This information is current as
of June 16, 2017.
Human Follicular Dendritic Cells Express
Prostacyclin Synthase: A Novel Mechanism to
Control T Cell Numbers in the Germinal
Center
In Yong Lee, Eun-Mi Ko, Sang-Hyun Kim, Doo-Il Jeoung
and Jongseon Choe
J Immunol 2005; 175:1658-1664; ;
doi: 10.4049/jimmunol.175.3.1658
http://www.jimmunol.org/content/175/3/1658
Subscription
Permissions
Email Alerts
This article cites 41 articles, 18 of which you can access for free at:
http://www.jimmunol.org/content/175/3/1658.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2005 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
References
The Journal of Immunology
Human Follicular Dendritic Cells Express Prostacyclin
Synthase: A Novel Mechanism to Control T Cell Numbers in
the Germinal Center1
In Yong Lee,*† Eun-Mi Ko,*† Sang-Hyun Kim,* Doo-Il Jeoung,†‡ and Jongseon Choe2*†
T
cell-dependent humoral immune responses culminate in
the germinal centers (GCs)3 of secondary lymphoid follicles. B cells are the major constituent of the GC and
found closely associated with fine dendritic structures of specialized stromal cells called follicular dendritic cells (FDCs) (1). FDC
is thought to provide B cells with essential survival signals because
B cells detached from FDC undergo rapid apoptosis (2, 3). The
essential requirement of FDCs for normal immune responses has
been demonstrated in vivo with lymphotoxin-␣ knockout animals.
Mice deficient in lymphotoxin-␣ lack the FDC network and the
ability to mount isotype-switched memory Ig response (4 – 6). Accumulated evidence suggests that FDCs play central roles in B cell
survival, proliferation, and differentiation into memory cells (1,
7–9). FDCs also appear to interact with another cellular component
of the GC, T cells, which are found much less frequently than B
cells (10). T cells stimulate proliferation of FDC-like cells (11). In
contrast, FDCs inhibit T cell proliferation (12). However, little is
known of the molecular basis of these cellular interactions.
FDCs constitute only a minor cellular component of the GC,
which makes extensive functional studies problematic due to the
difficulty of isolating cells to a significant purity and maintaining
them in culture. Several research groups including ours have de-
*Department of Microbiology and Immunology, Kangwon National University College of Medicine, and †Vascular System Research Center, Kangwon National University, Chunchon, Kangwon, Republic of Korea; and ‡Division of Life Sciences,
Kangwon National University College of Natural Sciences, Chunchon, Kangwon,
Republic of Korea
Received for publication March 7, 2005. Accepted for publication May 24, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Vascular System Research Center grant and a grant
(R05-2003-000-11631-0) from the Korea Science and Engineering Foundation.
2
Address correspondence and reprint requests to Dr. Jongseon Choe, Department
of Microbiology and Immunology, Kangwon National University College of Medicine, Chunchon, Kangwon 200-701, Republic of Korea. E-mail address:
[email protected]
3
Abbreviations used in this paper: GC, germinal center; FDC, follicular dendritic
cell; PGIS, PGI2 synthase; AP, alkaline phosphatase; MALDI-TOF-MS, MALDITOF mass spectrometry; siRNA, small interfering RNA.
Copyright © 2005 by The American Association of Immunologists, Inc.
veloped mAbs against FDC for the purpose of using them in immunohistological studies and in purification of FDCs (13–21).
Most FDC-reactive mAbs developed to date cross-react with bone
marrow-derived cells. For example, DRC-1, Ki-M4, 7D6, and HJ2
stain B cells (13, 16, 18), while 12B1 cross-reacts with monocytes
(15), AS02 with blood stem cells (22), and CNA4.2 with T cells
(17). In addition, FDC-SP, a recently discovered novel FDC molecule, is induced on cultured PBMC (23). Because mAb 3C8 that
was developed in our laboratory did not display cross-reactivity
with bone marrow-derived cells but specifically recognized FDCs
in the GC (24, 25), we reasoned that identification of the Ag would
reveal novel molecular mechanisms to explain biological functions
of FDC. In this study, we have attempted molecular identification
of the 3C8 Ag by expression cloning and proteome analyses,
which led to the conclusion that human FDC expresses PGI2 synthase (PGIS, EC 5.3.99.4). PGIS belongs to the cytochrome P450
superfamily. This enzyme is localized in the endoplasmic reticulum and converts PGH2 into PGI2 or prostacyclin, the most potent
endogenous inhibitor of platelet aggregation and a strong vasodilator (26, 27). With regard to the biological function of PGIS in the
GC, we found that prostacyclin inhibits proliferation and spontaneous apoptosis of T cells.
Materials and Methods
Abs and reagents
The mAb 3C8 was developed as previously described (21), purified, and
conjugated with biotin using a biotinylation kit (Pierce) according to the
manufacturer’s instructions. Abs and reagents used in this work were anti␤-actin, biotin-conjugated mouse IgG1, streptavidin-FITC (DAKO),
mouse IgG1, PGI2 (Sigma-Aldrich), alkaline phosphatase (AP)-conjugated
goat anti-mouse IgG1, beraprost, PGH2, rabbit anti-human PGIS (Cayman
Chemical), unconjugated anti-human CD3 (64.1, Dr. Y. S. Choi, Ochsner
Medical Foundation, New Orleans, LA), and HRP-conjugated anti-rabbit
Ig (Pierce).
Culture of HK cells and T cells
HK cells were prepared and maintained as described by Kim et al. (11).
Synovial fibroblasts were a kind gift from Dr. C.-S. Cho (Division of Rheumatology, Center for Rheumatic Diseases in St. Mary’s Hospital, Seoul,
Korea). HK cells and fibroblasts were grown in RPMI 1640 (Invitrogen
0022-1767/05/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Stromal cells in the lymphoid organs provide a microenvironment where lymphocytes undergo various biological processes such
as development, homing, clonal expansion, and differentiation. Follicular dendritic cells (FDCs) in the primary and secondary
follicles of the peripheral lymphoid tissues interact with lymphocytes by contacting directly or producing diffusible molecules. To
understand the biological role of human FDC at the molecular level, we developed a mAb, 3C8, that recognizes FDC but not bone
marrow-derived cells. Through expression cloning and proteome analysis, we identified the protein that is recognized by 3C8 mAb,
which revealed that FDC expresses prostacyclin synthase. The 3C8 protein purified from FDC-like cells indeed displayed the
enzymatic activity of prostacyclin synthase and converted PGH2 into prostacyclin. In addition, prostacyclin significantly inhibited
proliferation of T cells but delayed their spontaneous apoptosis. These findings may help explain why T cells constitute only a
minor population compared with B cells in the germinal center. The Journal of Immunology, 2005, 175: 1658 –1664.
The Journal of Immunology
Life Technologies) supplemented with 10% FBS, 2 mM L-glutamine (Invitrogen Life Technologies), and 80 ␮g/ml gentamicin (Sigma-Aldrich). T
cells were isolated from tonsillar mononuclear cells by rosetting with
SRBC; the resulting cells contained ⬎98% CD3⫹ cells as analyzed by
FACScan (BD Biosciences). T cells were cultured under the various conditions as described in the figure legends in the presence or absence of PGI2
and beraprost. For the proliferation assay, 96-well flat-bottom microtiter
plates were coated with 10 ␮g/ml 64.1 Ab, followed by incubation with T
cells for the indicated periods of time. The degree of cellular proliferation
was measured by using cell-counting kit-8 reagent (Dojindo Laboratories)
according to the manufacturer’s instructions. The cellular proliferation was
also measured by pulsing with 0.5 ␮Ci [3H]thymidine (DuPont NEN) during the last 16 h culture period. The cultures were harvested onto glassfiber filters, and [3H]thymidine incorporation was measured by a liquid
scintillation counter (Packard). In the survival assay, T cell survival was
measured with the cell-counting kit-8 reagent or by directly counting viable
cells after trypan blue staining.
Expression cloning of 3C8 Ag
DNA sequencing and identification of the clone
Sequencing of the insert cDNA was performed using a BioDye Terminator
version 3.1 Cycle Sequencing kit (Applied Biosystems) and an automatic
sequencer ABI 3730xl DNA Analyzer (Applied Biosystems). The sequencing primers used were the standard pBluescript T3 (5⬘-AATTAACCCT
CACTAAAGGG-3⬘) and T7 (5⬘-AATACGACTCACTATAG-3⬘) universal primers and the internal custom-synthesized primer (5⬘-GATGT
CTTCCACACCTTTCG-3⬘). For identification, sequences were manually
edited to remove vector sequences and compared with the National Center
for Biotechnology Information (NCBI) nonredundant GenBank nucleotide
database. Sequence alignments were conducted using the ClustalW alignment procedure (28) with the BioEdit software.
Western blotting and immunoprecipitation for MALDI-TOF
mass spectrometry (MALDI-TOF-MS) analysis
Western blotting was conducted as previously described (24). 3C8 Ags
were immunoprecipitated and visualized on 10% SDS-PAGE gel by Coomassie blue-staining. The 3C8 Ag band was excised from the gel with a
sterile scalpel, subjected to in situ digestion with trypsin, and analyzed by
MALDI-TOF-MS. The peptide mass fingerprints obtained were allowed to
search against a mammalian subset in the NCBI nonredundant protein database. Sequence alignments were conducted using the ClustalW program.
Measurement of PGIS enzymatic activity
To measure PGIS enzymatic activity of 3C8 Ags, 3C8-immunoprecipitate
and its supernatant were incubated with PGH2 for 30 min. The produced
amount of 6-keto-PGF1␣, chemically stable metabolite of prostacyclin, was
measured using an enzyme immunoassay kit (Cayman) according to the
manufacturer’s instructions.
Preparation of PGIS small interfering RNA (siRNA) duplexes
and transfection of HK cells
In the survival assay, T cells were cultured with HK cells that were transfected with PGIS-specific siRNA. The construction of siRNA was conducted according to the manufacturer’s instructions (Ambion). For the construction of PGIS siRNA, sense-(5⬘-AAGGTTCTAGACAGCACAC
CTCCTGTCTC-3⬘) and antisense-(5⬘-AAAGGTGTGCTGTCTAGAAC
CCCTGTCTC-3⬘) oligonucleotides were synthesized. The sequences of
control siRNA with random PGIS nucleotides are as follows; sense- (5⬘AAACTGACTCTTCTCCACAGACCTGTCTC-3⬘) and antisense-(5⬘AATCTGTGGAGAAGAGTCAGTCCTGTCTC-3⬘). These siRNAs were
transfected into HK cells by using FuGENE6 transfection reagent (Boehringer Mannheim) according to the manufacturer’s directions. The degree of
gene silencing was assayed by Western blotting.
Results
3C8 Ag is prostacyclin synthase
Because the 3C8 Ag was expressed in synovial fibroblasts as well
as in FDC (24, 25), we attempted to isolate 3C8 cDNA from the
synoviocyte cDNA library by expression cloning. A 3.2-kb cDNA
insert was obtained from a positive plaque (Fig. 1). The sequence
of the 3C8 cDNA insert matched exactly that of PGIS (data not
shown).
To rule out the possibility that 3C8 Ab bound the isolated cDNA
clone by cross-reactivity, we purified 3C8 protein from HK cells
by immunoprecipitation for biochemical analysis of the 3C8 Ag.
FIGURE 1. Expression cloning with cDNA library of synovial fibroblasts reveals the molecular identity of the 3C8 Ag. With a cDNA library of human
rheumatoid arthritis positive synoviocytes, expression cloning was conducted by immunoscreening with 3C8 mAb. A positive plaque (A) was subjected
to the second (B) or third (C) rounds of immunoblotting. A replica of B was immunoblotted with isotype-matched control Ab (D).
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
A Uni-ZAP XR expression cDNA library of human rheumatoid arthritispositive synoviocyte was purchased from Stratagene. The cDNA library
was immunoscreened according to the manufacturer’s protocols. Briefly,
the library at the concentration of 1 ⫻ 104 plaques in 1 ␮l of SM buffer
(100 mM NaCl, 10 mM MgSO4, 50 mM, Tris-HCl, pH 7.5) was preincubated with 600 ␮l of XL1-Blue Escherichia coli (OD600 ⫽ 0.5) resuspended in 10 mM MgSO4 for 15 min at room temperature. Then 7 ml of
top agar was added, and the mixture was plated immediately into a 150-mm
agar plate. Plates were incubated at 37°C until small size plaques appeared.
Nitrocellulose membranes (Schleicher & Schuell) impregnated in 10 mM
isopropyl ␤-D-thiogalactoside (USB) were overlaid on the agar surface for
at least 5 h at 37°C. The membranes were removed, washed in TTNT (10
mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20), and blocked with
3% skim milk in TTNT for 1 h at room temperature. The membranes were
then incubated overnight at 4°C with 3C8 ascites diluted 1/1000 in blocking buffer. After thorough washing three times for 10 min in TTNT, APconjugated goat anti-mouse IgG1 (0.3 mg/ml diluted 1/5000 in blocking
buffer) was applied for 2 h at room temperature. The membranes were
washed five times and preincubated in AP buffer (0.1M Tris-HCl, pH 9.5,
0.1M NaCl, 5 mM MgCl2 䡠 6H2O) for 5 min. Color development was
conducted at the dark within 20 min by immersing in NBT/5-bromo-4chloro-3-indolyl phosphate substrate solution (0.3 and 0.15 mg/ml, respectively, in AP buffer). A positive plaque was cored from the agar plate and
transferred to 500 ␮l of SM buffer containing 20 ␮l of chloroform. To
release the plaque particles into the buffer, the plaque was vortexed vigorously and incubated overnight at 4°C. After the third round of screening,
a clone was obtained. And then, 250 ␮l of the positive phage stock containing over 1 ⫻ 105 phage particles was combined with 200 ␮l of XL1Blue bacteria (A600 ⫽ 1.0) and 1 ␮l of ExAssist helper phage containing
over 1 ⫻ 106 PFU. The mixture was incubated at 37°C for 15 min, followed by the addition of 3 ml of Luria-Bertani broth and further incubation
for 3 h at 37°C with shaking. After heating at 70°C for 20 min, the mixture
was centrifuged at 1000 ⫻ g for 15 min. To plate the excised phagemid,
100 ␮l of the supernatant was added to 200 ␮l of freshly grown SOLR cells
(A600 ⫽ 1.0) for incubation at 37°C for 15 min. Two hundred microliters
of the mixture was then spread on the Luria-Bertani/ampicillin (50 ␮g/ml)
plates for incubation overnight at 37°C. Colonies appearing on the plate
contain the pBluescript phagemid with the cloned cDNA insert. Phagemid
DNA was extracted from the 3C8 clone using the Plasmid Mini kit (Qiagen) according to the manufacturer’s protocols. The insert size was determined on an agarose gel after digesting phagemid DNA with EcoRI and
XhoI.
1659
FDC EXPRESSION OF PROSTACYCLIN SYNTHASE
1660
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
The Journal of Immunology
1661
SDS-PAGE followed by immunoblotting with 3C8 Ab revealed
two specific bands (Fig. 2A), a major and a minor band with molecular masses of around 55 kDa. The nominal molecular mass of
PGIS is 57 kDa. These two specific bands were identified both as
PGIS by MALDI-TOF-MS analysis (Fig. 2, B and C), confirming
the expression cloning result. The appearance of 3C8 as a doublet
may have been resulted from protein fragmentation during immunoprecipitation procedure since a single band was obtained with
Western blot analysis that was performed with 3C8 Ab and commercially available polyclonal Ab against PGIS (Fig. 3A). Immunoprecipitation with 3C8 Ab followed by immunoblotting with
PGIS polyclonal Ab further confirmed that 3C8 mAb and antiPGIS polyclonal Ab recognize the same molecule (Fig. 3B). Finally, because 3C8 Ag was successfully isolated in pure forms by
immunoprecipitation, we investigated whether the isolated Ag
would exhibit the enzymatic activity of PGIS, that is, conversion
of PGH2 into PGI2. After incubation of 3C8 Ab with HK cell
lysates followed by precipitation with protein A-Sepharose beads,
we measured the PGIS activity of precipitates and supernatants.
Compared with the control precipitate that had been incubated
with isotype-matched control Ab, the 3C8 precipitate converted 15
times more PGH2 into PGI2 (Fig. 4A). At the same time, the PGIS
activity of the control supernatant that had been incubated with
control Ab was comparable to that of 3C8 precipitate while 3C8
supernatant lost most of the PGIS activity after incubation with
3C8 Ab (Fig. 4A). The production of PGI2 by 3C8 precipitate was
dependent on the concentration of PGH2. However, increased addition of PGH2 to control precipitate did not result in a significant
increase of PGI2 production (Fig. 4B). These results suggest that
FIGURE 4. Immunoprecipitated 3C8 Ag exhibits PGIS activity. A, After incubation of HK cell lysates with control or 3C8 mAb, the ability of
supernatants and precipitates to convert PGH2 to 6-keto PGF1␣ was measured by ELISA. B, Dose-response result of control or 3C8 precipitates.
The results are expressed as the ability of each fraction relative to control
precipitate and in terms of the mean ⫾ SEM of three independent experiments. Asterisks indicate significant difference compared with controls
(ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001).
the protein that precipitated with 3C8 Ab is indeed the
enzyme PGIS.
Prostacyclin inhibits the proliferation of T cells but promotes
their survival
Widespread distribution of PGIS expression in the GC suggests its
critical role in the GC reaction. The report that indomethicin reversed FDC-induced inhibition of T cell proliferation (12)
prompted us to investigate the effect of prostacyclin on T cell proliferation. Because prostacyclin is a very unstable molecule (29),
FIGURE 2. MALDI-TOF-MS analysis of immunoprecipitated 3C8 protein confirms the cloning result. A, 3C8 molecules were purified from HK cells
by immunoprecipitation, and then the presence of 3C8 molecules in the supernatants (lanes 1, 2, 5, 6) or precipitates (lanes 3, 4, 7, 8) was examined by
Coomassie blue staining (lanes 1– 4) and Western blotting (lanes 5– 8) after SDS-PAGE under the nonreducing condition. The supernatants or precipitates
were obtained after incubation with control Ab (lanes 1, 3, 5, 7) or 3C8 mAb (lanes 2, 4, 6, 8). Arrows indicate specific bands obtained with 3C8 mAb.
B, MALDI-TOF mass spectrum of a digest of the upper band of the gel (band 1) with trypsin was obtained as described in Materials and Methods. C, The
amino acid sequences of eight selected peptides of the two 3C8-specific bands were aligned in boxes with the sequence of human PGIS protein.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 3. mAb 3C8 and anti-PGIS polyclonal Ab recognize the same
molecule. A, Immunoblotting of HK cell lysate was conducted with 3C8
mAb (lane 1) or anti-PGIS polyclonal Ab (lane 2) after SDS-PAGE under
the reducing condition. B, Immunoblotting with anti-PGIS polyclonal Ab
after immunoprecipitation of HK cell lysate with control (lane 1) or 3C8
mAb (lane 2). SDS-PAGE was conducted under the nonreducing condition
1662
we also examined the effect of beraprost, a more stable prostacyclin analog. T cells were stimulated to proliferate on the plate
coated with anti-CD3 Ab and continuously proliferated up to 80 h
poststimulation (Fig. 5A). Addition of prostacyclin resulted in a
slight decrease of T cell proliferation, whereas beraprost significantly reduced the proliferation from 48 h (Fig. 5C). The inhibition
of T cell proliferation was dose-dependent, which was more evident with beraprost than prostacyclin at the equivalent concentrations (Fig. 5, B and D). This inhibitory effect of prostacyclin and
beraprost was confirmed because addition of beraprost gave rise to
⬃50% inhibition of [3H]thymidine uptake by proliferating T cells
(Fig. 5E).
We next examined the effect of beraprost on T cell survival. T
cells underwent spontaneous apoptosis, and only small numbers of
viable cells were observed after 48 h of the culture. In contrast to
its inhibitory effect on T cell proliferation, beraprost enhanced T
cell survival during the 3-day culture period. The presence of bera-
prost resulted in significantly higher number of viable cells at 24
and 48 h (Fig. 6A). Beraprost enhanced the T cell survival in a
dose-dependent manner, and significant difference was obtained at
the concentration of 1 ␮M (Fig. 6B). Because HK cells express
PGIS and beraprost enhances T cell survival, we investigated
whether knockdown of PGIS from HK cells would modulate the
effect of HK cells on T cell survival. The PGIS gene was silenced
by transfection with siRNA against PGIS. The HK cells that were
transfected with 100 nM PGIS siRNA expressed significantly reduced PGIS proteins, whereas endogenous expression of PGIS
protein was slightly modulated by transfection with control siRNA
(Fig. 6C). Freshly isolated T cells were cultured with equal numbers of normal or transfected HK cells. T cells cultured in the
absence of HK cells exhibited 48% of viable cell recovery at the
end of 48 h culture. Both normal HK cells and HK cells that were
transfected with control siRNA increased the recovery to 57%.
However, PGIS siRNA-transfected HK cells did not support the T
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 5. Prostacyclin and beraprost suppress T
cell proliferation. Kinetics and dose response of the effect of PGI2 (A and B) or beraprost (C and D) on tonsillar T cell (1 ⫻ 105 cells/well) proliferation stimulated
by immobilized anti-CD3 Ab. The degree of cellular
proliferation was measured colorimetrically by cell
counting kit-8 at the indicated time points or at the end
of 72 h culture (B and D). E, The effect of prostacyclin
and beraprost on the [3H]thymidine uptake by proliferating T cells. Tonsillar T cells (1 ⫻ 105 cells/well) were
cultured in the presence or absence of prostacyclin or
beraprost for 72 h, and [3H]thymidine incorporation during the last 16 h was measured. Results are presented as
the mean ⫾ SEM of three independent experiments. Asterisks indicate significantly different effect compared
with controls (ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍
0.001).
FDC EXPRESSION OF PROSTACYCLIN SYNTHASE
The Journal of Immunology
1663
cell survival and yielded 49% of viable cell recovery (Fig. 6D).
This result suggests that HK cells enhance T cell survival by producing prostacyclin. The effect of transfected HK cells on the T
cell proliferation was not examined because the coating of culture
plate with anti-CD3 Ab was technically problematic due to the
presence of adherent HK cells.
Discussion
Based on the following results presented previously (25, 26) and in
this paper, we conclude that human FDCs express prostacyclin
synthase: 1) FDCs express 3C8 Ag. 2) Molecular cloning and proteome analysis reveal that 3C8 Ag is prostacyclin synthase. 3) 3C8
Ag is recognized by anti-PGIS polyclonal Ab. 4) Purified 3C8
protein displays PGIS activity.
Although PGs, particularly PGE2, have been suggested as potent
modulators of immunity (30), their cellular source and biological
functions at the culminating site of the immune response, the GC,
were not studied. Strong expression of PGIS throughout the GC
suggests that prostacyclin is produced by FDC and is the major PG
species in this microenvironment. This is, to our best knowledge,
the first report that FDC expresses PGIS. Therefore, 3C8 is the first
mAb raised against human PGIS. Furthermore, the result that prostacyclin and beraprost inhibited T cell proliferation is extending
the previous report by Butch et al. (12) that PGs are involved in
FDC-mediated inhibition of T cell proliferation and specifies the
molecular identity of the responsible PG. Our results may explain
how T cell numbers are controlled in the GC. GC T cells are
mostly CD4⫹ and specific to the inducing Ag (31, 32), suggesting
cognate interactions between T and B cells. Considering that B
cells are efficient APCs to T cells, it has been puzzling that GC T
cells do not normally proliferate and are defective for IL-2 production in response to Ag stimulation (12, 33). Our current data
and recent results by Tenner-Racz and colleagues (31) that showed
70 –90% GC T cells express CTLA-4 suggest that the number of T
cells in the GC appears to be tightly regulated by FDC and B cells.
B cells may induce negative signals in T cells during the cognate
interactions through CTLA-4. At the same time, FDCs in close
contact with T cells inhibit T cell proliferation by producing
prostacyclin.
FDC expression of PGIS is more evidence supporting the view
that FDC originates from local fibroblast as reviewed by Heinen
and Bosseloir (34). Ultrastructural (35, 36), enzymological (35,
37), and functional studies (38, 39) support this view. We demonstrated recently that fibroblasts as well as FDCs express 3C8 Ag
(25). Because 3C8 Ag has turned out to be PGIS in the current
study, fibroblast and FDC exhibit another common feature of expressing PGIS. Fibroblasts are the major producer of prostacyclin
(40). When the requirement for FDC increases in the GC during
immune response, fibroblasts in connective tissues may migrate to
lymphoid follicles in response to certain chemokines and differentiate to FDCs as a result of interaction with GC B cells. FDC
may produce prostacyclin, which controls the number of T cells by
inhibiting their proliferation but promoting their survival. Lindhout
et al. (41) have demonstrated that synovial fibroblasts could be
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 6. Beraprost enhances T
cell survival, which is impaired by silencing of PGIS. Resting T cells (1 ⫻
105 cells/well) were cultured in the
presence or absence of beraprost, and
the kinetics (A) and dose response (B
at 24 h) of the effect of beraprost on
the survival were measured by cellcounting kit 8 as described in Materials and Methods. HK cells were
transfected with control or PGIS
siRNA for 72 h, and immunoblotting
was conducted to examine the effect
of transfection on the endogenous expression of PGIS (C). The effect of
PGIS knockdown on the T cell survival was assayed by culturing resting T cells (5 ⫻ 105 cells/well) for
48 h with or without HK cells that
were nontreated or transfected with
control or PGIS siRNA. The number
of viable cells was counted after
staining with trypan blue (D). Results
are presented as the mean ⫾ SEM of
three independent experiments. An
asterisk indicates significantly different effect compared with controls (ⴱ,
p ⬍ 0.05).
1664
induced with IL-1␤ and TNF-␣ to express the FDC phenotype.
Because fibroblasts are ubiquitous cells, it remains to be elucidated
whether all kinds of fibroblasts are capable of differentiating to
FDCs or whether this property is limited to certain types of fibroblasts. The distinct staining pattern of 3C8 Ab from those of other
fibroblast-specific Abs (25) implies that only a subset of fibroblasts
may differentiate to FDCs.
Disclosures
The authors have no financial conflict of interest.
References
18. Liu, Y.-J., J. Xu, O. de Bouteiller, C. L. Parham, G. Grouard, O. Djossou,
B. de Saint-Vis, S. Lebecque, J. Banchereau, and K. W. Moore. 1997. Follicular
dendritic cells specifically express the long CR2/CD21 isoform. J. Exp. Med. 185:
165–170.
19. Li, L., X. Zhang, S. Kovacic, A. J. Long, K. Bourque, C. R. Wood, and
Y. S. Choi. 2000. Identification of a human follicular dendritic cell molecule that
stimulates germinal center B cell growth. J. Exp. Med. 191: 1077–1084.
20. Bofill, M., A. Akbar, and P. Amlot. 2000. Follicular dendritic cells share a membrane-bound protein with fibroblasts. J. Pathol. 191: 217–226.
21. Lee, I. Y., J. Lee, W. S. Park, E.-C. Nam, Y. Shin, and J. Choe. 2001. 3C8, a new
monoclonal antibody directed against a follicular dendritic cell line, HK. Immune
Network 1: 26 –31.
22. Saalbach, A., R. Kraft, K. Herrmann, U.-F. Haustein, and U. Anderegg. 1998.
The monoclonal antibody AS02 recognizes a protein on human fibroblasts being
highly homologous to Thy-1. Arch. Dermatol. 290: 360 –366.
23. Marshall, A. J., Q. Du, K. E. Draves, Y. Shikishima, K. T. HayGlass, and
E. A. Clark. 2002. FDC-SP, a novel secreted protein expressed by follicular
dendritic cells. J. Immunol. 169: 2381–2389.
24. Lee, I. Y., K. S. Ha, and J. Choe. 2003. 3C8 antigen is a novel protein expressed
by human follicular dendritic cells. Biochem. Biophys. Res. Commun. 303:
624 – 630.
25. Lee, I. Y., and J. Choe. 2003. Human follicular dendritic cells and fibroblasts
share the 3C8 antigen. Biochem. Biophys. Res. Commun. 304: 701–707.
26. Yokoyama, C., T. Yabuki, H. Inoue, Y. Tone, S. Hara, T. Hatae, M. Nagata, E.-I.
Takahashi, and T. Tanabe. 1996. Human gene encoding prostacyclin synthase
(PTGIS): genomic organization, chromosomal localization, and promoter activity. Genomics 36: 296 –304.
27. Chevalier, D., C. Cauffiez, C. Bernard, J.-M. Lo-Guidice, D. Allorge, F. Fazio,
N. Ferrari, C. Libersa, M. Lhermitte, J.-C. D’Halluin, and F. Broly. 2001. Characterization of new mutations in the coding sequence and 5⬘-untranslated region
of the human prostacyclin synthase gene (CYP8A1). Hum. Genet. 108: 148 –155.
28. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence
weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673– 4680.
29. Pola, R., E. Gaetani, A. Flex, T. R. Aprahamian, M. Bosch-Marce,
D. W. Losordo, R. C. Smith, and P. Pola. 2004. Comparative analysis of the in
vivo angiogenic properties of stable prostacyclin analogs: a possible role for
peroxisome proliferator-activated receptors. J. Mol. Cell Cardiol. 36: 363–370.
30. Harris, S. G., J. Padilla, L. Koumas, D. Ray, and R. P. Phipps. 2002. Prostaglandins as modulators of immunity. Trends Immunol. 23: 144 –150.
31. Castan, J., K. Tenner-Racz, P. Racz, B. Fleischer, and B. M. Broker. 1997. Accumulation of CTLA-4 expressing T lymphocytes in the germinal centres of
human lymphoid tissues. Immunology 90: 265–271.
32. Fuller, K. A., O. Kanagawa, and M. H. Nahm. 1993. T cells within germinal
centers are specific for the immunizing antigen. J. Immunol. 151: 4505– 4512.
33. Velardi, A., M. C. Mingari, L. Moretta, and C. E. Grossi. 1986. Functional analysis of cloned germinal center CD4⫹ cells with natural killer cell-related features:
divergence from typical T helper cells. J. Immunol. 137: 2808 –2813.
34. Heinen, E., and A. Bosseloir. 1994. Follicular dendritic cells: whose children?
Immunol. Today 15: 201–204.
35. Heusermann, U., K. H. Zurborn, L. Schroeder, and H. J. Stutte. 1980. The origin
of the dendritic reticulum cell: an experimental enzyme-histochemical and electron microscopic study on the rabbit spleen. Cell Tissue Res. 209: 279 –294.
36. Maeda, K., M. Matsuda, and Y. Imai. 1995. Follicular dendritic cells: structure as
related to function. Curr. Top. Microbiol. Immunol. 201: 119 –139.
37. Rademakers, L. H. P. M., R. A. de Weger, and P. J. M. Roholl. 1989. Identification of alkaline phosphatase positive cells in human germinal centers as follicular dendritic cells. Adv. Exp. Med. Biol. 237: 165–169.
38. Humphrey, J. H., P. Grennan, and A. Sundaram. 1984. The origin of follicular
dendritic cells in the mouse and the mechanism of trapping of immune complexes
on them. Eur. J. Immunol. 14: 859 – 864.
39. Heinen, E., M. Braun, P. G. Coulie, J. van Snick, M. Moeremans, N. Cormann,
C. Kinet-Denoel, and L. J. Simar. 1986. Transfer of immune complexes from
lymphocytes to follicular dendritic cells. Eur. J. Immunol. 16: 167–172.
40. Harvey, W., F. Guat-Chen, D. Gordon, S. Meghji, A. Evans, and M. Harris. 1984.
Evidence for fibroblasts as the major source of prostacyclin and prostaglandin
synthesis in dental cyst in man. Arch. Oral Biol. 29: 223–229.
41. Lindhout, E., M. van Eijk, M. van Pel, J. Lindeman, H. Dinant, and C. de Groot.
1999. Fibroblast-like synoviocytes from rheumatoid arthritis patients have intrinsic properties of follicular dendritic cells. J. Immunol. 162: 5949 –5956.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
1. MacLennan, I. C. M. 1994. Germinal centers. Annu. Rev. Immunol. 12: 117–139.
2. Kosco, M. H., E. Pflugfelder, and D. Gray. 1992. Follicular dendritic cell-dependent adhesion and proliferation of B cells in vitro. J. Immunol. 148: 2331–2339.
3. Koopman, G., R. M. J. Keehnen, E. Lindhout, W. Newman, Y. Shimizu,
G. A. van Seventer, C. de Groot, and S. T. Pals. 1994. Adhesion through the
LFA-1 (CD11a/CD18)-ICAM-1 (CD54) and the VLA-4 (CD49d)-VCAM-1
(CD106) pathways prevents apoptosis of germinal center B cells. J. Immunol.
152: 3760 –3767.
4. Hir, M. L., H. Bluethmann, M. H. Kosco-Vilbois, M. Muller, F. di Padova,
M. Moore, B. Ryffel, and H.-P. Eugster. 1996. Differentiation of follicular dendritic cells and full antibody responses require tumor necrosis factor receptor-1
signaling. J. Exp. Med. 183: 2367–2372.
5. Fu, Y.-X., H. Molina, M. Matsumoto, G. Huang, J. Min, and D. D. Chaplin. 1997.
Lymphotoxin-␣ (LT␣) supports development of splenic follicular structure that is
required for IgG responses. J. Exp. Med. 185: 2111–2120.
6. Fu, Y.-X., G. Huang, Y. Wang, and D. D. Chaplin. 2000. Lymphotoxin-␣-dependent spleen microenvironment supports the generation of memory B cells and
is required for their subsequent antigen-induced activation. J. Immunol. 164:
2508 –2514.
7. Choe, J., H.-S. Kim, R. J. Armitage, and Y. S. Choi. 1997. The functional role of
BCR stimulation and IL-4 in the generation of human memory B cells from
germinal center B cells. J. Immunol. 159: 3757–3766.
8. Choe, J., L. Li, X. Zhang, C. D. Gregory, and Y. S. Choi. 2000. Distinct role of
follicular dendritic cells and T cells in the proliferation, differentiation, and apoptosis of a centroblast cell line, L3055. J. Immunol. 164: 56 – 63.
9. Tew, J. G., J. Wu, M. Fakher, A. K. Szakal, and D. Qin. 2001. Follicular dendritic
cells: beyond the necessity of T-cell help. Trends Immunol. 22: 361–367.
10. Jacobson, E. B., L. H. Caporale, and G. J. Thorbecke. 1974. Effect of thymus cell
injections on germinal center formation in lymphoid tissues of nude (thymusless)
mice. Cell Immunol. 13: 416 – 430.
11. Kim, H.-S., X. Zhang, and Y. S. Choi. 1994. Activation and proliferation of
follicular dendritic cell-like cells by activated T lymphocytes. J. Immunol. 153:
2951–2961.
12. Butch, A. W., K. A. Kelly, M. S. Willbanks, and X. Yu. 1999. Human follicular
dendritic cells inhibit superantigen-induced T-cell proliferation by distinct mechanisms. Blood 94: 216 –224.
13. Naiem, M., J. Gerdes, Z. Abdulaziz, H. Stein, and D. Y. Mason. 1983. Production
of a monoclonal antibody reactive with human dendritic reticulum cells and its
use in the immunohistological analysis of lymphoid tissue. J. Clin. Pathol. 36:
167–175.
14. Parwaresch, M. R., H. J. Radzun, M.-L. Hansmann, and K.-P. Peters. 1983.
Monoclonal antibody Ki-M4 specifically recognizes human dendritic reticulum
cells (follicular dendritic cells) and their possible precursor in blood. Blood 62:
585–590.
15. Farace, F., N. Kieffer, S. Caillou, W. Vainchenker, T. Tursz, and M. C. Dokhelar.
1986. A 93-kDa glycoprotein expressed on human cultured monocytes and dendritic reticulum cells defined by an anti-K562 monoclonal antibody. Eur. J. Immunol. 16: 1521–1526.
16. Butch, A. W., B. A. Hug, and M. H. Nahm. 1994. Properties of human follicular
dendritic cells purified with HJ2, a new monoclonal antibody. Cell Immunol. 155:
27– 41.
17. Raymond, I., T. A. Saati, J. Tkaczuk, S. Chittal, and G. Delsol. 1997. CNA. 42,
a new monoclonal antibody directed against a fixative-resistant antigen of follicular dendritic reticulum cells. Am. J. Pathol. 151: 1577–1585.
FDC EXPRESSION OF PROSTACYCLIN SYNTHASE