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IMMUNOBIOLOGY
From Creagene Research Institute, Creagene,
Tanbang-dong, Seo-gu; and Department of Microbiology, Hannam
University, 133 Ojung-dong, Daedeok-gu, Daejeon, South Korea.
Recent studies on dendritic cell (DC)-associated genes have been
performed using monocyte-derived DCs (MoDCs) in different maturation stages. In our approach, to uncover the novel
DC-associated genes and their expression profiles among the different
DC subsets, we constructed a subtracted DC-cDNA library from
CD1a+, CD14+, and CD11c Dendritic cells (DCs) are specialized to
modulate T-cell immunity, either by priming or tolerizing antigen
(Ag)-specific T cells, depending on the exact physiological
conditions, such as the nature and amount of Ag and the presence of
DC-maturating stress signals.1-4 While constituting less
than 1% of the total mononuclear cells in mouse spleen and
human peripheral blood, DCs are present ubiquitously in all tissues,
even in the human central nervous system.5 Unlike other
immune cells, DCs arise, upon different signals, from many different
progenitor cells of myeloid or lymphoid origin.6-8 The
heterogeneity of the DC population is well demonstrated by the multiple
DC subsets in human blood and mouse spleens. Although the ontogeny of
each type of DC remains unclear, the presence of multiple distinct DC
lineages in both humans and mice has raised the possibility that
distinct DC subsets might have unique functions in recruiting distinct
types of immune responses.9-12 Intriguingly, even for a
given type of DC, there is considerable plasticity in DC functions
depending on the maturation stage and the duration of Ag exposure,
resulting in different outcomes of DC-mediated immune
triggering.13-17
Due to their pivotal role in immune induction and tolerance, DCs have
been explored for their usefulness in the control of malignant cancers
and autoimmune diseases in mouse models.18,19 However,
considering the heterogeneity of naturally occurring DCs, the current
DC study in association with human immunotherapy might have been skewed
in monocyte-derived DCs (MoDCs). Indeed, many clinical trials using
MoDCs are being undertaken by independent workers to elicit
tumor-specific immunities.20,21 Increasing pressure from
translational research, however, necessitates the study of other human
DCs, which might be useful to control harmful immune responses, such as
autoimmunity and graft rejection.
In the last few years, advances in methodology have enabled us to
access various human DCs of high purity and good
quantity.22-25 To have better insight into the unique
capacities of distinct DC subsets, attempts have been made to disclose
DC-associated genes and their expression patterns using a
high-throughput analysis system. Several independent approaches have
been made to reveal the genes highly expressed in MoDCs by employing
sequential analysis of gene expression (SAGE)26,27 or a
cDNA microarray system.28,29 The expression profiles from
these studies are generally in good agreement with each other and are
sufficient to arrive at a consensus about genes that are highly
expressed in MoDCs. However, it remains to be seen whether these genes
are also prominent in other types of DCs.
In the present study, we attempted to pool out the
"DC-associated genes" from 3 different DC subsets, namely,
CD11c Cell and RNA preparations
CD11c T lymphocytes were purified from peripheral blood mononuclear cells
(PBMCs) by immunoaffinity depletion using a T-cell isolation kit (Pierce, Rockford, IL). B lymphocytes were obtained from whole blood using RossettSep (StemCell Technologies, Vancouver, BC, Canada).
Monocytes were purified from PBMCs based on their tendency to adhere to
human MoDCs were generated from adherent mononuclear cells. PBMCs were seeded
in 6-well culture plates at a density of 5 × 106/mL,
allowed to adhere for 1 hour at 37°C, and nonadherent cells were
washed away with prewarmed RPMI 1640. Adherent cells were cultured for
7 days in RPMI 1640 medium supplemented with 10% autologous human
serum and 1000 U/mL each of IL-4 (Endogen) and GM-CSF (LG Chem). Media
were refreshed on days 3 and 5. On day 7, nonadherent cells were
collected as immature MoDCs by moderately vigorous agitation. For
matured MoDCs, the nonadherent cells of day 7 were cultured for 2 more
days in a monocyte-conditioned medium (final concentration 50%,
vol/vol) supplemented with 10 ng/mL TNF- The total RNA was extracted from each DC subset using Trizol reagent
(Life Technologies, Carlsbad, CA), and mRNA was affinity-purified by a
polyATtrack system (Promega, Madison, WI).
Generation of the subtracted DC-cDNA library
Colony PCR and microarray fabrication
Based on the result of DC/BMT differential analysis on DC chips, another DC microarray (HI380 chip; Creagene, Daejeon, Korea) was fabricated with 71 DC-specific genes of high significance. In addition to the DC-specific genes, it included 307 known genes encoding CD antigens, cytokines, chemokines, cytokine receptors, and chemokine receptors, either purchased from Incyte or cloned in this laboratory. The genes were mounted in duplicate. The complete list of the genes can be accessed at http://www.creagene.com/Ncreagene/dnachip/genelist.html. Microarray analysis In DC/BMT differential analysis on DC chips, the probes were the forward- and the reverse-subtracted cDNAs. In DC subset-specific analysis on HI380 chips, the probes were the amplified cDNA of each DC subset.The probes were prepared as follows: 1 µg of the cDNA was mixed with 20 to 100 pg of the plant spike DNAs and then fluorescently labeled with either Cy3 or Cy5 dye by the random priming method using Klenow fragment (NEB, Beverly, MA). The labeled cDNAs were purified through ethanol precipitation at room temperature with 2 volumes of ethanol and resuspended in 40 µL of 4 × SSC, 0.2% sodium dodecyl sulfate (SDS), 0.1 µg/µL poly(dA), 0.1 µg/µL yeast tRNA, and 0.25 µg/µL Cot1 DNA. Finally, the labeled probes were denatured at 100°C for 5 minutes and applied to the microarray for hybridization at 55°C for 12 to 16 hours and then followed by several washings. Fluorescent images of hybridized microarrays were obtained using a Scanarray 4000 microarray scanner (GSI Lumonics, Northville, MI), and the images were analyzed with GenePix Pro 3.0 (Axon Instruments, Union City, CA). The photomultiplier tube (PMT) and the laser value for scanning were tuned by equalizing the intensities of Cy3 and Cy5 on a spike gene. Fluorescence ratios were calibrated by applying normalization factors calculated from the mean intensity of spike genes (over 6 spots on each microarray). Back-hybridization For the clones randomly pooled from the subtracted DC-cDNA library, the redundancy of each clone was examined by its frequency of identification in the sequencing analysis. The clones of high redundancy were then PCR amplified with primers flanking the T-vector insertion site (sense 5'-TGCTCCCGGCCGCCAT, antisense 5'-CGGCCGCGAATTCACTAG). The amplified clones were collected, labeled with Cy3 or Cy5, and then hybridized with the DC microarray (DC chip, Creagene). The single-stranded vector DNA was prepared by asymmetric PCR with lab1 primer using self-ligated pGEM T-Easy PCR product (lab1- and lab2-primed) as a template. To minimize the background hybridization between vector sequences, the single-stranded vector DNA was included in the hybridization reaction as a blocking DNA. Clones identified with an intensity value of higher than 10 000 were screened out.Sequence analysis Following back-hybridization, the nonredundant DC-specific clones were recovered from the cell stock and each insert in the pGEM T-Easy was amplified with M13 forward and reverse primers. The PCR products were then sequenced with the Big Dye Terminator Kit (Perkin-Elmer, Boston, MA) and analyzed with a 377 ABI automated 96-lane sequencer (Perkin-Elmer). Approximately 200 to 700 bp sequences were trimmed for vector sequence with Seqman (DNAstar, Madison, WI) and were analyzed with an Advanced BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/).Quantitative PCR The initial cDNA content in each sample was normalized with an amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Quantitative PCR reactions were performed in a 50 µL volume using 4 ng of each cDNA on a Perkin-Elmer DNA thermocycler 9600 Prism for 30 cycles (15 seconds at 94°C, 20 seconds at 55°C, and 1 minute at 72°C). To evaluate the specificity of each message semiquantitatively, 10 µL each of the PCR product was withdrawn from 25 cycles and from 30 cycles, respectively, and then run simultaneously on 1.1% agarose gels. PCR primers to each selected clone were designed with PrimerSelect (DNAstar). The expected sizes of the PCR products were 300 to 600 bp, and the optimal annealing temperature ranged from 55°C to 65°C. The sequence of the RT-PCR primers is available from the authors upon request.
Immunophenotypes of purified dendritic cells The purity of CD1a+ DCs, CD14+ DCs, or CD11c DCs for the construction of the subtracted
DC-cDNA library was 90% ± 4%, and the purities of each DC subset
in the additional experiments were more than 98% after cell sorting or
isolation (Figure 1A). CD1a+ DCs and CD14+ DCs
at day 18 were strikingly distinguished not only by their surface
phenotypes but also by their morphologies with and without well-developed dendrites, respectively (Figure 1B). However, these 2 DCs were very similar in their levels of HLA-DR, CD83, and CD86 expression. The expression of DC-Lamp was observed only in
CD1a+ DCs but not in CD14+ DCs at day 18. In
this specific batch of CD14+ DCs (Figure 1B), the
up-regulation of CD83 was observed in parallel with an unusual decrease
in the levels of HLA-DR and CD86 during their development between days
8 and 18. While Langerin staining was expected in CD1a+ DCs
between days 8 and 12,35 CD1a+ DCs at day 8 were not stained by Langerin monoclonal antibody, probably indicating a
kinetic variation between different cultures of CD1a+ DCs.
The expression of Langerin at day 18 in CD1a+ DCs was not
apparent in their immunostaining, although the microarray analysis on
the same DCs revealed the up-regulated Langerin expression at day 18 in
CD1a+ DCs (Table 2). Thus, CD1a+ DCs at day 18 of their maturation were considerably nonexpressive of the
Langerin-positive phenotype.35 Unlike these
cytokine-induced DCs, CD11c DCs freshly isolated from
peripheral blood barely expressed CD86 on their cell surfaces and were
relatively small and even in size (Figure 1B). On the other hand,
CD11c DCs at day 5 expressed significant levels of CD86
as well as of DC-Lamp. Interestingly, the absence of CD83 up-regulation
in the 5-day maturation period of CD11c DCs was in good
contrast with the maturation-associated expression of CD83 in the other
DC subsets. The representative phenotypes of MoDCs were
CD1a+/CD83/DC-Lamp at day 7 and
CD1a/CD83+/DC-Lamp+ at day 9. However, regarding the level of CD1a expression in MoDCs, there was
some degree of difference, depending on the donor.
The subtracted DC-cDNA library was very specific to the DC subset To gain direct access to DC-specific genes without being hampered by highly abundant messages shared by most leukocytes, we have employed a DC cDNA subtraction strategy followed by microarray analysis (Figure 2). A subtracted DC-cDNA library was constructed by subtracting B-cell, monocyte, and T-cell messages concurrently from the combined ones of CD1a+, CD11c, and CD14+ DCs. In this subtraction, we
carried out the modified subtractive hybridization, termed
PCR-Select,36 which exploits suppressive PCR to
selectively enrich the subtracted genes. To compare the specificity of
subtraction, not only forward (DCs subtracted by BMT) but also reverse
subtraction (BMT subtracted by DCs) was performed in parallel. The
profile of the PCR products from either subtraction revealed a unique
pattern of discrete bands on agarose gel that was absent in the
nonsubtracted control (data not shown.) To examine the integrity of the
subtracted DC-cDNA library, 8 clones were randomly selected and
sequenced. Sequence analysis revealed 2 cDNAs that
corresponded with the sequences from the MMP12 gene, 3 cDNAs from the
mitochondrial genes, and 2 novel expressed sequence tags
(ESTs). The high efficacy of subtraction was indicated by the
absence of cDNAs corresponding to well-known housekeeping genes among
these randomly picked clones. To further assess the integrity of
subtraction, the forward- and reverse-subtracted cDNAs were labeled
with Cy3 and Cy5, respectively, and then hybridized on a microarray
with 2000 known genes (a generous gift from Dr J. H. Park at Korea
Research Institute of Bioscience and Bioengineering, Daejeon, Korea) in
double-blind approaches. Most of the spots developed single
fluorescence of either Cy3 or Cy5, and few developed dual fluorescence
of both Cy3 and Cy5. These results suggest that the subtraction was
successfully performed for the depletion of the common messages in the
2 cDNA populations, so that the subtracted cDNA was acceptable as a
specific probe for each population in the following microarray
analysis.
Several DC-associated genes were newly identified by microarray analysis To identify DC-associated genes, 1920 clones from the subtracted DC library and 181 cDNAs of CD and cytokine genes were immobilized on a glass slide and subjected to differential hybridization using cDNA probes manipulated as follows: The forward-subtracted (DC-specific) and the reverse-subtracted (BMT-specific) cDNAs were labeled differentially with Cy3 or Cy5 and then cohybridized with the cDNAs on the same microarray. To normalize the intrinsic signal differences coming from Cy3 and Cy5 labeling, another hybridization was set up for reverse labeling with Cy3 and Cy5. As expected, quick visual inspection of the hybridization signals revealed most of the spots originating from the subtracted DC library were DC specific, so they were not detected among the dual-labeled ones but strongly hybridized with DC-specific probes. In contrast, most of the known CD genes were not DC specific in the sense that they were barely detected with DC-specific probes, and only a few were strongly labeled by BMT-specific probes.Of the 1920 clones, 1140 were selected for their propensity to adopt
highly DC-specific signals (threshold intensity ratio of DC/BMT > 3).
To minimize the number of clones to be analyzed, redundant clones had
to be screened out. For this purpose, 74 clones were randomly selected
and sequenced. Of the 74 clones sequenced, 31 were unique genes. The
following genes were most frequently identified: immunoglobulin (Ig)
superfamily Z39Ig, mitochondrial genes (COI and COIII, 12S rRNA, 16S
rRNA, and cytochrome b), MHC class II DR
Each DC subset shows its own expression profile for DC-associated genes The DC-associated genes identified in the DC/BMT differential microarray analysis were then further examined for their expression profiles in different DC subsets by using another microarray, HI380, (Table 2) and by semiquantitative RT-PCR (Figure 3). Some other genes of special interest, such as CCR1, CCR7, DC-Lamp, E-cadherin, Langerin, and others, were also included in the subset-specific expression analysis. As shown in Table 2 and Figure 3 and as summarized in Figure 4, the results from the microarray were in good agreement with the results revealed in the quantitative RT-PCR.
As expected from their lineage differences, the most striking
difference was seen between CD11c While the difference was not as remarkable as that shown in
CD11c Expression of DC-associated genes in different maturation stages To answer the important question of how the differentiation/maturation stages of DCs affect the difference in DC-associated gene expression among the 4 DC subsets, we undertook similar microarray analyses for the immature myeloid DCs and the matured CD11c DCs (Table
3). After maturation by culturing for 5 days under the influence of CD40L and IL-3, CD11c DCs did
not overexpress the messages commonly up-regulated in fully
differentiated DCs of myeloid origin. Thus, genes such as  - and
 -tubulin, Eta-1, GPNMB, MCP4, lysosomal acid lipase, enolase 1, thymosin 4, ferritin L-chain, annexin A2, VAMP8, and GABARAP were
considered to be truly myeloid DC associated. Interestingly, the high
expression of FLAP, implicated in allergic
inflammations,42 was not restricted to a certain stage of
maturation but was relatively common to the DC subclasses, including
CD11c DCs. The exceptional absence of the FLAP
overexpression in MoDCs at day 9 was repeatedly observed in the ones
derived from a different donor (data not shown). The IRF4 expression in
CD1a+ DCs, CD11c DCs, and CD14+
DCs was markedly down-regulated upon DC maturation but was completely reversed in MoDCs (Table 3), suggesting that the control of IRF4 expression might be cell type specific, even among the myeloid DCs.
However, in the other set of experiments, the IRF4 expression was
considerably higher in CD1a+ DCs at day 18, suggesting that
the control of IRF4 expression might not simply be maturation related
but might vary depending upon the multiple signals, at least in
CD1a+ DCs (data not shown). At a glance, the expression of
most DC-associated genes was not remarkable in the case of immature DCs
and seemed to be associated with the maturity of the relevant DC
subsets. For example, MMP12, Z39Ig, GPNMB, Eta-1, and others showed a
DC/BMT ratio lower than 1 in immature DCs. However, there were genes constitutively overexpressed in the immature stages of the relevant DCs
(DC/BMT > 1), while the exact level of overexpression often varied
substantially following their maturation. These genes included TARC in
MoDCs, MHC class II DR in all of 4 DC subclasses, CD1b in MoDCs and
CD1a+ DCs, CD20-like precursors and MRC1 in the 2 CD34+-derived DCs, lysosomal acid lipase and TGFBI in
MoDCs, and others. For the 2 DCs derived from the CD34+
cells, the DC subset-specific control appeared in the early stages of
DC development. Thus, there was differential expression of MCP1 in
CD14+ DCs, just as in the case of DC-Lamp in
CD1a+ DCs at day 8, indicating their development through
distinct pathways from the same precursor.
Expression of DC-associated genes in different donors To examine how donor difference affects the results, we prepared another set of 4 DC subclasses from different donors and undertook microarray analyses with cDNA probes freshly derived from the second set of DCs. As shown in Table 4, most of the representative genes generally showed "relative consistency" in their expression profiles for different donors. A profound relative consistency was found in the expression of TARC, Ig superfamily (Z39Ig), MCP1, TGFBI, CCR1, DC-Lamp, E-cadherin, and DEC205. However, the relative consistency of the expression of Eta-1, MRC1, and IRF4 was not as strong as in those mentioned above. Among the DC-associated genes newly identified, MDL-1 was consistently distinguished by its marginal DC association in both donor sets. Briefly, even though the DC/BMT ratios for the selected genes were not exactly conserved, the relative consistency between different donors for their specificity to each DC subset was present for most of the selected genes.
The heterogeneity of DCs has been manifested as distinct
surface phenotypes, differences in their tissue homing, and at the level of their development. To understand the functional heterogeneity of human DCs, we attempted to explore the differences in the
transcriptional profiles of different DC subclasses. Because previous
work has revealed specific gene expression in MoDCs, we aimed to
delineate the gene expression related to the remainder of the subsets
of human DCs, namely CD11c In an effort to pool DC-associated genes, we combined the cDNAs derived
from the 3 DC subsets (CD1a+ DCs, CD11c The DC-associated genes identified in this study were further
examined for their expression profiles among the different DC subsets
using an HI380 microarray. Consistent with the result of DC/BMT
differential screening, most of the DC-associated genes showed their
specificity to one or more of the DC subsets (Table 2). However, in
contrast to the results shown in Table 2, 5 genes Although this study was quite limited in uncovering the genes
selectively expressed in the lymphoid DCs, our results revealing the
total absence of the myeloid DC-related messages in CD11c Human myeloid DCs can be developed from various sources, including blood monocytes and CD34+ hematopoietic stem cells. Likely reflecting the heterogeneous DCs developed from different precursors under different tissue-cytokine microenvironments, the 3 DC subclasses of myeloid origin (CD1a+ DCs, CD14+ DCs, and MoDCs) appeared to be somewhat different in their gene regulations. According to our data shown in Table 2, the apparent differences were in the range of "from the subtle to the profound," indicating the presence of shared as well as unique functions. The most outstanding difference was revealed in their expression levels of chemokine and chemokine receptors, implying their remarkable differences in trafficking properties. Based on the previous findings,47 MoDCs and CD1a+ DCs, which showed the unique up-regulation of TARC and CCR7, fitted into the authentic migratory DC pattern of traveling from tissue to the draining lymph nodes to convey Ag to T cells. On the other hand, the results from the subset-specific expression analysis indicated that CD14+ DCs were more likely to be tissue resident DCs with a transcription profile of "no CCR7 but more CCR1" and "no TARC but more MCP1 and Eta-1." The preferential expression of Eta-1 (osteopontin)48 and MCP1 in CD14+ DCs tends to suggest that CD14+ DCs play multiple roles in the regulation of inflammation and tissue remodeling. The outstanding expression of factor XIIIa in CD14+ DCs further supports the likely connection of CD14+ DCs with dermal DCs. Our finding of Eta-1 overexpression by CD14+ DCs certainly provides the missing link between factor XIIIa+ dermal DCs and wound healing, angiogenic, and fibrogenic processes.49-51 The absence or the lower level of DC-Lamp expression has been described
in the immature forms of MoDCs,52 CD1a+
DCs,53 and CD11c TGF- The up-regulation of some DC-associated genes is likely to explain the
connection of DC subtypes with certain human diseases. These genes
include high-affinity IgE receptor These findings provide a great deal of important molecular information about each DC subset, which will be useful in further functional studies of DC subsets. Although the biologic significance of these findings is still unclear, further studies in vitro and in vivo may elucidate the functional milieu of the genes.
We are very grateful to Drs Y. I. Yeom, J. H. Park, K. A. Yoon, and S. J. Yang of The Center for Functional Analysis of the Human Genome at the Korea Research Institute of Bioscience and Bioengineering for their kind help in microarray fabrication and analysis. We thank Drs S. Y. Kim and S. R. Nam of the Faculty of Medicine, Chungnam National University, Dr K. J. Baek of the Motae Obstetrician's Office, Dr J. I. Lee of the Shina Obstetrician's Office, and Dr I. S. Kim of the Sungae Hospital for their generous supplies of cord blood and PBMC. We are also grateful to L. Waldron for her generous editorial help.
Submitted August 13, 2001; accepted April 17, 2002.
Supported by the Korea Ministry of Health and Welfare grant 01-PJ1-PG4-01PT02-0003 and in part by the Korea Science and Engineering Foundation grant 2000-1-20200-002-3.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Yong Soo Bae, Department of Microbiology, Hannam University, 133 Ojung-dong-, Daedeok-gu, Daejeon 306-791, South Korea; e-mail: ysbae{at}mail.hannam.ac.kr.
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Top
Abstract
Introduction
globulin-coated plates.
) for expression detectable only after 30 cycles of PCR and
marginally detectable even after 30 cycles of PCR, respectively. GAPDH
was used for normalization of each cDNA amount. CD19, CD14, and CD28
were used as control genes for B cells, monocytes, and T cells,
respectively. The ratio of DC/BMT represents the degree of DC
specificity of each clone as determined by microarray analysis and as
described in Table 
that is,
Ag-uptake/processing/presentation and cell metamorphosis/migration. In
addition, throughout the analyses there were frequent appearances of
the mitochondrial genes (such as MTATP6, MTCYB, COII) and the genes for
Ca++ binding proteins (such as S100B, calmodulin). The
up-regulation of these genes seems to be necessary for the 2 typical DC
functions requiring a high level of adenosine triphosphate
(ATP) and Ca++ modulation. Lastly, the other class of the
genes differentially expressed in the DCs was the chemokine genes, such
as TARC, MCP1, and MCP4. The identification of such genes was in
accordance with the previous findings on MoDCs.
1) in any of the 4 DC subsets. This could be due to the
hypersensitivity of the subtracted cDNA probes used for screening the
DC microarray, which resulted in the amplification of the trivial
differences between the DCs and the BMTs.