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*
Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia;
Cooperative Research Centre for Vaccine Technology, Brisbane, Queensland, Australia;
Medical Microbiology, Immunology and Hygiene, Technischen Universitat Munchen, Germany; and
Austin Research Institute, Heidelberg, Victoria, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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has been used to divide
murine DC into two general types, CD8+ and
CD8-. More recently, CD4 has been used to
further subdivide the CD8- DC to produce three
splenic DC subsets:
CD8+CD4-,
CD8-CD4+, and
CD8-CD4-
(8). These appear to be the product of separate
developmental streams (9). Many functional differences
between CD8+ and CD8- DC
have been described (5, 10, 11, 12, 13). It has been proposed that
CD8+ DC are involved in maintaining
self-tolerance, whereas the CD8- DC are involved
in inducing immunity (3, 6, 14, 15). Certainly, in vitro
these subpopulations differ in their ability to stimulate T cells. The
CD8- DC are more potent at inducing cytokine
production and consequently inducing a more prolonged proliferative
response (16, 17), a difference not attributable to
variations in levels of surface MHC or known costimulatory molecules
(18). A further crucial difference is that
CD8+ DC produce more IL-12, and can bias an
immune response toward an inflammatory Th1 reaction (10, 13, 19, 20). Thus, DC subtypes may posses differentially expressed
surface signaling molecules, and these could be targets for immune
modulation strategies. To investigate such differences we screened CD8+ and CD8- DC for novel gene expression using representational difference analysis (RDA) (21). RDA is a technique that combines subtractive hybridization of the genes that are common with PCR amplification of the products that are unique to the nominated population. We isolated several gene fragments that were preferentially expressed by CD8- DC. Of particular interest was a gene coding for a cell surface protein similar to the mouse macrophage marker, F4/80 (22). In this study we describe the identification and characterization of this F4/80-like receptor (FIRE). FIRE is expressed on the surface of a subset of CD8- DC and of macrophages, as well as on blood monocytes, the precursors of these APC, but it is down-regulated on activation or maturation. We have developed several mAbs against FIRE that are immediately useful for DC subtype characterization and that point to future use of FIRE as a target for modifying immune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6J wehi mice were bred under specific pathogen-free conditions at the Walter and Eliza Hall Institute (WEHI).
Cloning of mouse FIRE
RDA was conducted using cDNA obtained from DC subtypes
segregated as
CD8+CD4-Mac-1-
and
CD8-CD4-Mac-1+.
Briefly, RNA was extracted using QuickPrep Micro mRNA Purification kit
(Pharmacia Biotech, Uppsala, Sweden) and cDNA was synthesized (cDNA
Synthesis kit, Boehringer Mannheim Biochemica, Indianapolis, IN)
according to the manufacturer’s instruction. The cDNA RDA method was
essentially as described previously (21). Totals of 5
x 105
CD8-CD4- DC and
1.8 x 106
CD8+CD4- DC were used
to extract mRNA. The synthesized double-stranded cDNA was digested with
DpnII and was ligated to the R-Bgl-24 and R-Bgl-12 linkers.
The ligated cDNA was diluted to equivalent input levels and then
amplified with 20 PCR cycles using R-Bgl-24. One
CD8- RDA product sharing sequence similarity
with F4/80 and henceforth referred to as FIRE was selected for further
study. The complete cDNA sequence of FIRE was obtained by using the
RDA-derived fragment to make PCR oligonucleotides (5'-TCC GTC GAC TCA
TCC TTC CCA ATG GAC ACA G and 5'-CGA CGC GTC TAG AAG CTG GCA ACA ACA C)
and identify a mouse thymus cDNA library (Uni Zap Lamda Vector;
Stratagene, La Jolla, CA) that contained the transcript. This library
was then probed with [32P]-labeled cDNA of FIRE
(hybridized in 0.2% BSA, 0.2% Ficoll, 0.2% polyvinylpyrrolidone, 0.1
mM ATP, 0.05 mg/l tRNA, 2 mM NaPP, 0.05 mg/l herring sperm DNA, and
0.02% sodium azide) overnight at 42°C, then washed at 67°C, once
with 2x SSC/0.1% SDS, and then three times with 0.2x SSC/0.1%
SDS.
Northern blot analysis
Total RNA was isolated from tissue using TRIzol LS (Life Technologies, Rockville, MD) and from DC using RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturers’ guidelines. The RNA was fractionated on formaldehyde agarose gels and transferred to a Hybond-N nylon membrane (Amersham, Arlington Heights, IL). The nylon membrane was prehybridized for 1 h at 42°C in a buffer containing 1% SDS, 50 mM Tris-HCl (pH 7.5), 0.2% BSA, 0.1% tetra-sodium pyrophosphate (Na4PO4), 0.2% polyvinylpyrrolidone-40 (m.w. 40,000), 0.2% Ficoll 400, 50% formamide, 10% dextran sulfate, and 1 M NaCl. The [32P]-labeled cDNA probe was hybridized overnight at 42°C, then washed as described above.
Generating mAbs
FIRE and a control protein were expressed as FLAG-tagged proteins on the surface of Chinese hamster ovary (CHO) cells. Briefly, primers (5'-TAG TAG ACG CGT ATA TTA CAA ATG ATG AAT ATT and 5'-TAG TAG ACG CGT TCA ATC ACT AAT AGT TCT GCT) were designed to amplify FIRE and allow its cloning into a pEF-BOS vector (provided by Dr. T. Willson; http://www.wehi.edu.au/willsonvectors) that had been modified to contain the IL-3 leader sequence followed by the FLAG epitope. This construct resulted in the surface expression of FIRE protein that contained the FLAG epitope at the extracellular N terminus. Using FuGENE 6 Transfection Reagent (Boehringer Mannheim), CHO cells were cotransfected with the pEF-BOS-FIRE and a pCI-neo plasmid containing the neomycin phosphotransferase gene (Promega, Madison, WI), at a ratio of 10:1. Transfectants were allowed to recover for 24 h before selection with 750 µg/ml G418 (Geneticin, Life Technologies). FIRE-positive cells were stained with anti-FLAG mAb (IC7; provided by Dr. N. Nicola, WEHI), followed by a PE-conjugated anti-mouse Ig (SILENUS Labs, Boronia, Australia), and they were then isolated by sorting on a MoFlow Instrument (Cytomation, Fort Collins, CO). After two rounds of enrichment, a pool of stable transfectants was established. To produce soluble FIRE, the external portion of FIRE was amplified (primers: 5'-CGG GAT CCT CCT CAT GGG GTA GAG CC and 5'-CGG GTA CCA CCA TGG GAA GCA GGT GCC TTC TGC) and then fused to the human IgG1 Fc domain and expressed in the Cigh vector. The construct was cotransfected with the pCI-neo plasmid into CHO cells. Transfectants were cloned by limiting dilution, and clones that produced the Fc-fusion protein were selected using an anti-human Ig ELISA. Fc-FIRE was purified and enriched using an anti-human IgG agarose column (Sigma, St. Louis, MO). The fusion protein was used in ELISA where an anti rat-Ig-HRP (Chemicon International, Temecula, CA) Ab was used to detect sera that bound to Fc-FIRE. To generate mAb, rats were immunized four times with 5 x 106 CHO cells expressing FIRE-FLAG, and they were then given a final boost 4 days before fusion. Hybridomas secreting specific mAb were identified by ELISA using the Fc-FIRE fusion protein and were confirmed by flow cytometric analysis of supernatants using CHO-FIRE-FLAG or CHO-Neo.
Isolation of DC, macrophages, and peripheral blood monocytes
The isolation of DC subpopulations has been described (23). Briefly, tissues were chopped, digested with collagenase and DNase, and treated with EDTA. Low-density cells were enriched by density centrifugation, then the non-DC-lineage cells were removed by immunomagnetic bead depletion. The remaining cells were stained with fluorochrome-conjugated mAb and populations of >95% pure CD11c+CD8+CD4-, CD11c+CD8-CD4+, and CD11c+CD8-CD4- DC were isolated by sorting. Normal or activated macrophages were obtained from the peritoneal cavity of normal or thioglycolate-injected mice and were identified as CD11b+ cells. Splenic macrophages were isolated from spleen cell suspensions by first removing dead cells and erythrocytes, then removing irrelevant cells by immunomagnetic bead depletion of cells bearing CD3, CD4, CD8, B220, and the erythrocyte marker TER119. This enriched fraction was then stained with anti-CD11b (M1/70) and anti-F4/80, and double-positive cells considered macrophages. To obtain blood mononuclear cells, mice were bled by cardiac puncture into tubes containing heparinized buffered saline solution. Mononuclear cells were isolated from heparinized mouse blood by density centrifugation using Lympholyte M (Cedarlane Laboratories, Hornby, Ontario, Canada), then cells bearing CD3, Thy-1, B220, Gr-1, and the erythrocyte marker TER119 removed by immunomagnetic bead depletion. Dead cells were excluded from analysis based on their uptake of propidium iodide.
Immunohistochemical analysis
Frozen spleen sections (5 µm) were cut and fixed using ice-cold acetone. Sections were rehydrated in PBS, blocked with mouse and goat sera (5%), and then incubated with either MOMA-1, mucosal addressin cell adhesion molecule-1 (MadCAM-1) (BD PharMingen, San Diego, CA), or anti-CD11c (N418) mAb followed by alkaline phosphatase-conjugated goat anti-rat specific polyclonal Ab (PharMingen) or alkaline phosphatase-conjugated anti-armenian hamster polyclonal (The Jackson Laboratory, Bar Harbor, ME). Further nonspecific binding to the anti-rat polyclonal was prevented by an incubation with rat Ig (100 µg/ml). Sections were then incubated with biotinylated anti-FIRE (6F12) or biotylated isotype control mAb (IgG2a; PharMingen) and the signal was amplified using Vecta Stain Elite ABC kit (Vector Laboratories, Burlingame, CA). Alkaline phosphatase and horseradish peroxidase activity were revealed using NovaBlue and NovaRed (Vector Laboratories), respectively.
RT-PCR analysis of FIRE mRNA expression
RNA was extracted from isolated DC subsets and cDNA was
synthesized using random hexameric primers and Moloney murine leukemia
virus reverse transcriptase (Promega) as recommended by the
manufacturer. Primers specific for FIRE (5'-GCT GCA GGT GGA GTG TCG and
5'-CGA CGC GTC TAG AAG CTG GCA ACA ACA C) and 
-actin (5'-GTG GGC CGC
TCT AGG CAC CAA and 5'-CTC TTT GAT GTC ACG CAC GAT TTC) were
synthesized. PCR was performed (94°C for 3 min denaturing, then
various cycles at 94°C for 30 s; annealing at 60°C for 30
s; extension at 72°C for 30 s) and the products were visualized
on ethidium bromide gels.
DC activation
Isolated DC subsets (1 x 105) were
cultured for 20 h in 200 µl of modified RPMI 1640 medium
containing 10% FCS, antibiotics, 10-4 M 2-ME,
GM-CSF (50 U/ml), IFN-
(100 ng/ml), anti-CD40 (25 µg/ml), and
LPS (50 ng/ml).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the functional differences between
CD8-CD4- DC and
CD8+CD4- DC, RDA was used
to examine differential gene expression. We isolated many novel gene
fragments that were preferentially expressed by
CD8-CD4- DC, a
surprisingly large proportion of which coded for surface membrane
proteins. Of particular interest was a novel gene fragment whose
sequence showed similarity with mouse F4/80. Using the partial gene
fragment as a probe and conventional hybridizing techniques, the
complete cDNA was obtained by the isolation of a 3.25-kb clone from a
mouse thymus cDNA library. The sequence and proposed structure of this
molecule termed FIRE are shown in Fig. 1
, A and B.
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B). FIRE has a leader sequence of 
19
residues followed by two extracellular epidermal growth factor
(EGF)-like domains. The second EGF-like domain has the consensus
sequence Asp/Asn, Asp/Asn, Gln/Glu, Asp/Asn, and Tyr/Phe, which
predicts calcium-binding capacity (24, 25). Between the
second EGF-like domain and the predicted transmembrane regions there
are 216 residues that serve as a hinge. This region is rich in serine
and threonine residues, which are potential sites for
O-linked glycosylation. The seven-transmembrane region is
composed of 251 residues followed by an intracellular tail of 93
residues (Fig. 1B).
The FIRE sequence shows that it is a novel member of the recently
described EGF-transmembrane-7 (TM7) subfamily of seven-transmembrane
proteins (26, 27). FIRE shows a striking homology with
F4/80 (EMR1), CD97, and EMR2, displaying 45%, 38%, and 44% identity,
respectively (calculated using the ALIGN program PAM 250 mutation data
matrix, break penalty = 6; Ref. 28). Sequence
similarity is distributed throughout the
variousregions of these complex molecules. FIRE, like
other members of the EGF-TM7 subfamily, has EGF domains at the N
terminus. There is also significant sequence conservation in the hinge
region (data not shown). Fig. 2 shows an
alignment of FIRE with other members of the EGF-TM7 subfamily and
demonstrates the significant sequence homology in the Cys-Box
(26), and in the seven-transmembrane domains.
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Northern blot analysis revealed that FIRE has an extremely
restricted expression pattern. FIRE was preferentially expressed
by CD8-CD4- DC;
when standardized for the amount of RNA loaded, the
CD8-CD4- DC expressed
12-fold more FIRE mRNA than did
CD8+CD4- DC. There was no
detectable expression of FIRE in heart, brain, kidney, thymus, and
liver, but low levels were evident in spleen (Fig. 3A) and very low levels could
sometimes be detected in the lung. Neither freshly isolated T and B
cells nor a panel of T and B cell lines expressed FIRE (Fig. 3A). Nor was FIRE was expressed by several myeloid and
macrophage cell lines. Only one cell line, GB2, which was of
ProB/myeloid origin and could give rise to DC (L. Harrison, WEHI,
unpublished observations), expressed FIRE mRNA (Fig. 3B).
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Four hybridomas produced mAb that recognized CHO-FIRE-FLAG on the
cell surface by flow cytometry, but failed to recognize control
transfectants that expressed an irrelevant FLAG-tagged protein or the
neomycin resistance gene only. Three of the hybridomas, 4E9, 6F12, and
3H7, produced IgG2a mAb, while 9B12 produced an IgM mAb. Fig. 4 shows that 6F12 and 3H7 specifically
stain CHO-FIRE-FLAG transfectants, but fail to stain CHO-Neo control
cells. None of these mAb were efficient at Western blot or
immunoprecipitation analysis, but 6F12 allowed visualization by Western
blot analysis of a 100-kDa protein band and a 65-kDa presumed breakdown
product in the CHO-FIRE-FLAG transfected cells. No bands were detected
in the above control transfectants, confirming specificity. Using these
mAb, we analyzed the surface expression of FIRE on various cell types.
Splenic T cells and most B cells (Fig. 4) failed to express FIRE. By
contrast, a proportion of splenic DC, lymph node DC, and splenic and
peritoneal macrophages all expressed FIRE protein on their surface
(Fig. 4). As predicted by the RDA and Northern blot analysis, a much
larger proportion of
CD8-CD4- DC than
CD8+CD4- DC expressed FIRE
protein (Fig. 4).
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). CD11c+ cells
(seen in blue, Fig. 5C), the classical marker for DC, is not
coexpressed by most FIRE+ cells in the red pulp,
and FIRE is not coexpressed by the CD11c+ cells
in the T cell area, which are predominantly CD8+.
However, in the marginal zones and in some regions of the red pulp of
the spleen, there appears to be such coexpression because both markers
stain cells in these compartments and some double-positive cells are
evident. Conclusive evidence that a subpopulation of
CD11c+ DC also express FIRE was obtained by flow
cytometry (Fig. 4). Costaining with MOMA-1 and anti-MadCAM-1 mAb
showed that FIRE+ cells reside in the marginal
zone of spleen. MOMA-1 is expressed by metalophilic macrophages and is
visualized as a blue stain (Fig. 5D), but when
counterstained with anti-FIRE mAb (revealed in red), this blue
stain becomes purple, indicating that at least a subset of MOMA-1 cells
also coexpress FIRE (Fig. 5E). Thus, at least a subset of
metalophillic macrophages express FIRE. MadCAM-1 is expressed by
reticular cells found in the marginal zones (seen in blue; Fig. 5F) and when costained for FIRE (in red), single-stained
cells with either marker were seen, indicating that it is less likely
that MadCAM-1 cells also coexpress FIRE, rather
FIRE+ cells reside within close proximity of
MadCAM-1+ cells. However, coexpression of both
markers cannot be excluded by this analysis.
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H) and occasionally in the paracortex (Fig. 5I).
Macrophages are known to reside in the subcapsular sinus, whereas both
DC and macrophages are known to be present in the paracortex. Thus, the
staining pattern observed in the lymph nodes is similar to that in the
spleen, in that FIRE+ cells appear to colocalize
with macrophages and DC. Activation of DC and macrophages results in down-regulation of FIRE
To test whether activation affected the expression of FIRE,
purified DC subpopulations were stimulated in vitro with GM-CSF,
IFN-, anti-CD40, and LPS. Under these conditions, DC
down-regulated FIRE as determined by RT-PCR (Fig. 6) and surface staining (Fig. 4).
Likewise, activated peritoneal macrophages did not express FIRE (Fig. 4). Because the expression of FIRE appeared to be inversely related to
the activation or maturation status, we examined the expression of FIRE
at earlier stages of DC and macrophage development.
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Because FIRE appeared to be lost on activation or maturation, we
examined the expression of FIRE on the earlier stages of DC and
macrophage development. PBMC are frequently used to produce DC in
culture and can also give rise to macrophages (2, 29).
Isolated mouse blood monocytes were seen to be the richest source of
FIRE-positive cells (Fig. 4). The specificity of this staining was
confirmed by the near complete blocking of FIRE fluorescence by an
excess of FIRE-Fc fusion protein, while staining for the related marker
F4/80 was undiminished. Looking still earlier at BM, which provides the
precursors of cells that populate the blood, a proportion of
FIRE-positive cells were obtained (data not shown). These were mainly
among the Mac-1+F4/80+ BM
cells, but represented only a small proportion of
GR1+ cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using RDA, we identified FIRE, a gene differentially expressed by
CD8-CD4- DC, which may
serve as a model of new surface molecules detected on the basis of
differential expression on DC subsets. FIRE belongs to the EGF-TM7
subfamily of proteins, having a structure that predicts dual function
(26, 27, 37). The other members of the EGF-TM7 subfamily
are EMR2 (38), CD97, an early activation marker widely
expressed by leukocytes (39, 40, 41), and mouse F4/80 and its
likely human homologue EMR1 (42, 43). The
seven-transmembrane region of this group of proteins is closely related
to the G-protein-coupled receptors of the secretin/calcitonin receptor
superfamily, also known as the B family of G-protein-coupled receptors,
which can transduce signals via Gs, leading to activation of
adenylate cyclase (26, 44). FIRE, like these peptidic
hormone receptors, has conserved cysteine residues in the first and
second extracellular transmembrane loops that are thought to form a
disulfide bond and be critical in the tertiary structure. However,
unlike conventional G-protein-coupled receptors that have a short
extracellular region, members of the EGF/TM7 subfamily have a long
extracellular region (26). This region contains motifs
that may be involved in cell-cell interaction (26, 27, 37). The EGF domains of CD97 have been implicated in ligand
binding to the cell surface molecule CD55-DAF (45, 46).
CD97 and F4/80 have five and seven EGF-like domains, respectively, but
splice variants are known to occur (37, 42, 43, 45, 47).
CD97 and F4/80 also have an Arg-Gly-Asp motif, which may implicate
these molecules in binding to integrins (27). By contrast,
FIRE has two EGF-like domains, but lacks this Arg-Gly-Asp motif. The
second EGF-like domain, like several of the EGF domains found in CD97
and F4/80, has a calcium-binding consensus that can stabilize
conformation and mediate protein-protein interaction (24, 25). It is therefore probable that the EGF domains of FIRE also
mediate cell surface protein-protein interactions.
Although FIRE is a member of this emerging superfamily, it is the first
to be so clearly associated with DC. To emphasize this association, we
have recently isolated a homologue of FIRE from human cells and
demonstrated a similar expression by a subgroup of myeloid-related DC
(I. Caminschi and S. Vandenabeele, unpublished observations). The
relationship of FIRE to the macrophage marker F4/80 is of interest.
Both are expressed on myeloid cells that eventually give rise to DC
(Fig. 4 and Ref. 48), both are expressed at moderate
levels on DC expressing myeloid marker but not on
CD8+ DC (Fig. 4 and Ref. 8), and the
expression of both molecules declines as DC are activated to further
maturation (Fig. 6 and Refs. 49 and 50).
However, F4/80 is at higher levels on the
CD8-CD4+ DC subset
(8), whereas the
CD8-CD4- DC subset
contains more FIRE+ cells (Fig. 4). The biggest
difference is that F4/80 is expressed at much higher levels on mature
tissue macrophages (and so is considered as a macrophage rather than a
DC marker), whereas FIRE expression remains at modest levels on tissue
macrophages (Fig. 4), drops on stimulation, and is absent from most
macrophage cell lines (Fig. 3B). FIRE may best be described
as a marker of myeloid cells with a DC developmental potential.
At this stage, the function of FIRE is unknown. Because it is at higher levels on earlier stages of DC maturation, it is more likely to be involved in functions such as migration and tissue localization, maturation, or Ag uptake than in direct interaction of mature DC with T lymphocytes. The early expression of FIRE, especially at migratory DC stages such as in the blood, should make it an accessible target in selective immunomodulation strategies. The availability of a series of mAb that recognize FIRE will now assist in determining its function.
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Acknowledgments |
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Footnotes |
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2 The sequence presented in this article has been submitted to GenBank under accession number AF396935.
3 Address correspondence and reprint requests to Dr. Irina Caminschi, The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia. E-mail address: caminschi{at}wehi.edu.au
4 Abbreviations used in this paper: DC, dendritic cell; EGF, epidermal growth factor; TM7, transmembrane-7; FIRE, F4/80-like-receptor; RDA, representational difference analysis; CHO, Chinese hamster ovary; BM, bone marrow; MadCAM-1, mucosal addressin cell adhesion molecule-1.
Received for publication May 4, 2001. Accepted for publication July 17, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+ and CD8- subclasses of dendritic cells direct the development of distinct T helper cells in vivo. J. Exp. Med. 189:587.+ and CD8- dendritic cells in the induction of primary immune responses in vivo. J. Leukocyte Biol. 66:242.[Abstract]
+ dendritic cells generate an immune response after subcutaneous injection without homing to the draining lymph node. J. Exp. Med. 189:593.+ or CD8-/- dendritic cells. Eur. J. Immunol. 27:3350.[Medline]
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,
The position of introns as determined by genomic sequence
analysis.
