Department of Ophthalmology, University of Aberdeen Medical School Foresterhill, United Kingdom
Correspondence: Professor John V. Forrester or Dr. Hui-Rong Jiang, Department of Ophthalmology, University of Aberdeen Medical School Foresterhill, Aberdeen AB24 2ZD, Scotland, UK. E-mail: j.forrester{at}abdn.ac.uk or hr.jiang{at}abdn.ac.uk
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reduced the yield of DC but promoted the level of LPS-induced IL-10 production by lDC. Our data provide new evidence that ex vivo manipulation and the cytokine environment regulate DC maturation status and cytokine-secretor phenotype with implications for the control of T cell differentiation and function via DC-based immunotherapeutic strategies.
Key Words: bone marrow • cytokine-secretion profile • maturation status
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In rodents, DC with distinct functions in immunity and tolerance have been described [6 , 7 ]. During differentiation, DC up-regulate the expression of cell surface major histocompatibility complex (MHC) class II and costimulatory molecules and thus increase their efficiency as antigen-presenting cells (APC). After exposure to microbial agents, DC respond by producing immunostimulatory cytokines including interleukin (IL)-12 and further, by up-regulating the expression of MHC-II and costimulatory molecules [8 ]. Recently, new data have confirmed that the maturation status of DC plays an essential role in the determination of T cell differentiation and the immune response [9 10 11 12 ]. Thus, various factors including the microenvironment, the cytokines released by T cells or other cells in the vicinity, and the degree of DC differentiation may determine their subsequent function [4 , 9 , 13 , 14 ]. However, clear criteria defining the function as opposed to the phenotype of DC are still undefined.
In a previous study, we developed a method for generating bone marrow-derived DC (BMDC) capable of reducing inflammation in a mouse model of experimental autoimmune uveoretinitis (manuscript in revision for publication). During the progress of this work, it became apparent that additional, time-dependent events in the life cycle of cultured DC could be involved in polarizing the function of these cells toward tolerance or immunity. Here, we investigate this phenomenon and show that the timing of granulocyte macrophage-colony stimulating factor (GM-CSF)-generated BMDC exposure to bacterial lipolysaccharide (LPS) is critical to the cytokine pattern secreted. Immediate/early exposure of freshly purified (day 6) BMDC [early matured DC (eDC)] to LPS induced a prominent IL-10 response. In contrast, if the LPS stimulus was delayed for up to 22 h [later matured DC (lDC)], the BMDC generated a marked IL-12 response. Despite this divergent cytokine response, both DC populations exhibited similar levels of MHC-class II and costimulatory CD86 and CD40 expression. In addition, we show that this response is not fixed and can be changed in an inverse manner by the Th cytokines IL-4 or interferon- (IFN-).
By highlighting the importance of time in culture on subsequent cytokine secretion by BMDC receiving a maturation signal, our data provide a model for use by immunologists considering manipulation of DC function ex vivo for clinical applications.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Generation of BMDC
BMDC were prepared by a modification of the procedure described by Inaba et al. [15
]. In brief, a single-cell suspension of BM cells was depleted of B cells and T cells using a monoclonal antibody (mAb) mixture comprising rat immunoglobulin G anti-B220 (clone RA3-6B2), anti-CD4 (clone GK1.5), anti-CD8 (clone 53-6.7), and anti-MHC-II (clone P7/7). All antibodies were from BD Pharmingen UK Ltd. (Oxford) except MHC-II, which was from Serotec (Oxford, UK). The remaining cells were cultured at 7.5 x 105/ml in 12-well plates in RPMI 1640 (Gibco-BRL, Life Technologies, Paisley, UK) supplemented with 5% fetal calf serum, 2 mmol/l L-glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin, 5 x 10-5 mol/l 2-mercaptoethanol, 1 mmol/l sodium pyruvate, 0.1 mmol/l nonessential amino acids, and 5% GM-CSF supernatant. GM-CSF supernatant was prepared from the Ag8653 myeloma cell line transfected with murine GM-CSF cDNA, and the cell line was kindly given by Dr. Brigitta Stockinger (Division of Molecular Immunology, The National Institute for Medical Research, Mill Hill, London, U.K.) [16
]. From day 2, the cultures were fed daily by gently swirling the plates, aspirating 75% of the medium, and adding fresh medium with GM-CSF. Usually, the process of swirling and changing the medium removed nonadherent granulocytes, and clusters of developing DC remained loosely attached on a bed of firmly adherent macrophages. Six days after the culture, the loosely adherent clusters were collected, and the contaminating granulocytes were depleted using anti-mouse Gr-1 mAb (clone RB6-8C5; PharMingen, San Diego, CA) and Dyna-beads. A single-cell suspension of the purified cells was prepared and used for further experiments.
Administration of LPS to the purified BMDC culture
Purified BMDC were further seeded at 1 x 106/ml/well in 24-well plates with GM-CSF-supplemented medium as usual; then, 1 µg/ml LPS was added to the culture at various time points: Immediately (0 h), 2 h, 5 h, 10 h, or 22 h later, supernatant was collected from each well 22 h after the administration of LPS for cytokine measurement. Medium alone-treated DC were used as controls. Purified BMDC stimulated immediately with LPS after isolation (time 0 h) in culture were termed eDC, and BMDC cultured in medium alone for specific time periods (>2 h) after isolation prior to the addition of LPS were termed lDC.
To investigate the kinetics of cytokine production, purified BMDC were seeded in 24-well plates, and LPS was added immediately or after 22 h of culture in medium alone. Supernatant was collected at various time periods after addition of LPS (2 h, 5 h, 10 h, or 22 h) from eDC and lDC cultures for cytokine measurement. At various time points, cells were also collected for flow cytometric analysis.
To investigate the relationship between IL-10 and IL-12 secretion by DC, we undertook to block the production of IL-10 by eDC using anti-mouse IL-10 mAb (JES5-16E3, BD Pharmingen) added into the purified DC culture, after which LPS was added immediately or 20 h later as described above. Twenty-two hours after LPS administration, supernatant was collected for enzyme-linked immunosorbent assay (ELISA) assay.
Allogeneic mixed leukocyte reactions (MLRs)
B10RIII mouse BMDC were purified and then stimulated with or without LPS immediately or 10 h later for a further 10 h to generate immature and mature eDC and lDC, and their IL-10/IL-12 cytokine-secretion profiles were confirmed by ELISA assay of the supernatant. BALB/c mouse CD4+ T cells were purified to a final purity of >93% by using CD4+ antibody-coated magnetic microbeads (Miltenyi Biotec, Surrey, UK), which was followed by passing cells through a Mini Macs column in a magnetic field. The positively selected cells were collected and analyzed before coculture. Triplicate wells of 2 x 105 CD4+ T cells were seeded in a 96-well round-bottom plate, and titrated numbers of eDC or lDC were added. Cells were cultured for 4 days and pulsed with 1 µCi/well of thymidine during the last 16 h of culture.
Effect of IL-4 and IFN- on the DC maturation and cytokine profile
BM cells were cultured in GM-CSF alone or GM-CSF together with 1 ng/ml IL-4 or 2 ng/ml IFN- (both from PharMingen). These concentrations were optimized in preliminary experiments. On day 7, the clusters were collected and depleted of Gr-1+ cells as described above. The purified DC (GM-CSF alone, GM-CSF+IL-4, or GM-CSF+IFN-) were then cultured in 24-well plates with or without IL-4 or IFN-, and LPS was administered immediately (eDC) or after 18 h (lDC). Eighteen hours after LPS administration, supernatant was collected for ELISA measurement of cytokines, and cells were collected for flow cytometry.
Cytokine measurement
Cell culture supernatants were assayed for various cytokines including IL-6, IL-10, and IL-12 p70 using the optELISA kits from PharMingen and R&D Systems (Minneapolis, MN). Briefly, 96-well plates were coated with the appropriate anticytokine antibodies overnight. After blocking the plates with bovine serum albumin and a further 2 h incubation with supernatants or standard, the plates were developed using biotin-conjugated anticytokine antibodies. Then horseradish peroxidase-conjugated streptavidin was added before development with 3,4,5-trimethoxybenzoic acid substrate (BD Pharmingen).
Flow cytometric analysis
Cell surface markers were evaluated by double or triple immunofluorescence staining with the following mAb: anti-MHC-class II (clone P7/7), anti-DEC205 (clone NLDC-145), anti-CD11c-fluorescein isothiocyanate (clone HL3), anti-CD86-phycoerythrin (PE; clone GL-1), anti-CD40-PE (clone 3/23), and anti-CD11b (clone M1/70). MHC-II and DEC-205 Ab were from Serotec, and others were from BD Pharmingen. After incubating with antibodies for 30 min at 4°C, the cells were washed and resuspended in phosphate-buffered saline for flow cytometry analysis. As MHC-II and DEC-205 are purified Ab, anti-rat, biotinylated, secondary antibody and streptavidin-conjugated allophycocyanin antibody (both from Dako, Bucks, UK) were added following staining with the purified Ab. Matched isotypes were used as the negative controls.
Reproducibility and statistical analysis
Experiments were repeated at least three times and usually five or more times. The data were analyzed using the independent t test. Probability values of 
0.05 were considered significant.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-negative; positive, intracellular MHC-class II reactivity indicated the relatively immature status of the DC at this stage (H-R. Jiang, et al., submitted). These purified DC, when stimulated with LPS immediately after they were placed in the culture (i.e., from time zero; eDC) for up to 22 h, secreted high levels of IL-10 and minimal quantities of IL-12 (Fig. 1
, 0 h). However, with increasing delay in exposure of the cultured, immature DC to LPS, this cytokine-secretion profile progressively changed toward low IL-10 and high IL-12, reaching a peak after 10 h of culture (lDC; Fig. 1
, 10 h). This pattern was consistently observed in more than 15 experiments. Similar results were also obtained from C57BL/6- and BALB/c-derived BMDC and from ex vivo spleen-derived DC (data not shown). Therefore, for later experiments, we used time points between 10 and 20 h post-culture to stimulate lDC with LPS.
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Figure 1. Differential production of IL-10 and IL-12 by BMDC after stimulating with LPS. BM cells were cultured in the medium supplemented with GM-CSF for 6 days, and the clusters were collected and purified. LPS was added to the culture of purified BMDC immediately (0 h), 2 h, 5 h, 10 h, or 22 h later. Then, the supernatant was collected after 22 h incubation. Immediate administration of LPS to the purified BMDC (time 0) induced a high level of IL-10 (A) but low IL-12 production (B), and delayed administration of LPS reversed this cytokine production profile. When administration of LPS was delayed by 2 h, the DC were capable of secreting IL-12 and IL-10. However, after longer time delays, DC secreted higher levels of IL-12 but lower levels of IL-10. Results are mean ± 1 SD (n=three wells) from one experiment and are representative of 15 separate experiments.
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Figure 2. Kinetics of IL-10 and IL-12 production by eDC and lDC. Purified BMDC were cultured in 24 wells, and LPS was added to eDC immediately and to lDC after 22 h of culture in medium alone. Then, the supernatant was collected at 2 h, 5 h, 10 h, and 22 h after the administration of LPS in the culture. (A) IL-10 production by eDC is detectable after 2 h stimulation with LPS and peaks at 10 h. (B) IL-12 production by lDC after stimulation of LPS is detectable after 5 h, the level of IL-12 continuing to rise up to 22 h. (C) IL-6 levels were slightly higher in supernatant from eDC compared with lDC cultured in medium alone after 22 h, but the levels were markedly increased after stimulation with LPS in the culture. However, no significant difference was observed between eDC and lDC. Results are mean ± 1 SD (n=three wells) from one experiment representative of five.
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Figure 3. Effect of IL-10 on the secretion of IL-12 by early DC. Purified DC were cultured with or without anti-mouse IL-10 mAb and stimulated with LPS immediately or 20 h later. Then, the supernatant was collected 22 h following the LPS administration. The data show that although there was a reduction in the level of IL-10 detectable from BMDC in the presence of anti-IL-10 mAb, there was no effect on IL-12 production by this treatment. Results are mean ± 1 SD (n=three wells).
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Figure 4. Flow cytometric analysis of eDC and lDC phenotype. The data showed that eDC (thin line) and lDC (dotted line) cultured in medium only were immature with low expression of MHC-II and costimulatory markers CD86 and CD40. The administration of LPS immediately (eDC) or delayed (lDC) induced similar levels of maturation of eDC and lDC by up-regulation of MHC-II, CD86, and CD40. The isotype control is indicated by the shadow histogram. The data shown included the entire cell population.
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Figure 5. MLR-stimulating activity of immature (-LPS) and mature (+LPS) eDC and lDC. Medium-cultured and LPS-stimulated eDC and lDC were cocultured with allogeneic (BALB/c) CD4 T cells and cultured for 4 days. (Values are the means of triplicates with SD bars from one of the three representative experiments.) **, P < 0.01 comparing 2 x 103 DC with 2 x 102 DC per well as the control.
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inhibited DC yield and generated CD11c-negative DC on the BMDC generation and cytokine-secretion pattern. The Th2 cytokine IL-4 is commonly used in the culture of DC from BM and blood and has been considered as a maturation signal for DC [17
, 18
]. IFN- is a Th1 counterpart of IL-4 and is reported to increase IL-12 p40 production and may be involved in modulation of DC function [19
]. Primary BM cells were cultured in GM-CSF only, GM-CSF + IL-4, or GM-CSF + IFN--supplemented medium, respectively, for 6 days. We observed that IL-4 in the culture enhanced DC proliferation and cluster formation, and the presence of IFN- inhibited DC cluster formation in the culture compared with GM-CSF-cultured BMDC (Fig. 6
). As indicated in Figure 6D
, the DC yield was nearly doubled from 5.3 to 9.2 x 105 cells per well in the presence of IL-4. Surprisingly, despite the presence of GM-CSF in the culture medium, IFN- substantially reduced DC yield to 1.2 x 105 cells per well. The reduction in DC numbers in the cultures was attributed to the inhibition of BM cell proliferation, as during the later stages of culture, the cells harvested at day 6 were fully viable.
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Figure 6. Phase-contrast micrographs illustrating the development of BMDC with the supplement of GM-CSF, GM-CSF + IL-4, or GM-CSF + IFN-. (A) DC clusters formed in the culture medium supplemented with GM-CSF. (B) IFN- presence inhibited BMDC proliferation with only a few small clusters developing in the culture. (C) Increased size and number of clusters generated in cultures with GM-CSF and IL-4. (D) DC yield per well (12-well plate) showing IL-4-enhanced DC proliferation (*, P=0.02) and IFN--inhibited DC yield (**, P=0.01) compared with GM-CSF-cultured DC. The data are one of three representative experiments.
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(data not shown). GM-CSF and IL-4 + GM-CSF-cultured DC were CD11c-positive, but IL-4 in the culture promoted DC maturation with higher expression of MHC-class II, CD86, and CD40. In contrast, IFN--generated BMDC were CD11c-negative but with slightly higher levels of DEC205, suggesting that these DC were differentiating along a different pathway. Also, slightly higher levels of MHC-II and similar levels of costimulatory molecules were expressed by IFN- + GM-CSF-generated DC.
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Figure 7. FACS analysis of DC phenotype cultured in different cytokine milieu. GM-CSF (thin line)- and GM-CSF + IL-4 (dark line)-generated BMDC are CD11c-positive; however, IL-4 enhanced BMDC maturation with higher expression of MHC-II and costimulatory molecules. IFN- generated CD11c-negative DC (dotted line), which have a moderate level of MHC-II and costimulatory molecule expression.
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up-regulates IL-10 secretion by lDC, and IL-4 down-regulates IL-10 secretion by eDC versus GM-CSF-cultured BMDC on the induction of IL-10higheDC/IL-12highlDC by LPS. eDC and lDC were prepared from each set of cultures with immediate incubation of LPS (eDC) or after a delay of 18 h (lDC). Our data (Fig. 8
) showed that BMDC cultured in GM-CSF alone exhibited the eDC/IL-10 and lDC/IL-12 pattern as previously observed. However, in the presence of IL-4, there was a profound suppression of IL-10 production by eDC (GM-CSF+IL-4 DC; Fig. 8A
). Moreover, DC generated in the presence of IL-4 had slightly higher levels of IL-12 production by lDC compared with GM-CSF-cultured DC (Fig. 8C)
.
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Figure 8. Influence of Th1 and Th2 cytokines on the DC cytokine profile. BMDC cultured in GM-CSF alone or GM-CSF together with IL-4 or IFN- for 6 days. Equal numbers of purified DC from the clusters were then reseeded in 24-well plates in the presence or absence of IL-4 or IFN- with the stimulation of LPS immediately (eDC) or 18 h later (lDC). The data (A and B, IL-10; C and D, IL-12; E and F, IL-6) show that the presence of IL-4 during the generation of BMDC inhibited IL-10 production (GM-CSF+IL-4) by the eDC. In contrast, the presence of IFN- (GM-CSF+IFN-) altered the eDC/lDC pattern, particularly by increasing IL-10 production by lDC. *, P 0.05; **, P 0.01 refers to the difference between GM-CSF versus GM-CSF + IL-4 or GM-CSF versus GM-CSF + IFN--generated BMDC groups.
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had the opposite effect on the eDC/lDC cytokine profile. Generation of DC in the presence of IFN- (GM-CSF+IFN-) promoted IL-10 production by eDC and lDC (Fig. 8B)
and slightly reduced IL-12 production by lDC (Fig. 8D)
. No effect was observed on the IL-6 production in any of the groups (Fig. 8E
and 8F
) indicating that the IL-4 and IFN--cultured, GM-CSF-generated DC were fully functional with respect to cytokine production and secretion. Thus, the presence of IFN- skewed eDC/lDC cytokine polarization, particularly with regard to the higher level of IL-10 production by lDC (GM-CSF+IFN-; Fig. 8B
). FACS analysis of DC cultured under the three different conditions indicated that LPS administration induced DC maturation as defined by higher MHC-II and costimulatory molecule expression but with no significant differences between the eDC and lDC cultured under the same conditions (data not shown).
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DC activation is a critical event for the induction of immune responses, and the activation event can be separated into two distinct processes: maturation and survival. LPS induces survival and maturation of DC but by different signaling pathways [21 ]. This is in good agreement with our data that DC maturation as shown by phenotype analysis probably involved a different signaling system from that involved in DC functional activation and cytokine secretion. However, the mechanisms of eDC-IL-10/lDC-IL-12 differentiation and the signaling pathways involved are not clear. In parallel experiments (data not shown), we have investigated the expression level of mRNA of Toll-like receptor 4, a receptor for LPS, but did not observe obvious difference; further investigations of differences in signaling pathways are in progress.
It has been shown that the immunostimulatory properties of DC are linked to their maturation state [9
]. Also, it is well accepted that cells of the monocyte/DC lineage, particularly activated DC, direct adaptive T cell response toward a Th1 or Th2 pattern by secreting the cross-regulatory cytokines IL-12 and IL-10 [22
]. IL-12 is the most crucial cytokine that drives the development of naïve T cells into Th1 cells, producing high levels of IFN- in vitro and in vivo [23
]. In contrast, IL-10 is involved in down-regulating DC antigen-presenting function and inducing T cell tolerance [24
]. In addition, IL-10 can inhibit the release of IL-12 and the effect of IL-12 on T cells, thus down-regulating Th1 responses [25
]. The balance between IL-10 and IL-12 is therefore considered important in the induction of T cell immunity or tolerance. Our model of IL-10 and IL-12-polarized DC, which appeared equally mature by the criteria of MHC-class II and costimulatory molecule expression, allowed us to investigate a number of factors involved in the DC-induced immune response. Our MLR results suggested a functional differentiation between eDC and lDC by showing that IL-12-producing lDC induced a much higher level of allogeneic, naïve T cell proliferation compared with IL-10-producing eDC, an effect enhanced by LPS stimulation. Our data suggest that the induction of tolerance versus immunity may be regulated by the degree of maturation as well as the cytokine-secretion pattern of DC [26
, 27
]. This aspect of eDC versus lDC effects on Th cell differentiation and function is now under investigation.
Our eDC/lDC model has also allowed us to further understand the roles of classical Th cytokines in DC generation and function. The Th2 cytokine IL-4 is commonly used as a "maturation" factor in DC culture [27
, 28
], but its role in DC generation and function is less clear. However, we found that the Th cytokines IL-4 and IFN- played important roles in DC generation and cytokine profile. In particular, IL-4 promotes DC development with higher levels of MHC-class II and costimulatory molecule expression, and these DC are more efficient in the MLR proliferation [18
, 29
, 30
]. The cytokine profile of IL-4-generated DC in our data further suggests that IL-4 promoted DC immune-stimulatory capacity, not only by enhancing DC maturation but also by altering the DC cytokine profile of down-regulating IL-10 secretion by eDC. In contrast, the Th1 cytokine IFN- inhibited DC growth in the culture and induced lDC, secreting higher levels of IL-10. A previous study [31
] of human DC showed that the Th2 cytokine IL-4 enhanced DC1 maturation but killed precursor DC2, and this IL-4 effect was blocked by IFN-. These authors further suggested that a feedback loop from the mature Th cells is likely to selectively inhibit prolonged Th1 or Th2 responses by regulating survival of the appropriate DC subset. Most recently, Hochrein et al. [19
] also reported that IL-4 is a major regulatory cytokine governing bioactive IL-12 production by mouse and human DC. We have now added important new evidence to support a general view that there is a negative feedback loop in which IL-4 and IFN- negatively regulate Th1 and Th2 development, respectively, by controlling DC yield and by altering DC phenotype and their potential cytokine profile. This may represent an indirect mechanism to balance the immune response in vivo and represents another example of antagonism between IL-4 and IFN- [32
].
The phenotype change of DC generated in the presence of IFN- is of particular interest. The data reported here confirm the enhancement of DC maturation by IL-4, as reported by others [17
, 33
]. However, we were surprised to find that the addition of IFN- in the BM culture down-regulated CD11c expression despite slightly enhanced DEC 205 expression, generating lDC capable of secreting substantial quantities of IL-10 as well as IL-12 on LPS stimulation. CD11c has been regarded as an important marker for murine DC, but in a study using human DC, it has been reported that plasmacytoid T cells with a CD4+CD11c-CD3- phenotype can develop into DC [34
]. The role of murine CD11c-negative DC in vivo merits further investigation.
As the potential of DC is for inducing immunity as well as tolerance in vivo, there are many protocols for the generation of DC and manipulation in vitro to optimize DC in vivo function. Our data provide important evidence that ex vivo manipulation of DC, particularly when using microbial stimuli to induce DC maturation, will affect DC signaling and further change their in vivo function. Vaccination strategies aimed at promoting Th1 or Th2 responses must now consider the appropriate resting time of DC before antigen or other stimulus pulsing. Further, it is important to consider the indirect proinflammatory or anti-inflammatory effects of IL-4 and IFN- on DC, particularly in vivo in inflammatory situations. Thus, our data add an important new dimension—time—to our current understanding of DC terminal differentiation relative to function, and there are important clinical implications for the development of DC-based immunotherapeutic strategies.
Received March 29, 2002; revised July 1, 2002; accepted July 10, 2002.
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