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Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 39-47
CHEMOKINES
Activation of C-C  -chemokines in human peripheral blood
  T cells by isopentenyl pyrophosphate and regulation by
cytokines
Barbara Cipriani,
Giovanna Borsellino,
Fabrizio Poccia,
Roberta Placido,
Daniela Tramonti,
Simona Bach,
Luca Battistini, and
Celia F. Brosnan
From the I.R.C.C.S., Santa Lucia, Laboratory of Neuroimmunology,
Rome, Italy; the Department of Biology, University of Rome, Tor
Vergata, Rome, Italy; the Department of Pathology, Albert Einstein
College of Medicine, Bronx, NY; and the International Center for AIDS
and Emerging Infection Research Center, L. Spallanzani Institute for
Infectious Disease, Rome, Italy.
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Abstract |
Human   T lymphocytes respond to viral, bacterial, protozoal,
and tumoral antigens, but their precise function remains unknown. In
adults the major circulating T-cell subset expresses the V9V2 T-cell receptor and responds to protease-resistant
phosphorylated derivatives found in many pathogens. In this study we
show that activation of V2+ cells with the nonpeptidic
antigen isopentenyl pyrophosphate (IPP) rapidly induces (within 4-12 hours) the C-C chemokines MIP-1 , MIP-1 , and lymphotactin but not
MCP-1. The most robust response was obtained for MIP-1. IPP
induction of MIP-1 and MIP-1 was not affected by costimulation
with interleukin-4 (IL-4), IL-10, TGF-, or interferon- (INF-).
However, IL-12 significantly enhanced IPP-induced expression and
release of MIP-1 that was down-regulated by TGF- whereas the
induction of MIP-1 by IPP+IL-12 was refractory to cotreatment
with TGF indicating that these chemokines are differentially
regulated by these cytokines. V2+ T cells also
expressed a wide range of C-C chemokine receptors including CCR1, CCR5,
and CCR8, all of which were down-regulated following activation. We
conclude that V2+ cells can be rapidly induced by
components of bacterial cell walls to express high levels of
proinflammatory chemokines, supporting an important role for these
cells in the early stages of the inflammatory responses to many common
pathogens. (Blood. 2000, 95:39-47)
© 2000 by The American Society of Hematology.
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Introduction |
T cells that express the T-cell receptor (TCR)
form a minor component of the peripheral circulating T-cell
pool.1,2 In human peripheral blood, 2 main populations of
T cells have been identified based on the TCR composition. The
predominant subset expresses the V2 chain associated with V9 and
represents 70% of the circulating T cells in adults, while a
minor subset (approximately 30%) expresses a V1-J 1 chain linked
to a chain different from V9. At birth the V1 population
predominates, whereas V2 T lymphocytes are almost completely
absent.3 It has been proposed that the shift from V1
predominance in the blood of newborns to V2-expressing cells in the
blood of adults may be due to a selective response to environmental
stimuli such as commonly encountered bacteria.4,5
T cells that express the V2/V9 rearrangement of the TCR
are known to respond, in a major histocompatibility complex (MHC) independent manner,6,7 to antigens that differ from
conventional peptidic antigens recognized by  T
lymphocytes.The nature of these compounds, which are characterized
by a low molecular weight (100-160 Da) and the presence of phosphate
groups, have recently been summarized .8
Studies that have addressed whether the diversity of the TCR CDR3
region contributes to the fine specificity of the V2/V9 T cells,
allowing them to discriminate between stimulatory metabolites, have
shown that different V2/V9 T-cell clones present the same pattern
of cross-reactivity toward these compounds. However, although these
cells are broadly cross-reactive, they are also highly specific for
ligand structure, since the number and position of phosphate groups are
important for T-cell activation.9 The expansion of these
cells during the first years of life is thought to reflect a selective
response of this T-cell subset to these nonpeptidic antigens
associated with common pathogens. Little is known about the function or
the ligands recognized by V1 T cells, although their expansion has
been observed in several pathological conditions, especially in
patients with human immunodeficiency virus (HIV).10,11
Because the V2/V9 T cells are prevalent in human peripheral blood
and lymphoid organs and react to phosphorylated protease-resistant bacterial antigens, it has been suggested that they could perform a
sentinel function by responding to either products released by bacteria
or to ligands released by autologous damaged cells (infected or
necrotic).8 Previously we have shown that V2 V9+ T cells from peripheral blood of healthy donors
can be induced to release proinflammatory cytokines, particularly TNF-
and INF- when challenged in vitro with
either phosphoantigens or with PMA + ionomycin
.12,13 These cells have also been shown to possess potent
cytotoxic activity and to kill via a perforin-dependent process.
In this study we address the role of these phosphoantigens in inducing
chemokine production in V2V9+ cells. Interest in
studying chemokines has relevance not only to the potential role of
T cells in inflammatory reactions but also to certain
infections, such as HIV type 1 (HIV-1), because chemokine receptors
have been shown to function as obligate coreceptors for
HIV-1.14,15
Chemokines are small cytokines that are classified into different
subfamilies depending upon the positioning of 4 N-terminal conserved
cysteine residues involved in disulfide bond formation.16 The presence or absence of an intervening amino acid in the first 2 cysteine residues defines the 2 main chemokine families. Chemokines with a C-X-C structure ( -chemokines) are potent chemoattractants for neutrophils, while chemokines with a C-C structure (-chemokines) preferentially attract monocytes but not neutrophils. In addition, certain C-C chemokines, such as MIP-1, MIP-1, and RANTES, induce the migration of activated T lymphocytes.17-20 More
recently 2 other families of chemokines have been identified:
lymphotactin,21 which lacks 2 of the 4 cysteine residues
and is a powerful attractant for T lymphocytes, and fractalkine, which
contains 3 amino acids between the first 2 cysteines.22
Chemokines are known to regulate leukocyte movement in development,
homeostasis, and inflammation by binding to specific G-protein-coupled cell-surface receptors on target cells.23,24 Triggering of chemokine receptors leads to the generation of biochemical signaling events, such as release of intracellular calcium and activation of
protein kinase C, which regulates specific directional migration. Some
chemokine receptors are restricted to particular cell types, while
others are widely expressed or may be constitutively expressed on some
cells and inducible in others.
Activation of TCR+ T cells has also been shown to
lead to the production of chemokines. Mitogenic stimuli generally leads to a low-level transient expression of chemokines, such as MIP-1 and
MIP-1, and ligation of CD3, particularly when there is costimulation with CD28, which leads to a more sustained stimulation and
release.25
In this study we have examined the ability of phosphoantigens to
activate chemokine expression in V2+ T cells. We chose
to focus our efforts on expression of the C-C chemokines and their
receptors because of the potential contribution of these cells and
their response to bacterial cell products, such as phosphoantigens. In
the transition from the innate to the acquired immune response, the
ability to induce the chemoattraction of specific lymphocyte subsets
could play an important role in the development of antigen-specific
responses. Our data show that phosphoantigen-activation of
V2+ T cells induces synthesis and releases the
-chemokines MIP-1 and MIP-1 (but not MCP-1) by
phosphoantigens. They express a wide range of C-C chemokine receptors.
The data also show that chemokine induction by isopentenyl
pyrophosphate (IPP) can be further enhanced by the addition of
interleukin 12 (IL-12) and, for MIP-1 but not MIP-1, can be
down-regulated by TGF. The immunomodulatory cytokines IL-4, IL-10,
and interferon (INF) are without effect.
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Materials and methods |
Cell preparation and stimulation
Peripheral blood mononuclear cells (PBMCs) were isolated from
heparinized blood obtained from healthy donors by
gradient centrifugation (Ficoll-Hypaque; Pharmacia Biotech, Uppsala,
Sweden). PBMCs were cultured at 106 cells/mL in medium
composed of RPMI 1640 supplemented with 10% (vol/vol) heat-inactivated
fetal bovine serum, 2 mmol/L L-glutamine, 20 mmol/L HEPES, and 10 units/mL penicillin and streptomycin (Life Technologies, Grand Island,
NY). Long-term cultures (20-30 days) were maintained with 50 units/mL
human recombinant IL-2 (SIGMA, St.Louis, MO). Phosphoantigen-specific
stimulation of V2+ T cells was performed using the
synthetic compound isopentenyl pyrophosphate (IPP; SIGMA) at 30 µg/mL
final concentration. V2+ T cells were expanded with IPP
for approximately 4 weeks to obtain V2+ T cell lines. FACS analysis was performed to
determine the percentage of V2+ and V1+
cells in the cultures by using the B6 monoclonal antibody (mAb) (IgG1;
PharMingen, San Diego, CA) coupled with PE and the TS8.2 mAb (IgG1;
Endogen, Woburn, MA) coupled to FITC. Lipopolysaccharide (SIGMA) was
also used in some cultures at 10 ng/mL. We used the cytokines INF,
TGF, IL-4, and IL-10 (R & D Systems, Minneapolis, MN).
V2+ T cell lines and clones
PBMCs were stained with PE-conjugated mAb to anti-V2 (see above)
by incubation for 30 minutes at 4°C. After 2 washes in
phosphate-buffered saline, positive cells were sorted at 1 or 10 cells/well into 96 well plates (Costar, Cambridge,
MA) using a cell sorter (MoFlo High Speed Cell
Sorter; Cytomation, Fort Collins, CO).
Cells were cultured in RPMI 1640 (Life Technologies) supplemented with
10% human serum (BioWhittaker, Walkersville, MD), 5% heat-inactivated
FCS (HyClone, Logan, UT), 200 mmol/L L-glutamine, 100 µg/mL MEM
nonessential amino acids, 0.5 mg/mL 2-ME (Life Technologies), 10 µg/mL penn-strept, 1 mg/mL MEM sodium pyruvate (Life Technologies), 0.5 µg/mL PHA (Murex, Dartford, England) and 100 units/mL human recombinant IL-2 (Boehringer Mannheim, Mannheim, Germany).
Cells were expanded with IL-2 and restimulated every 2 weeks with PHA
and irradiated feeder cells (3000 rad) according to standard
procedures. Cells were activated with plate-bound antibodies to CD3,
the TCR, and CD28, as described previously.13
Detection of chemokine production by sandwich ELISA
For detection of chemokines, freshly isolated PBMCs were plated in
96 well plates at 1 × 105 cells/well in the
presence of the following stimuli: 50 units/mL IL-2, 30 µg/mL IPP + IL-2, or 10 ng/mL LPS + IL-2. Cells were stimulated every 7 days. On
days 1, 7, and 14, supernatants were harvested at time 0 (immediately
after IPP or LPS was added); 30 minutes; and hours 1, 2, 6, 20, 24, or
48. For V2+ T cell lines and clones, cells were cultured
in 96 well plates, and supernatants were harvested 48 hours after stimulation.
To quantify the amount of chemokines secreted in the medium, sandwich
enzyme-linked immunosorbent assay (ELISA) was performed using matched
antibody pairs, 13 as previously described, and a standard
curve was established using human recombinant
chemokines (R&D System).
Modulation of chemokine production by cytokines
V2+ T cell lines were obtained by stimulating freshly
isolated human PBMCs, derived from 3 healthy donors, with 30 µg/mL of IPP and culturing the cells for 1 month in the presence of 50 units/mL
of human recombinant IL-2 (SIGMA). Enriched V2+ T cells
were cultured in 96 well plates at 2 × 105
cells/well and divided into 2 groups: 1 was stimulated with IPP for a
second time (IPP 2 × ), and the other was incubated in medium alone and used as a control group. IL-2 (10 units/mL) was given to both
groups during the experiment. Cells were then cultured for 48 hours in
the absence or presence of the following human recombinant cytokines
(given alone or in combination): IL-4 (2.5 ng/mL), IL-10 (5 ng/mL),
TGF- (10 ng/mL), IL-12 (2 units/mL), or INF- (100 units/mL).
Supernatants were harvested, and chemokine ELISA was performed as
described above.
Ribonuclease protection assay
Chemokine and chemokine receptor mRNA expression was determined
using multiprobe ribonuclease protection assay26,27 (RPA) (Riboquant; PharMingen, San Diego, CA). Cells were harvested and washed
twice in phosphate-buffered solution. Total RNA was extracted according
to standard procedures (RNAzol B; TEL-TEST, Frienswood, TX) according
to standard procedures. Twenty µg of RNA was hybridized overnight at
43°C to specific probe sets containing 32P-UTP labeled
transcripts using the RPA kit (RPA II kit; Ambion, Austin, TX) per
manufacturers instructions. Single-stranded RNA was digested with RNase
A/T1 mixture (Ambion), and the hybrids were analyzed on denaturing
urea/polyacrylamide denaturing gels. Bands were detected by
autoradiography and were quantified by phosphoimaging with a scanner
and software package (Storm 860, ImageQuaNT 3.01; Molecular Dynamics,
San Francisco, CA). Results were calculated as a ratio of the volume of
the band of interest to the sum of the bands for the housekeeping
genes. The housekeeping genes were large ribosomal protein subunit L32
and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). To compare data
for different chemokines, gel imaging data were also corrected for the
number of 32P-dUTP incorporation sites
for each of the chemokines examined. Differences between data sets were
analyzed by ANOVA, using P < 0.05 as the significant measurement.
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Results |
MIP-1, MIP-1, and RANTES induction by IPP stimulation
We and others have shown that IPP exclusively stimulates proliferation
of the V2V9 T-cell subset (Figure 1)
and also induces cytokine production in the same T-cell
population.7,9,12,28 To determine whether IPP selectively
stimulated chemokines within the V2+ population, freshly
isolated PBMCs from 2 healthy donors were cultured with either IL-2,
IL-2 + IPP, or IL-2 + LPS. LPS was used as a positive control
since it is known to induce the expression of both MIP-1 and MCP-1
in PBMCs.
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| Fig 1.
FACS analysis of PBMC after stimulation with IPP.
PBMC isolated by density centrifugation (Ficoll-Hypaque) from a healthy
donor were stained to detect the percentage of V2+ and
V1+ T cells before (B) and after culture for 21 days in
vitro with either IL-2 alone (C) or IL-2+IPP (D).Cells were gated for
the expression of the gene product V2 or V1. The gates are
indicated by boxes, and the numbers indicate the percentage of positive
cells within the total T-cell population. Panel A shows reactivity for
the isotype control antibodies. Data shown are representative of 3 separate donors.
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Cells were stimulated 3 times at weekly intervals to expand T
cells. At the time of each stimulation, supernatants were harvested,
and ELISA determined the release of chemokines MIP-1, MIP-1,
RANTES, and MCP-1 into the medium. FACS analysis was performed to
determine the percentage of cells present in the culture that expressed
the V2 gene product. Culture with IPP led to a significant increase
in the number of V2+ T cells. By 15 days in culture,
approximately 50% of the total lymphocyte population was positive for
V2 antigen (data not shown). No change was noted in the percent
representation of V2+ cells in cultures incubated in
IL-2 or LPS. None of the culture conditions altered the representation
of V1 cells in these cultures, which was always <10% (data not
shown). In these 2 donors the initial percentage of V2 cells was
0.8% for LP3 and 15% for XC.
Following the first stimulation (Figure 2),
only treatment with LPS led to the induction and release of
MIP-1 MIP-1, and to a lesser extent, MCP-1. RANTES was produced
at low levels in all culture conditions. After the second stimulation
on day 7, exposure to IPP led to the release of MIP-1, MIP-1, and
RANTES in both donors, which was equivalent to levels induced by LPS. In contrast, levels of MCP-1 remained low to nondetectable in both
donors following stimulation with IPP. Stimulation with LPS in these
same donors led to significant release of MCP-1 (data not shown).
Following the third stimulation on day 14, the amount of
MIP-1 MIP-1, and RANTES increased further in IPP-stimulated cells, and in 1 donor exceeded that induced by LPS (Figure 2). MCP-1
was found at high levels (12-13 ng/mL) in supernatants from LPS-stimulated cells, but it was almost undetectable following the
third IPP stimulation.
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| Fig 2.
Chemokine expression as determined by ELISA in PBMC
cultured in vitro in IL-2 or IL-2+IPP.
PBMCs were cultured in medium IL-2 (open bars), IL-2+IPP (hatched
bars), or LPS (closed bars) either once (day 1) or 3 times (day 14).
Supernatants were harvested at the times shown. The presence of
MIP-1, MIP-1, RANTES, and MCP-1 in these supernatants was
determined by ELISA. Data shown are for cultures that were challenged
either once (day 1) or that had been stimulated with these agents at
weekly intervals (day 14).
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These data indicate that IPP selectively stimulates MIP-1, MIP-1,
and RANTES but not MCP-1 production in the V2 subpopulation of T
cells. It also suggests that IPP does not lead to the release of
chemokines from other subsets of mononuclear cells. This suggests that
IPP selectively stimulates MIP-1b MIP-1, and RANTES production but does not stimulate MCP-1 production in the V2 subpopulation of T
cells. It also suggests that IPP does not lead to the release of
chemokines from other subsets of mononuclear cells.
To investigate the potential role of antigen presenting cells (APC) in
this response, we performed 2 additional experiments. In the first, we
sorted V2+ cells from the total peripheral blood
population and activated them with either IPP or PHA in the presence or
absence of APC. Activation with IPP for 24 hours led to the release of
MIP-1 and IFN, which did not require the presence of APC;
although an enhanced response was noted when APC were present (Figure
3). In contrast, no response to PHA was
noted in the absence of APC. APC alone did not release MIP-1 or
IFN in response to either IPP or PHA.
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| Fig 3.
Chemokine and cytokine expression in freshly sorted
V2+ cells in response to IPP and PHA.
V2+ cells were FACS sorted from the total PBMCs, seeded
at 40 000 cells per well, activated for 24 hours with IPP (30 µg/mL)
or PHA (0.5 µg/mL) in the presence or absence of autologous
irradiated APC (10 000 cells/well), and supernatants collected.
Autologous irradiated APC were also cultured alone or with either IPP
or PHA. The levels of MIP-1 and IFN in the supernatants were
determined by ELISA. Data shown are from 1 representative donor of 2 tested.
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In the second experiment, V2+ clones were prepared and
activated with IPP in the presence or absence of APC. In these cells the presence of APC had little to no effect on the release of MIP-1
or IFN. For example, in 4 different clones the levels of MIP-1 in
the cell supernatant at 6 hours following IPP stimulation without/with
APC were 2310/3200, 2330/2270, 1800/2700, and 3650/3550 pg/mL. At 24 hours, the levels without/with APC were 10160/8060, 2140/2590,
4410/4370, and 6490/3700 pg/mL. APC alone did not secrete MIP-1
following stimulation with IPP. From these data we conclude that the
induction of chemokines in V2+ T cells in response to
IPP does not require the presence of APC.
Analysis of chemokine production by T cell lines
To further investigate the possibility that V2+ T
cells can be induced to express certain members of the C-C chemokine
family following activation, we prepared highly enriched lines of
V2+ T cells from healthy donors by FACS sorting. We then
stimulated the cells through the TCR using plate-bound mAbs to CD3 and
CD28 or the TCR. Cells were activated overnight, and the amount of MIP-1, RANTES, and MCP-1 in the supernatants were measured by ELISA.
The results showed that compared to unstimulated controls,
V2+ T cell lines produced significant amounts of
MIP-1 and RANTES (Figure 4). In
contrast, MCP-1 was expressed at very low levels, and this did not
change following stimulation through the TCR (data not shown). These
data support the conclusion that activated V2+ T cells
are a potent source of chemokines such as MIP-1 but not MCP-1.
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| Fig 4.
Chemokine expression as determined by ELISA for
V2+ T cell lines.
V2+ T cell lines were established by FACS sorting at 10 cells per well and expanded in vitro using PHA and IL-2. Cells were
then activated by plate-bound anti-CD3 or anti- TCR and anti-CD28
for 24 hours. Control cells were maintained in IL-2 alone. Supernatants
were harvested, and chemokine expression was determined by ELISA. Data
for 8 different V2+ T cell lines are shown. All of the
lines produced MIP-1 and RANTES when stimulated with anti-CD3 + anti-CD28. However, MCP-1 was not detected in the same supernatants
(data not shown).
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Chemokine and chemokine receptor expression by RPA
It has been suggested that the expression of MIP-1 and its
receptors (CCR5 and CCR1) by activated T cells is characteristic of a
response that has been biased toward a Th1 cytokine
profile,29 whereas expression of MCP-1 and its receptor
(CCR2) may be more characteristic of a Th2 response. To investigate
whether incubation with IPP led to the selective expression of
chemokines, we assessed the expression of additional members of the C-C
and CXC chemokine families by T cell lines using multiprobe RPA.
V2+ T cells were expanded for 4 weeks in the presence of
IL-2 and IPP (IPP × 1), at which time T cells
represented approximately 90% of the lymphocyte population. The T
cells were then activated again with IPP (IPP × 2);
supernatants were collected; and mRNA was extracted at 4, 12, 24, and
48 hours post-challenge. Cells cultured with IL-2 alone were examined
as the control population. The results showed that following exposure
to IPP, there was a rapid up-regulation of the mRNA expression for
lymphotactin, MIP-1, MIP-1, and RANTES, whereas the expression of
a protected band for IP-10 was variable from 1 donor to another.
Protected bands for MCP-1, IL-8, and I-309 were not visible (Figure
5A).
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| Fig 5.
Chemokine mRNA expression induced by IPP in
IPP-expanded V2+ T cell lines as determined by RPA.
V2+ T cell lines were established by culturing PBMCs
from a healthy donor for 4 weeks in vitro, following a single
stimulation with IPP (IPP 1 × ). A population of the same PBMCs
was also maintained in IL-2 alone (IL-2). The cells that had been
stimulated 4 weeks previously were then stimulated again with IPP (IPP
2 × ), and RNA was extracted at 4, 12, 24, and 48 hours
post-stimulation. (A) The result of the RPA analysis for chemokine mRNA
expression using a multiprobe RPA system (hCK5). The undigested probe
set (U) and the digested probe set (D) are shown in the first 2 lanes
respectively, and the control RNA (ctr) provided with the kit is shown
in the extreme right-hand lane. (B) Quantitative analysis of the bands
by phosphoimaging are shown and are expressed as a ratio of the gene of
interest to the sum of the housekeeping genes L32 and GAPDH. Data shown
are representative of 3 different healthy donors.
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The gels were then subjected to phosphoimaging. Differences in
expression of the mRNA over time were determined as a ratio of the
protected band for each chemokine to the sum of the protected bands for
L32 and GAPDH. Gel imaging data were also corrected for the number of
32P-dUTP incorporation sites for each of the chemokines
examined. The results showed that peak expression of mRNA for these
chemokines was observed at 4 hour post-challenge (Figure 5B), and that
values had returned to near baseline levels by 48 hour post-challenge. The most robust response was noted for MIP-1. These data show, therefore, that exposure to IPP leads to the induction of these C-C
chemokines in V2+ cells. However, no differences were
noted in the overall pattern of chemokine mRNA expression from that
observed in the same population of cells that had been incubated for
the same length of time in high-dose (50 units/mL) IL-2, indicating
that the effect of IPP was quantitative rather than qualitative.
ELISA data for MIP-1, MIP-1, and RANTES in these culture
supernatants are shown in Figure 6.
Consistent with the mRNA data, levels of MIP-1 were higher than for
the other chemokines in all 3 donors. In addition, MIP-1 levels were
elevated earlier (at 12 hours) than MIP-1. No release of MIP-1 or
MIP-1 into the medium was found in the cells cultured with IL-2 or
stimulated with IPP 4 weeks earlier (IPP × 1). In contrast,
RANTES was expressed at low levels in all of these cultures, and this
increased only slightly following exposure to IPP.
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| Fig 6.
Chemokine expression induced by IPP in IPP-expanded
V2+ T cell lines as determined by ELISA.
The supernatants from the cultures shown in Figure 4 were analyzed for
expression of MIP-1, MIP-1, and RANTES by ELISA. Pooled data for
all 3 donors are shown (mean ± SD).
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The effect of immunomodulatory cytokines on the induction of
chemokine expression in V2+ cells
Studies in the mouse have shown that the induction of the chemokines
MIP-1 and MIP-1 by LPS can be differentially regulated by the
immunomodulatory cytokines INF, IL-10, IL-4, and
TGF.30 To determine whether these cytokines modulated
IPP-induced chemokine production, V2-enriched T cell lines were
established from 3 healthy donors by activating with IPP
(IPP × 1) and culturing for an additional 4 weeks. At the end
of this culture period, V2+ cells represented 93%,
90%, and 70%, respectively, of the total T-cell population, as
assessed by FACS analysis (data not shown). The cells were then
stimulated again with IPP (IPP × 2) either alone (IL-2 at 50 U/ml) or in the presence of cytokines IL-4, IL-10, TGF, or INF.
After 48 hours the supernatants were harvested, and MIP-1 and
MIP-1 release was determined by ELISA. Cells that had been activated
4 weeks previously and maintained in IL-2-containing medium showed no
chemokine release (Figure 7). However,
following activation again with IPP (IPP × 2), high-level
release of both MIP-1 and MIP-1 was observed for all 3 donors.
Co-culture with IL-4, IL-10, TGF, or INF did not substantially
affect chemokine release induced by IPP. In an additional experiment,
we also tested whether pretreatment with INF (100 U/ml) for 2h
modulated chemokine release induced by IPP. No effect was observed
(data not shown). These data show that the activatory effects of IPP
for MIP-1 and MIP-1 expression are not altered by regulatory
cytokines.
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| Fig 7.
Cytokine regulation of IPP-induced chemokine expression
as determined by ELISA.
V2+-enriched cultures were established by stimulating
with IPP and culturing in IL-2 for 4 weeks. Cells were then activated
again with IPP (IPP × 2) in the presence
of IL-2 alone and IL-2 +IL-4, +IL-10, +TGF,
and +INF. Culture supernatants were harvested 48 hours later, and
MIP-1 and MIP-1 were released into the medium as determined by
ELISA. Data shown represent the mean ± SD of 3 healthy donors.
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In previous studies we have shown that V2+ T cells
prominently express both IL-12r1 and IL-12R2 and can be shown to
up-regulate cell surface expression of the C-type lectin NKR-P1A in
response to IL-12.31 Therefore, we tested whether culture
with IL-12 affected the production of MIP-1 and MIP-1 in these
cultures and if this could be modulated by co-culture with TGF. In
resting cultures maintained with low-dose IL-2 (10 U/ml), culture with IL-12 led to low-level release of both MIP-1 and MIP-1 in all 3 donors tested. This was down-regulated by co-culture with TGF (Figure 8). In cultures activated again
with IPP (IPP × 2), co-culture with IL-12 led to a significant
increase (P < .01) in the levels of MIP-1, whereas the
release of MIP-1 was increased to a lesser extent. In agreement with
the data shown in Figure 7, co-culture with TGF had no effect on
IPP-induced MIP-1 or MIP-1 production, demonstrating that
differences in the levels of IL-2 in the medium (10 units/mL for the
data shown in Figure 8 and 50 units/mL for the data shown in Figure 7)
did not influence this result.
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| Fig 8.
Regulation by IL-12 of IPP-induced chemokine expression
as determined by ELISA.
V2-enriched cultures were established as described in Figure 6 and
cultured with IL-12 and TGF, either alone or in combination, in
control cultures maintained in IL-2 (IPP × 1) or in cultures
that had been stimulated again with IPP (IPP × 2). Supernatants
were harvested at 48 hours, and MIP-1 and MIP-1 expression was
determined by ELISA. Data shown represent the mean ± SD of 3 healthy donors. The values for IPP × 2,
IPP × 2 + IL-12, and IPP × 2 + IL-12 + TGF were
significantly different from each, with a P > .01.
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Interestingly, however, the presence of both IL-12 and TGF in the
culture medium differentially affected IPP-induced release of MIP-1
and MIP-1. TGF significantly (P < .01) down-modulated the IL-12-induced augmentation of IPP-induced MIP-1, but co-culture with TGF had no effect on the IL-12-induced augmentation of
IPP-induced MIP-1 (Figure 8). These data support the conclusion that
these 2 chemokines, although highly homologous, are under different regulatory controls, as has been previously documented in mouse macrophages activated with LPS.30 Co-culture with IL-4,
IL-10, and INF had no significant effect on the levels of either
MIP-1 or MIP-1 in cultures stimulated with IPP+IL-12 (data not shown).
The effect of IPP on chemokine receptor expression
To determine whether activation with IPP led to altered expression
of chemokine receptors, the RNA samples shown in Figure 4 were studied
by RPA for the expression of C-C chemokine receptors (Figure
9A). Quantitation of these data by
phosphoimaging of the gels is shown in Figure 9B. The data showed that
resting V2+ cells, previously expanded by challenge
with IPP and analyzed after 4 weeks, expressed predominantly CCR5 and
also CCR1, CCR4, CCR8, and CCR2a+b (Figure 9A, black bars).
Re-exposure to IPP (IPP × 2) led to rapid down-regulation of
the mRNA signal for all of these chemokine receptors; gradual recovery
occurred over time. In the donor shown in Figure 9A, no signal for CCR3 was detected, whereas it was possible to detect it in other donors (data not shown), which may reflect polymorphism within this
receptor.32
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| Fig 9.
Chemokine mRNA receptor analysis as determined by RPA.
(A)The samples shown in Figure 4 were also analyzed by the RPA
multiprobe system for chemokine receptor expression. (B) Quantitative
analysis of the data determined by phosphoimaging of the gels are shown
and are expressed as a ratio of the band of interest to the sum of the
housekeeping genes L32 and GAPDH. Following the second activation with
IPP expression, all of the chemokine receptors
were rapidly down-regulated but gradually recovered over time. Data
shown are representative of 3 donors.
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Discussion |
Peripheral circulating human T cells have been shown to
respond to viral, bacterial, protozoal, and tumoral antigens, but the specific nature of the antigens involved remains to be
defined.4-8,33,34 Because these cells share many
features in common with both natural killer cells as well as B and T
lymphocytes, it has been suggested that they might form a
bridge between the innate and acquired immune response by functioning
as a source of cytokines involved in activation of specific
arms of the immune response.
Two observations are consistent with such a notion. The T-cell
subset expresses the V2 V9+
CD3+CD4 CD8phenotype and
represents the major T-cell subset found in the circulation of
most normal adults. As such, the T-cell subset specifically
responds to nonprotein compounds, such as prenyl pyrophosphate
derivatives, by rapidly producing high levels of the cytokines
INF and TNF.8,12,28 These nonprotein
ligands are components of the cell wall of many common pathogens and, as such, would fit well into the hypothesis of Janeway and
colleagues35,36 that components of the innate immune
response are specialized to recognize and respond rapidly to conserved
molecular patterns found in microorganisms.
In this study we have investigated whether V2+ T cells
from human peripheral blood can also be induced to secrete C-C
chemokines in response to prenyl pyrophosphate derivatives such as IPP,
a synthetic phosphoantigen. The results show that these cells are specifically activated by IPP to release large quantities of the -chemokines MIP-1 and MIP-1. Studies at both the mRNA and
protein levels indicated that the most robust response was obtained for MIP-1. We also detected differences in the kinetics of release, with
MIP-1 being induced and released rapidly and MIP-1 induction occurring more gradually.
Although MIP-1 and MIP-1 share significant sequence homology and
may use the same receptor,37 they are known to have
distinct and sometimes opposing properties.38 In vitro,
human MIP-1 and MIP-1 recruit different populations of T cells,
with MIP-1 attracting mainly CD4+ T cells and MIP-1
inducing chemotaxis of predominantly CD8+ T
cells.18,19 Consistent with this are the observations that MIP-1 peptides display differential agonist activity for different chemokine receptors. MIP-1 activates CCR1, CCR5, and perhaps CCR4,
whereas MIP-1 more selectively interacts with CCR5. Furthermore, MIP-1 has been shown to activate macrophages, eosinophils, and basophils, whereas MIP-1 lacks this activity.39
The production of MIP-1 and MIP-1, as well as INF and TNF,
in response to IPP stimulation would add further support to the
conclusion that IPP activates a Th1-type response in these V2+ T cells.12,28 IL-12 is a potent
proinflammatory cytokine and acts on activated T cells and NK cells to
stimulate cytokine production and cytotoxicity.40 We have
previously shown that V2+ T cells express both
IL-12r1 and IL-12r2, and they respond to IL-12 by up-regulation
of the activation marker NKR-P1A.31 In V2+ T
cells stimulated with IPP, IL-12 has been shown to increase the number
of cells expressing INF.28 We now show that IL-12 also
induces the expression of MIP-1 and MIP-1 in these cells and
augments the induction of these chemokines by IPP. IL-12 is induced in
macrophages following phagocytosis of different intracellular organisms, including mycobacteria, which are a potent source of these
phosphorylated ligands.6,7 As a result, chemokine
expression by V2+ T cells could significantly contribute
to the proinflammatory microenvironment at sites of infection.
In T cells the regulatory cytokine TGF has been shown to
down-regulate IL-12 responsiveness by inhibiting the early signaling events essential to IL-12-induced gene expression.41 In
V2+ T cells, we found that TGF strikingly
reduced the effect of IL-12 on MIP-1 production while sparing the
IPP+IL-12-induced expression of MIP-1. This differential effect of
regulatory cytokines on MIP-1 and MIP-1 expression has been
previously noted in mouse macrophages activated with LPS.30
The authors of that report speculate that this may reflect a response
to different roles that these chemokines play in the immune response.
MIP-1, in addition to its potent chemotactic activity, is a potent
activator of the immune response, which could become detrimental over
time, once repair mechanisms have become activated. In contrast, the production of MIP-1, which lacks the cellular activating functions characteristic of MIP-1, could be maintained and still contribute to
the wound-healing process at later stages of the response. However, in
contrast to the report where the cytokines IL-4, IL-10, TGF, and
INF all down-regulated LPS induction of MIP-1 in
macrophages,30 we failed to find a regulatory effect of
these cytokines on IPP-induced expression in T cells. At the
present time, the IPP-activated signaling pathway, which is involved in
proinflammatory gene expression, has yet to be defined, and thus
potential regulatory pathways involved in this response must await
further study.
In addition to these chemokines, the RPA data showed that in |
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Abstract
Introduction