium and MDP, anti-IFN-{gamma} mAb completely abrogated MGC generation, and both anti-GM-CSF and IL-3 mAbs inhibited LGC generation. These findings indicated that IFN-{gamma} is necessary to induce MGCs and that IL-3 and GM-CSF are important factors for induction of LGCs. However, because about 30% of the FI of LGCs was still observed when GM-CSF or IL-3 was added in the culture with MDP, other LGC-related cytokines may be released from monocytes stimulated with MDP together with conditioned medium. Byrd [14 ] reported that IFN-{gamma} and IL-3 promote LGC formation and dense growth of M. tuberculosis surrounded by a ring of nuclei localized to the centers of LGCs and supposed that physical sequestration of M. tuberculosis by LGCs might limit the spread of this pathogen, thereby restricting growth. Since MDP is a component of cell walls of M. tuberculosis, there is a possibility that MDP is a key factor in the pathogen which determines the distribution of nuclei of MGCs.

Previous studies have shown that the cytoskeletal systems are involved in the movement and orientation of nuclei in MGCs [48 49 50 51 ]. In vitro incubation with cytochalasin B and/or colchicine disrupts the structure of LGCs and generates a cytoarchitecture that is more typical of FGCs [37 ]. There were no differences in monocyte-macrophage markers and adhesion molecules on the cell surface between LGCs and FGCs. Therefore, MDP was considered to affect MGCs morphologically but not phenotypically. Further investigation on the effects of MDP on parts of the cytoskeletal system such as the microtubule structure is necessary.

Two subpopulations of monocytes are distinguished in human peripheral blood based on CD14 and CD16 expression. CD14++/CD16- monocytes are the major population, and CD14+/CD16+ monocytes are considered to resemble tissue macrophages [52 ]. The mRNA levels for IL-10, phagocytosis, and reactive-oxygen production in CD14+/CD16+ monocytes are lower than those in CD14++/CD16- monocytes [15 ]. Our studies showed that fusion rates of MGCs and LGCs were the same between whole monocytes and CD14++/CD16- monocytes. On the other hand, MGCs were not produced from CD14+/CD16+ monocytes. MDP enhanced LGC formation from whole monocytes or CD14++/CD16- monocytes but did not affect MGC formation from CD14+/CD16+ monocytes. Monocytes can also be differentiated into two types of immunocompetent cells, macrophages and dendritic cells, by cytokines such as M-CSF, GM-CSF, and IL-4 [35 , 36 , 53 , 54 ]. Möst et al. [13 ] examined the influence of monocyte-to-macrophage maturation on the ability of human monocytes-macrophages to fuse with each other. In their report, fusion rates gradually decreased with the stage of monocyte differentiation to macrophages. Our study used dendritic cells and macrophages from monocytes induced by GM-CSF plus IL-4 and M-CSF or GM-CSF, respectively. MGCs were not generated from dendritic cells and only weakly generated from macrophages by stimulation with conditioned medium and MDP. Therefore, precursor cells of MGCs induced by conditioned medium with or without MDP were CD14++/CD16- monocytes.

In conclusion, MDP predominantly induced LGCs from human monocytes stimulated together with peak production at day 3 of culture. Their precursor cells were CD14++/CD16- monocytes but not CD14+/CD16+ monocytes or differentiated cells from monocytes such as macrophages and dendritic cells. Not only is MDP a component of cell walls of M. tuberculosis, but it is also detected in epithelioid cells and MGCs of granulomatous lesions [55 ]. Therefore, these data raise the possibility that in granulomatous disorders such as tuberculosis and sarcoidosis, CD14++/CD16- monocytes may infiltrate into the lesional tissues and be fused to form LGCs by inflammatory mediators and MDP derived from the pathogens of the disorders.


   ACKNOWLEDGEMENTS
 
This work was supported by grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (11670855) and by grants from Kansai Medical University (research grant D). We thank Mr. Kazumi Kobayashi and Miss Sachiko Miura for their excellent technical assistance.

Received September 27, 2000; revised April 9, 2001; accepted April 16, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Langhans, T. (1868) Ueber Riesenzellen mit wandstandigen Kernen in Tuberkeln und die fibrose Forn des Tuberkels Virchows Arch. Pathol. Anat 42,382-404
  2. Postlethwaite, A. E., Jackson, B. K., Beachey, E. H., Kang, A. H. (1982) Formation of multinucleated giant cells from human monocyte precursors: mediation by a soluble protein from antigen- and mitogen-stimulated lymphocytes J. Exp. Med 155,168-178[Abstract/Free Full Text]
  3. Kreipe, H., Radzun, H. J., Rudolph, P., Barth, J., Hansmann, M., Heidorn, K., Parwaresch, M. R. (1988) Multinucleated giant cells generated in vitro: terminally differentiated macrophages with down-regulated c-fms expression Am. J. Pathol 130,232-243[Abstract]
  4. Möst, J., Neumayer, H. P., Dierich, M. P. (1990) Cytokine-induced generation of multinucleated giant cells in vitro requires interferon-{gamma} and expression of LFA-1 Eur. J. Immunol 20,1661-1667[Medline]
  5. Enelow, R. I., Sullivan, G. W., Carper, H. T., Mandell, G. L. (1992) Induction of multinucleated giant cell formation from in vitro culture ofhuman monocytes with interleukin-3 and interferon-{gamma}: comparison with other stimulating factors Am. J. Respir. Cell Mol. Biol 6,57-62
  6. Takashima, T., Ohnishi, K., Tsuyuguchi, I., Kishimoto, S. (1993) Differential regulation of formation of multinucleated giant cells from concanavalin A-stimulated human blood monocytes by IFN-{gamma} and IL-4 J. Immunol 150,3002-3010[Abstract]
  7. Kazazi, F., Chang, J., Lopez, A., Vadas, M., Cunningham, A. L. (1994) Interleukin 4 and human immunodeficiency virus stimulate LFA-1-ICAM-1-mediated aggregation of monocytes and subsequent giant cell formation J. Gen. Virol 75,2795-2802[Abstract/Free Full Text]
  8. Fais, S., Burgio, V. L., Silvestri, M., Capobianchi, M. R., Pacchiarotti, A., Pallone, F. (1994) Multinucleated giant cells generation induced by interferon-{gamma}: changes in the expression and distribution of the intercellular adhesion molecule-1 during macrophage fusion and multinucleated giant cell formation Lab. Invest 71,737-744[Medline]
  9. McNally, A. K., Anderson, J. M. (1995) Interleukin-4 induces foreign body giant cells from human monocytes/macrophages: differential lymphokine regulation of macrophage fusion leads to morphological variants of multinucleated giant cells Am. J. Pathol 147,1487-1499[Abstract]
  10. Matsuda, S., Akagawa, K., Honda, M., Yokota, Y., Takebe, Y., Takemori, T. (1995) Suppression of HIV replication in human monocyte-derived macrophages induced by granulocyte/macrophage colony-stimulating factor AIDS Res. Hum. Retrovir 11,1031-1038[Medline]
  11. Akagawa, K., Takasuka, N., Nozaki, Y., Komuro, I., Azuma, M., Ueda, M., Naito, M., Takahashi, K. (1996) Generation of CD1+ RelB+ dendritic cells and tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated giant cells from human monocytes Blood 88,4029-4039[Abstract/Free Full Text]
  12. Ikeda, T., Ikeda, K., Sasaki, K., Kawakami, K., Hatake, K., Kaji, Y., Norimatsu, H., Harada, M., Takahara, J. (1998) IL-13 as well as IL-4 induces monocytes/macrophages and a monoblastic cell line (UG3) to differentiate into multinucleated giant cells in the presence of M-CSF Biochem. Biophys. Res. Commun 253,265-272[Medline]
  13. Möst, J., Spötl, L., Mayr, D., Gasser, A., Sarti, A., Dierich, M. P. (1997) Formation of multinucleated giant cells in vitro is dependent on the stage of monocyte to macrophage maturation Blood 89,662-671[Abstract/Free Full Text]
  14. Byrd, T. F. (1998) Multinucleated giant cell formation induced by IFN-{gamma}/IL-3 is associated with restriction of virulent Mycobacterium tuberculosis cell to cell invasion in human monocyte monolayers Cell. Immunol 188,89-96[Medline]
  15. Ziegler-Heitbrock, H. W. (1996) Heterogeneity of human blood monocytes: the CD14+CD16+ subpopulation Immunol. Today 17,424-428[Medline]
  16. Passlick, B., Flieger, D., Ziegler-Heitbrock, H. W. L. (1989) Identification and characterization of a novel monocyte subpopulation in human peripheral blood Blood 74,2527-2534[Abstract/Free Full Text]
  17. Fingerle, G., Pforte, A., Passlick, B., Blumenstein, M., Ströbel, M., Ziegler-Heitbrock, H. W. L. (1993) The novel subset of CD14+/CD16+ blood monocytes is expanded in sepsis patients Blood 82,3170-3176[Abstract/Free Full Text]
  18. Allen, J. B., Wong, H. L., Guyre, P. M., Simon, G. L., Wahl, S. M. (1991) Association of circulating receptor Fc gamma RIII-positive monocytes in AIDS patients with elevated levels of transforming growth factor-beta J. Clin. Invest 87,1773-1779
  19. Capsoni, F., Minonzio, F., Ongari, A. M., Bonara, P., Pinto, G., Carbonelli, V., Lazzarin, A., Zanussi, C. (1994) Fc receptors expression and function in mononuclear phagocytes from AIDS patients: modulation by IFN-gamma Scand. J. Immunol 39,45-50[Medline]
  20. Nockher, W. A., Bergmann, L., Scherberich, J. E. (1994) Increased soluble CD14 serum levels and altered CD14 expression of peripheral blood monocytes in HIV-infected patients Clin. Exp. Immunol 98,369-374[Medline]
  21. Locher, C., Vanham, G., Kestens, L., Kruger, M., Ceuppens, J. L., Vingerhoets, J., Gigase, P. (1994) Expression patterns of Fc gamma receptors, HLA-DR and selected adhesion molecules on monocytes from normal and HIV-infected individuals Clin. Exp. Immunol 98,115-122[Medline]
  22. Vanham, G., Edmonds, K., Qing, L., Hom, D., Toossi, Z., Jones, B., Daley, C. L., Huebner, B., Kestens, L., Gigase, P, Ellner, J. J. (1996) Generalized immune activation in pulmonary tuberculosis: co-activation with HIV infection Clin. Exp. Immunol 103,30-34[Medline]
  23. Katayama, K., Matsubara, T., Fujiwara, M., Koga, M., Furukawa, S. (2000) CD14+ CD16+ monocyte subpopulation in Kawasaki disease Clin. Exp. Immunol 121,566-570[Medline]
  24. Ziegler-Heitbrock, H. W. L., Strobel, M., Kieper, D., Fingerle, G., Schlunck, T., Petersmann, I., Ellwart, J., Blumenstein, M., Haas, J. G. (1992) Differential expression of cytokines in human blood monocyte subpopulations Blood 79,503-511[Abstract/Free Full Text]
  25. Frankenberger, M., Sternsdorf, T., Pechumer, H., Pforte, A., Ziegler-Heitbrock, H. W. L. (1996) Differential cytokine expression in human blood monocyte subpopulations: a polymerase chain reaction analysis Blood 87,373-377[Abstract/Free Full Text]
  26. Ellouz, F., Adam, A., Ciorbaru, R., Lederer, E. (1974) Minimal structural requirements for adjuvant activity of bacterial peptidoglycan derivatives Biochem. Biophys. Res. Commun 59,1317-1325[Medline]
  27. Kotani, S., Watanabe, Y., Kinoshita, F., Shimono, T., Morisaki, I., Shiba, T., Kusumoto, S., Tarumi, Y., Ikenaka, K. (1975) Immunoadjuvant activities of synthetic N-acetylmuramyl peptides or -amino acids Biken J 18,105-111[Medline]
  28. Tanaka, A., Nagao, S., Saito, R., Kotani, S., Kusumoto, S., Shiba, T. (1977) Correlation of stereochemically specific structure in muramyl dipeptide between macrophage activation and adjuvant activity Biochem. Biophys. Res. Commun 77,621-627[Medline]
  29. Fevrier, M., Birrien, J. L., Leclerc, C., Chedid, L., Liacopoulos, P. (1978) The macrophage, target cell of the synthetic adjuvant muramyl dipeptide Eur. J. Immunol 8,558-562[Medline]
  30. Wahl, S. M., Wahl, L. M., McCarthy, J. B., Chedid, L., Mergenhagen, S. E. (1979) Macrophage activation by mycobacterial water soluble compounds and synthetic muramyl dipeptide J. Immunol 122,2226-2231[Abstract/Free Full Text]
  31. Dinarello, C. A., Elin, R. J., Chedid, L., Wolff, S. M. (1978) The pyrogenicity of the synthetic adjuvant muramyl dipeptide and two structural analogues J. Infect. Dis 138,760-767[Medline]
  32. Tanaka, A., Emori, K. (1980) Epithelioid granuloma formation by a synthetic bacterial cell wall component, muramyl dipeptide (MDP) Am. J. Pathol 98,733-748[Abstract]
  33. Baba, T., Sakaguchi, N., Hotchi, M., Ohno, S. (1992) Three-dimensional study of epithelioid cells by quick-freezing and deep etching method in muramyl dipeptide-induced granulomas Virchows Arch. B Cell. Pathol 63,63-70[Medline]
  34. Darcissac, E. C. A., Bahr, G. M., Parant, M. A., Chedid, L. A., Riveau, G. J. (1996) Selective induction of CD11a,b,c/CD18 and CD54 expression at the cell surface of human leukocytes by muramyl peptide Cell. Immunol 169,294-301[Medline]
  35. Brand, U., Bellinghausen, I., Enk, A. H., Jonuleit, H., Becker, D., Knop, J., Saloga, J. (1998) Influence of extracellular matrix proteins on the development of cultured human dendritic cells Eur. J. Immunol 28,1673-1680[Medline]
  36. Becker, S., Warren, M. K., Haskill, S. (1987) Colony-stimulating factor-induced monocyte survival and differentiation into macrophages in serum-free cultures J. Immunol 139,3703-3709[Abstract]
  37. Rigby, P. J., Papadimitriou, J. M. (1984) Cytoskeletal control of nuclear arrangement in Langhans multinucleate giant cells J. Pathol 143,17-29[Medline]
  38. Allen, R. K. (1991) A review of angiotensin converting enzyme in health and disease Sarcoidosis 8,95-100[Medline]
  39. Chedid, L., Parant, M., Parant, F., Lefrancier, P., Choay, J., Lederer, E. (1977) Enhancement of nonspecific immunity to Klebsiella pneumoniae infection by a synthetic immunoadjuvant (N-acetylmuramyl-L-alanyl-D-isoglutamine) and several analogs Proc. Natl. Acad. Sci. USA 74,2089-2093[Abstract/Free Full Text]
  40. Kuroe, K., Haga, Y., Funakoshi, O., Mizuki, I., Kanazawa, K., Yoshida, Y. (1995) Rabbit granulomatous enterocolitis induced by injection of muramyl dipeptide emulsified with Freund’s incomplete adjuvant J. Gastroenterol 30,472-477[Medline]
  41. Fox, A., Hammer, M. E., Lill, P., Burch, T. G., Burrish, G. (1984) Experimental uveitis; elicited by peptidoglycan-polysaccharide complexes, lipopolysaccharide, and muramyl dipeptide Arch.Ophthalmol 102,1063-1067[Abstract]
  42. Akiyama, Y., Griffith, R., Miller, P., Stevenson, G. W., Lund, S., Kanapa, D. J., Stevenson, H. C. (1988) Effects of adherence, activation and distinct serum proteins on the in vitro human monocyte maturation process J. Leukoc. Biol 43,224-231[Abstract]
  43. Saha, B., Das, G., Vohra, H., Ganguly, N. K., Mishra, G. C. (1994) Macrophage-T cell interaction in experimental mycobacterial infection. Selective regulation of co-stimulatory molecules on Mycobacterium-infected macrophages and its implication in the suppression of cell-mediated immune response Eur. J. Immunol 24,2618-2624[Medline]
  44. Hildreth, J. E. K., Orentas, R. J. (1989) Involvement of a leukocyte adhesion receptor (LFA-1) in HIV-induced syncytium formation Science 244,1075-1078[Abstract/Free Full Text]
  45. Valentin, A., Lundin, K., Patarroyo, M., Asjö, B. (1990) The leukocyte adhesion glycoprotein CD18 participates in HIV-1-induced syncytia formation in monocytoid and T-cells J. Immunol 144,934-937[Abstract]
  46. Pantaleo, G., Butini, L., Graziosi, C., Poli, G., Schnittman, S. M., Greenhouse, J. J., Gallin, J. I., Fauci, A. S. (1991) Human immunodeficiency virus (HIV) infection in CD4+ T-lymphocytes genetically deficient in LFA-1; is required for HIV-mediated cell fusion but not for viral transmission J. Exp. Med 173,511-514[Abstract/Free Full Text]
  47. Heinzelmann, M., Mercer-Jones, M. A., Gardner, S.A, Wilson, M. A., Polk, H. C. (1997) Bacterial cell wall products increase monocyte HLA-DR and ICAM-1 without affecting lymphocyte CD18 expression Cell. Immunol 176,127-134[Medline]
  48. Cain, H., Kraus, B., Fringes, B., Osborn, M., Weber, K. (1981) Centrioles, microtubules and microfilaments in activated mononuclear and multinucleate macrophages from rat peritoneum: electron-microscopic and immunofluorescence microscopic studies J. Pathol 133,301-323[Medline]
  49. Holmes, K. V., Choppin, P. W. (1968) On the role of microtubules in movement and alignment of nuclei in virus-induced syncytia J. Cell. Biol 39,526-543[Abstract/Free Full Text]
  50. Sapp, J. P. (1976) An ultrastructural study of nuclear and centriolar configurations in multinucleated giant cells Lab. Invest 34,109-114[Medline]
  51. Wang, E., Cross, R. K., Choppin, P. W. (1979) Involvement of microtubules and 10-nm filaments in the movement and positioning of nuclei in syncytia J. Cell. Biol 83,320-337[Abstract/Free Full Text]
  52. Ziegler-Heitbrock, H.W, Fingerle, G., Strobel, M., Schraut, W., Stelter, F., Schutt, C., Passlick, B., Pforte, A. (1993) The novel subset of CD14+/CD16+ blood monocytes exhibits features of tissue macrophages Eur. J. Immunol 23,2053-2058[Medline]
  53. Zhou, L. J., Tedder, T. F. (1996) CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells Proc. Natl. Acad. Sci. USA 93,2588-2592[Abstract/Free Full Text]
  54. Laupeze, B., Fardel, O., Onno, M., Bertho, N., Drenou, B., Fauchet, R., Amiot, L. (1999) Differential expression of major histocompatibility complex class Ia, Ib, and II molecules on monocytes-derived dendritic and macrophagic cells Hum. Immunol 60,591-597[Medline]
  55. Eishi, Y. (1994) Seeking a causative agent of sarcoidosis Sarcoidosis 11,265-268



This article has been cited by other articles:


Home page
 J. Immunol.Home page
A. Krug, S. Rothenfusser, S. Selinger, C. Bock, M. Kerkmann, J. Battiany, A. Sarris, T. Giese, D. Speiser, S. Endres, et al.
CpG-A Oligonucleotides Induce a Monocyte-Derived Dendritic Cell-Like Phenotype That Preferentially Activates CD8 T Cells
J. Immunol., April 1, 2003; 170(7): 3468 - 3477.
[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
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mizuno, K.
Right arrow Articles by Horio, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mizuno, K.
Right arrow Articles by Horio, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS