Published online before print February 3, 2004
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Department of Biomolecular Science, Faculty of Science, Toho University, Chiba, Japan
1 Correspondence: Yoshiro Kobayashi, Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan. E-mail: yoshiro{at}biomol.sci.toho-u.ac.jp
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Key Words: phagocytosis • MIP-2
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Under pathological conditions, however, the number of apoptotic cells appearing may be too large for efficient phagocytosis, leading to an advanced stage of apoptosis, late apoptosis. We have demonstrated that coculturing macrophages, such as thioglycollate broth-induced peritoneal exudate cells (PEC) and Kupffer cells, with late apoptotic cytotoxic T (CTLL)-2 cells induce the production of proinflammatory cytokines macrophage-inflammatory protein (MIP)-2, a murine homologue of human IL-8, and tumor necrosis factor 
(TNF-) but not IL-1, IL-1ß, and IL-6 at the mRNA levels. MIP-2 but not TNF- was expressed as a protein in a coculture of macrophages with apoptotic cells [5
, 6
]. Moreover, transient infiltration of neutrophils was observed when late apoptotic CTLL-2 or P388 cells were injected into the peritoneal cavity [7
, 8
]. When macrophages phagocytosing apoptotic cells in vivo were isolated by a cell sorter, the macrophages produced MIP-2 at the protein level very close to that after coculturing PEC with late apoptotic cells [7
]. Consequently, MIP-2 production by a coculture of macrophages with late apoptotic cells may have biological significance under pathological conditions.
DCs are the sentinels of immune systems and play an important role in induction of immunity as well as maintenance of tolerance [9 ]. Although macrophages are well known as representative phagocytes, iDCs also phagocytose apoptotic cells, which are believed not to induce maturation of the iDCs [9 10 11 ], and exist in blood and many tissues, such as liver, thymus, skin, lung, and spleen [12 13 14 ]. Macrophages are also localized in many tissues, including liver, thymus, skin, lung, peritoneal cavity, and spleen [15 ]. In tissues such as liver, a close association or binding of Kupffer cells and iDCs is often observed in portal and sinusoidal areas [16 , 17 ].
We thus hypothesize that when macrophages, iDCs, and apoptotic cells are cocultured, they may cross-talk with each other. To substantiate this hypothesis, we focus on the production of MIP-2 and IL-6, as they are chiefly produced by macrophages and iDCs, respectively, during coculturing with apoptotic cells. Furthermore, we examine in detail whether the phagocytic functions of macrophages and iDCs are affected by a coculture of three types of cells, macrophages, iDCs, and apoptotic cells.
In this study, we show for the first time that the production of a proinflammatory cytokine, MIP-2, is decreased by iDCs via very small amounts of anti-inflammatory cytokines, IL-10 and TGF-ß, in a cell-to-cell contact-dependent manner.
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Preparation of macrophages
Mouse PEC, Kupffer cells, and alveolar macrophages were obtained according to the standard methods described previously [6
]. These cells were cultured on a 24-well plate or a four-well chamber slide in RPMI-1640 medium containing 10–20% heat-inactivated fetal bovine serum (FBS; Gibco BRL, Gaithersburg, MD) at the cell density of 50 x 104 cells/ml for 45 min at 37°C. Then, plastic adherent macrophages were obtained by washing with RPMI-1640 medium containing 5% FBS or phosphate-buffered saline (PBS; 20 mM PBS, pH 7.4, containing 14 mM Na2HPO4 and 6 mM KH2PO4) three times.
Preparation of iDCs
Murine bone marrow cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 2 mM L-glutamine, kanamycin sulfate, 50 µM 2-mercaptoethanol, and 10% FBS. DC complete medium contained 10–20% conditioned supernatant of a 3T3 cell line harboring a recombinant mouse granulocyte macrophage-colony stimulating factor (rmGM-CSF) expression vector (RIKEN, Bioresource Center, Tsukuba, Japan), resulting in a final concentration of 1000 U/ml GM-CSF. DCs were then obtained as described previously [18
]. Briefly, bone marrow was collected from the tibias and femurs of C57BL/6 male mice. Contaminating erythrocytes were lysed, and lymphocytes were depleted with a cocktail of antibodies [Ab; anti-B220: RA3-3A1/6.1; anti-Ly2: 2.43; anti-L3T4: GK1.5; and anti-Gr-1: RB6-8C5, all from American Type Culture Collection (ATCC), Manassas, VA] and rabbit complement on day 0. One million cells were placed on a 24-well plate in 1 ml DC complete medium. On day 3, 75% of the medium was aspirated off and replaced with fresh DC complete medium. On day 5, nonadherent cells were collected and resuspended at 1 x 106 cells/ml in fresh DC complete medium, and then replated on a new 24-well plate. On day 7, nonadherent cells were recovered as iDCs, 60–80% of which showed the surface phenotype and morphology of iDCs, granulocytes being the main contaminant. Then, the iDCs were washed three times with PBS and used for experiments. Maturation of iDCs was induced with 1 µg/ml lipopolysaccharide (Escherichia coli O55B5, Difco, Detroit, MI) for 24 h at 37°C.
Generation of apoptotic cells
An IL-2-dependent cytotoxic mouse T cell line, CTLL-2 cells, was washed with PBS by centrifugation at 1000 rpm for 10 min at 4°C, followed by incubation in IL-2 (Takeda Pharmaceutical Co., Osaka, Japan)-free RPMI-1640 medium containing 10% FBS for 28 h at 37°C at the cell density of 5 x 105 cells/ml [5
]. Thymocytes obtained from C57BL/6 mice were irradiated with X-rays (12 Gy; Hitachi, MBR-1505R2) and then incubated for 9 h at 37°C at the cell density of 106 cells/ml in culture medium. A murine leukemic cell line, P388, was adjusted to the cell density of 106 cells/ml in culture medium, followed by the addition of 1 mg/ml etoposide (Wako, Osaka, Japan), giving a final concentration of 1 µg/ml, and then the cells were incubated for 24 h at 37°C [7
]. Then the apoptotic cells were washed three times with PBS.
Phagocytosis assay
Apoptotic CTLL-2 cells were stained with PKH26 dye (Sigma-Aldrich, St. Louis, MO), followed by culturing with iDCs, macrophages, or a combination of iDCs and macrophages for 3 h at 37°C on a 24-well plate or a four-well chamber slide. Nonadherent cells were collected as iDCs and stained with fluorescein isothiocyanate (FITC)–anti-CD11c for 30 min at 4°C. Adherent cells were washed three times with PBS and then stained with FITC–anti-F4/80 for 30 min at 4°C. Phagocytosis was then examined by confocal microscopy using a Fluoview system (Olympus Inc., Tokyo, Japan). Phagocytic cells are defined as cells containing at least one PKH26-positive cell. The percentage of phagocytosis was determined by examination of at least 200 macrophages or iDCs. The phagocytic index of macrophages was determined by multiplying the percentage of phagocytosis by the average of the pixel areas corresponding to apoptotic cells phagocytosed by macrophages.
Measurement of cytokines
After coculturing, supernatants were harvested. Samples were stored at –80°C until the assay. The mouse IL-6, IL-10, and TGF-ß levels were determined using DuoSet enzyme-linked immunosorbent assay (ELISA) development systems (R&D Systems, Minneapolis, MN). The MIP-2 level was determined by means of a specific ELISA with the combination of rabbit anti-MIP-2 Ab (provided by Dr. Kouji Matsushima, The Tokyo University School of Medicine) and rMIP-2. The detection limits for MIP-2, IL-6, IL-10, and TGF-ß were 200 pg/ml, 7.8 pg/ml, 7.8 pg/ml, and 7.8 pg/ml, respectively.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was isolated from adherent cells (macrophages) or nonadherent cells (iDCs plus unphagocytosed apoptotic cells), and RT-PCR was performed as described previously [5
]. The primers (5' primer and 3' primer), annealing temperatures, concentrations of MgCl2, and predicted sizes were as reported previously [5
].
Ab and cytokines
Goat anti-IL-10 neutralizing Ab [immunoglobulin G (IgG)], chicken anti-TGF-ß neutralizing Ab (IgY), goat anti-IL-6 neutralizing Ab (IgG), normal goat IgG, and normal chicken IgY were purchased from R&D Systems. Rat hybridoma cell lines producing anti-Mac-1 (CD11b/CD18) monoclonal Ab (mAb; M1/70) or rat IgG2b (SFR8-B6) were purchased from ATCC. rIL-10 and rTGF-ß were obtained from R&D Systems.
Statistics
The significance of the data was evaluated by means of one factor ANOVA followed by Fisher’s PLSD test. A P value of less than 0.05 was considered statistically significant.
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) were cocultured for 3 h with late apoptotic (Apo) CTLL-2 cells in the ratio of 1:2 (M/Apo, 1:2), significant induction of MIP-2 production was observed (Fig. 1A
). Conversely, coculturing iDCs with apoptotic cells (iDC/Apo, 1:2) did not yield any MIP-2. We then examined the effect of the addition of iDCs to a coculture of macrophages and late apoptotic cells in the ratio of 1:1:2 (M/iDC/Apo, 1:1:2) on MIP-2 production. Compared with the MIP-2 production by a coculture of macrophages and apoptotic CTLL-2 cells (M/Apo, 1:2), the MIP-2 production in a three-cell coculture was significantly decreased from 3.9 ± 0.1 ng/ml to 2.6 ± 0.1 ng/ml (P<0.05; Fig. 1A
). When macrophages, iDCs, and apoptotic CTLL-2 cells were cultured alone, they produced almost no MIP-2.
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Figure 1. MIP-2 production by a three-cell coculture (macrophages, iDCs, and apoptotic cells). We cocultured thioglycollate broth-induced PEC, iDCs, and apoptotic CTLL-2 cells in various combinations for 3 h at 37°C. (A) MIP-2 production by various combinations of these three types of cells. The MIP-2 protein level in each supernatant was determined by means of a specific ELISA. Experiments were performed in triplicate. The results are expressed as means ± SE. (B) Effect of cell-to-cell contact on MIP-2 production. Some cocultures were performed in a double-chamber culture system (Millipore, Bedford, MA) to separate macrophages from iDCs or apoptotic CTLL-2 cells. The experiments were performed in triplicate. The MIP-2 protein level in each supernatant was determined by means of a specific ELISA. Experiments were performed in triplicate. The results are expressed as means ± SE. Brackets, Cultured in a double-chamber; N.D., not detected.
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MIP-2 production in a three-cell coculture of other apoptotic cells and/or other macrophages
We then examined whether such a decrease in MIP-2 production in a three-cell coculture was independent of the apoptotic cell and macrophage cell types. First, apoptotic thymocytes or apoptotic P388 cells were prepared according to the method described in Materials and Methods. When we cocultured macrophages, iDCs, and apoptotic cells, a significant decrease in MIP-2 production was observed (Table 1
), although MIP-2 production varied with the apoptotic cell type. We then used Kupffer cells and alveolar macrophages as resident tissue macrophages and cocultured them with apoptotic CTLL-2 or P388 cells. Compared with the MIP-2 production by a coculture of Kupffer cells and these apoptotic cells, the addition of iDCs to the coculture also decreased MIP-2 production (Table 2
). When alveolar macrophages were used as other tissue macrophages in a three-cell coculture, there was also slight inhibition of MIP-2 production (Table 3 ). These results suggested that inhibition of MIP-2 production on the addition of iDCs to a coculture of macrophages and apoptotic cells was independent of the apoptotic cell and macrophage cell types.
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Table 1. MIP-2 Productiona after Coculturing X-Irradiated Apoptotic Thymocytes or Etoposide-Induced Apoptotic P388 Cells with Macrophages and/or iDCs
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Table 2. MIP-2 Productiona after Coculturing Apoptotic CTLL-2 Cells or Apoptotic P388 Cells with Kupffer Cells
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Table 3. MIP-2 Productiona after Coculturing Apoptotic CTLL-2 Cells or Apoptotic P388 Cells with Alveolar Macrophage
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/Apo, 1:2) did not yield a significant amount of IL-6. We then examined the effect of the addition of macrophages to a coculture of iDCs with late apoptotic cells in the ratio of 1:1:2 (M/iDC/Apo, 1:1:2) on IL-6 production. Compared with the IL-6 production by a coculture of iDCs and apoptotic CTLL-2 cells (iDCs/Apo, 1:2), the IL-6 production in a three-cell coculture was significantly decreased from 288.4 ± 11 pg/ml to 90.4 ± 9.2 pg/ml (P<0.05; Fig. 2
). When macrophages, iDCs, and apoptotic CTLL-2 cells were cultured alone, they produced almost no IL-6.
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Figure 2. IL-6 production by a three-cell coculture (macrophages, iDCs, and apoptotic cells). We cocultured thioglycollate broth-induced PEC, iDCs, and apoptotic CTLL-2 cells in various combinations for 3 h at 37°C. (A) IL-6 production by various combinations of these three types of cells. The IL-6 protein level in each supernatant was determined by means of a specific ELISA. Experiments were performed in triplicate. The results are expressed as means ± SE. (B) Effect of cell-to-cell contact on IL-6 production. Some cocultures were performed in a double-chamber culture system (Millipore) to separate macrophages from iDCs or apoptotic CTLL-2 cells. The experiments were performed in triplicate. The IL-6 protein level in each supernatant was determined by means of a specific ELISA. Experiments were performed in triplicate. The results are expressed as means ± SE. Brackets, Cultured in a double-chamber; N.D., not detected.
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/Apo, 1:2), almost all the macrophages phagocytosed apoptotic cells, the percentage of phagocytosis being close to 100%. Conversely, iDCs showed lower phagocytic ability than macrophages [58.2±0.5% in the ratio of 1:2 (iDCs/Apo, 1:2)]. These results showed that macrophages have more potent phagocytic ability than iDCs (P<0.05). The phagocytosis by macrophages in a three-cell coculture in the ratio of 1:1:2 (M/iDC/Apo, 1:1:2/M) did not change as compared with that in a two-cell coculture in the ratio of 1:2 (M/Apo, 1:2). In contrast, the phagocytosis by iDCs in a three-cell coculture was much more significantly decreased than that in a two-cell coculture, irrespective of the ratio, namely from 58.2 ± 0.5% (iDCs/Apo, 1:2) to 29.6 ± 0.3% (M/iDC/Apo, 1:1:2/iDC; P<0.05). These results suggested that suppression of IL-6 production is caused by inhibition of phagocytosis of iDCs.
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Figure 3. Phagocytic ability of macrophages and iDCs in each coculture. We prepared PEC, iDCs, and apoptotic CTLL-2 cells as described in Materials and Methods. Apoptotic CTLL-2 cells were labeled with PKH26. After 3 h, two-cell cocultures, macrophages, and iDCs were stained with FITC–anti F4/80 mAb and FITC–anti-CD11c mAb, respectively, whereas after 3 h, three-cell cocultures, nonadherent cells as iDCs, and adherent cells as macrophages were collected and stained as mentioned before. (A) The percentages of phagocytosing macrophages and iDCs. We performed a two-cell coculture (left) and a three-cell coculture (right). Then we determined the percentages of phagocytosis of macrophages and iDCs by means of confocal laser microscopy. We performed three independent experiments, and the results are shown as means ± SE. (B) Phagocytic index of macrophages. We measured the areas corresponding to apoptotic cells engulfed by macrophages and then determined the phagocytic index. We performed three independent experiments, and the results are shown as means ± SE. (C) Images of the phagocytosis of macrophages. These are representative images, which show macrophages (green) and apoptotic CTLL-2 cells (red). (D) The correlation between MIP-2 production and the phagocytic index of macrophages. We plotted the phagocytic index (x-axis) and MIP-2 production (y-axis).
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/Apo, 1:2) to 409.4 ± 5.4 (M/iDC/Apo, 1:1:2/M; P<0.05). The increase in the phagocytic index is also shown by the images in Figure 3C
. Here, macrophages ingested more apoptotic cells in a three-cell coculture than in a two-cell coculture. We then confirmed that MIP-2 production is correlated with the phagocytic index in a two-cell coculture of macrophages and apoptotic cells (Fig. 3D)
. As the phagocytic index of macrophages increased in a three-cell coculture as compared with that in a two-cell coculture, these results suggested that the decrease in MIP-2 production in the three-cell coculture was not a result of inhibition of phagocytosis.
Requirement of cell-to-cell contact and anti-inflammatory cytokines IL-10 and TGF-ß for inhibition of MIP-2 production
To determine whether cell-to-cell contact is required for the inhibition of MIP-2 production in a three-cell coculture, we used a double-chamber culture system to separate the cells. As shown in Figure 1B
, when M/Apo were separated from iDCs or iDC/Apo with a double-chamber, there was no significant inhibition of MIP-2 production as compared with M/Apo. These results suggested that inhibition of MIP-2 production in a three-cell coculture depends on cell-to-cell contact.
Next, we examined whether the sequence of addition of iDCs can affect the MIP-2 production in a three-cell coculture. As illustrated in the upper part of Figure 4 , iDCs were added 30 min after starting the coculture of macrophages with apoptotic cells (Culture 1), iDCs were added at the same time of starting the coculture of macrophages with apoptotic cells (Culture 2), or iDCs were added to macrophages 30 min before starting the coculture with apoptotic cells (Culture 3). It should be noted that in either case, macrophages and apoptotic cells were cocultured for exactly 3 h. As shown in Figure 4 , MIP-2 production was decreased, as coculturing of iDCs with macrophages prolonged from Culture 1 (2.5 h) to Culture 2 (3 h) to Culture 3 (3.5 h).
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Figure 4. Effect of the sequence of addition of iDCs on the MIP-2 production in a three-cell coculture. iDCs were added 30 min after starting the coculture of macrophages with apoptotic cells (Culture 1), iDCs were added at the same time of starting the coculture of macrophages with apoptotic cells (Culture 2), or iDCs were added to macrophages 30 min before starting the coculture with apoptotic cells (Culture 3). MIP-2 levels in each supernatant were measured by means of a specific ELISA. Experiments were performed in triplicate. The results are expressed as means ± SE.
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/iDC/Apo, 1:0.5:2, 1:1:2, and 1:2:2). As shown in Figure 5
, MIP-2 production was decreased dependently on the increase in the number of iDCs.
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Figure 5. Effect of the increase in the number of iDCs on MIP-2 production in a three-cell coculture. We cocultured PEC with various numbers of iDCs and apoptotic CTLL-2 cells for 3 h at 37°C. Then, we measured MIP-2 levels in each supernatant by means of a specific ELISA. Experiments were performed in triplicate. The results are expressed as means ± SE.
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Figure 6. Effect of anti-Mac-1 mAb on MIP-2 production in a three-cell coculture. We cocultured PEC with iDCs and apoptotic CTLL-2 cells for 3 h at 37°C in the presence of anti-Mac-1 mAb or control rat IgG2b. Then, we measured MIP-2 levels in each supernatant by means of a specific ELISA. Experiments were performed in triplicate. The results are expressed as means ± SE.
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/iDC/Apo vs. M/iDC/Apo/anti-IL-10: 2.2±0.1 ng/ml vs. 2.9±0.04 ng/ml (P<0.05); M/iDC/Apo vs. M/iDC/Apo/anti-TGF-ß: 2.2±0.1 ng/ml vs. 2.8±0.1 ng/ml (P<0.05)], and the combination of these Ab reversed the suppression more significantly [M/iDC/Apo vs. M/iDC/Apo/anti-IL-10/anti-TGF-ß; 2.2±0.1 ng/ml vs. 3.5±0.2 ng/ml (P<0.05)]. Control Ab did not affect MIP-2 production. We also added anti-IL-6 Ab to a three-cell coculture (Fig. 7)
. Anti-IL-6 Ab showed a lower effect than that of anti-IL-10 Ab or anti-TGF-ß Ab on the suppression of MIP-2 production. The combination of anti-IL-10 Ab, anti-TGF-ß Ab, and anti-IL-6 Ab blocked the suppression of MIP-2 production to a similar extent to the combination of anti-IL-10 Ab and anti-TGF-ß Ab. To examine the possibility that MIP-2 production by macrophages is negatively regulated in an autocrine manner, we added anti-IL-10 Ab and anti-TGF-ß Ab to a two-cell coculture of macrophages and apoptotic cells. However, there was no effect on MIP-2 production (data not shown).
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Figure 7. Effects of anti-IL-10 Ab, anti-TGF-ß Ab, and anti-IL-6 Ab or combinations of them on iDCs-mediated suppression of MIP-2 production. PEC, iDCs, and apoptotic CTLL-2 cells were cocultured for 3 h at 37°C in the presence or absence of each Ab. The concentration of each Ab was 5 µg/ml. The MIP-2 protein in each supernatant was determined by means of a specific ELISA. We performed three independent experiments, and the results are shown as means ± SE.
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Figure 8. Effect of exogenouse IL-10 and TGF-ß or a combination of them on MIP-2 production in a two-cell coculture of macrophages and apoptotic CTLL-2 cells. We cocultured PEC with apoptotic CTLL-2 cells for 3 h at 37°C in the presence or absence of rIL-10 and rTGF-ß. Then, we measured MIP-2 levels in each supernatant by means of a specific ELISA. Experiments were performed in triplicate. The results are expressed as means ± SE.
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Figure 9. Cytokine mRNA expression in iDCs and macrophages in a three-cell coculture. By means of RT-PCR, the expression of TGF-ß, IL-10, MIP-2, and IL-6 mRNAs was examined after coculturing of macrophages, iDCs, and apoptotic cells for 3 h. ß2-Microglobulin (ß2m) was used as a control. The result shows representative data from three experiments.
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MIP-2 production correlated very closely with the phagocytic index of macrophages in a two-cell coculture, macrophages and apoptotic cells. However, the addition of iDCs to a coculture of macrophages and late apoptotic cells decreased MIP-2 production but augmented the phagocytic activity of macrophages in terms of the phagocytic index. This is not an unprecedented phenomenon, as it was found that glucocorticoids, including dexamethasone, promoted the ability of macrophages to phagocytose apoptotic cells and down-regulated the production of proinflammatory cytokines [20 21 22 ], although the underlying mechanisms may differ. In contrast, the addition of macrophages to a coculture of iDCs and late apoptotic cells decreased IL-6 production and percent of phagocytosis of iDCs. The latter suggested that the decrease in IL-6 production was a result of inhibition of phagocytosis of iDCs.
There has been the hypothesis that DCs do not present antigens derived from apoptotic cells, as macrophages are more powerful as to the phagocytosis of apoptotic cells [23 , 24 ]. The hypothesis is further supported by our results showing a decrease in phagocytosis of iDCs and an increase in phagocytosis of macrophages in a three-cell coculture.
As any three-cell cocultures, Kupffer cells, thioglycollate-induced macrophages, or alveolar macrophages as macrophages, and any three-cell cocultures including apoptotic thymocytes, apoptotic P388 cells, or apoptotic CTLL-2 cells as apoptotic cells led to the suppression of MIP-2 production to varying extents, the suppression appears to be independent of the macrophage and apoptotic cell types. It is not known at present, however, why iDCs caused slight suppression of MIP-2 production by alveolar macrophages in response to apoptotic cells.
Using a double-chamber culture system, we showed that the decrease in MIP-2 production in a three-cell coculture depended on cell-to-cell contact but not on soluble factor(s), if any, from iDCs phagocytosing apoptotic cells or iDCs alone. As the addition of anti-Mac-1 Ab to a three-cell coculture partially restored MIP-2 production, MIP-2 production may be suppressed through cell-to-cell contact involving ß2 integrin and possibly other adhesion molecules. It is likely that such a contact-dependent, inhibitory mechanism may operate in vivo, as it has been demonstrated that macrophages and iDCs coexist in tissues, including liver, thymus, skin, lung, and spleen, and as in a tissue such as liver, a close association between Kupffer cells and iDCs is often observed in portal and sinusoidal areas [16 , 17 ]. Moreover, in other tissues such as skin, macrophages and DCs are reportedly colocalized in the dermal area [25 , 26 ].
Although we could not detect anti-inflammatory cytokines IL-10 and TGF-ß in any cultures, anti-IL10 and anti-TGF-ß Ab could restore MIP-2 production in a three-cell coculture. In addition, exogenous IL-10 and TGF-ß, except at the lowest concentration (5 pg/ml), which is under detectable limits of ELISAs, could inhibit MIP-2 production in a two-cell coculture of macrophages and apoptotic cells. Consequently, these anti-inflammatory cytokines in the cell-to-cell contact area may exist at the high concentration, enough to inhibit MIP-2 production. Alternatively, membrane-bound TGF-ß might be involved in a three-cell coculture, as reported by others [27 , 28 ].
When cytokine mRNAs were measured by RT-PCR in macrophages or iDCs after a three-cell coculture, IL-10 and TGF-ß mRNAs were significantly up-regulated in iDCs as compared with in macrophages, whereas MIP-2 and IL-6 mRNAs were produced chiefly in macrophages and iDCs, respectively. This suggested that MIP-2 production in a three-cell coculture is suppressed by iDC-derived IL-10 and TGF-ß, although cell-to-cell contact may also deliver a negative signal to suppress MIP-2 production. As anti-IL-10 and anti-TGF-ß Ab did not affect MIP-2 production in a two-cell coculture, macrophages and apoptotic cells, it is rather unlikely that MIP-2 production is suppressed by macrophage-derived IL-10 and TGF-ß in an autocrine manner in a three-cell coculture. The notion that iDCs suppress MIP-2 production by macrophages in a three-cell coculture is further supported by the results that the increasing number of iDCs in a three-cell coculture resulted in a more significant decrease of MIP-2 production and that MIP-2 production was decreased as coculturing of iDCs with macrophages prolonged.
We favor the hypothesis that macrophages are likely to produce proinflammatory cytokines after the phagocytosis of apoptotic cells and that this production is prevented by unknown mechanism(s). As one such mechanism, we have reported that human serum, in particular normal IgG, can prevent proinflammatory cytokine production by macrophages after phagocytosis of apoptotic cells [19 ]. As another mechanism, we recently described that very early apoptotic cells can be phagocytosed by macrophages without MIP-2 production [29 ]. The latter observation strongly supports that the clearance of early apoptotic cells is essential for preventing inflammation in tissues and may explain why apoptotic cells could hardly be detected in normal tissues. This study provides a third mechanism for prevention of the production of inflammatory cytokines, when apoptosis is advanced, and proinflammatory cytokines are produced when there are few serum components near the macrophages.
In conclusion, MIP-2 and IL-6 production was inhibited by a three-cell coculture as compared with a two-cell coculture. Although the suppression of IL-6 production was associated with inhibition of phagocytosis by iDCs, the suppression of MIP-2 production was associated with up-regulation of phagocytosis by macrophages and needed cell-to-cell contact and very low concentrations of anti-inflammatory cytokines. Thus, our results reveal a third mechanism by which the production of MIP-2 is prevented. Further study is needed to reveal how inflammation is suppressed in vivo during the clearance of apoptotic cells.
Received October 9, 2003; revised December 6, 2003; accepted January 5, 2004.
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