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Division of Gastroenterology-Hepatology, Department of Internal Medicine, University of Iowa, Iowa City, Iowa 52242, USA
1Correspondence: Department of Internal Medicine, 4607 JCP, University of Iowa, 200 Hawkins Dr., Iowa City, IA 52242. E-mail: joel-weinstock{at}uiowa.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: Th1 • Th2 • CD4+ T cells • VIP receptor • VPAC2 • schistosomiasis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Vasoactive intestinal peptide (VIP), a member of the secretin family of
neuropeptides, is made by leukocytes within the granulomas of murine
schistosomiasis (1)
. Although the function of VIP in
schistosomiasis is poorly understood, various studies suggest that it
affects some aspects of immune responses such as lymphocyte
proliferation (2)
and trafficking (3
, 4)
,
cytokine production (5
, 6)
, antibody synthesis (7
, 8)
, natural killer cell activity (9)
, and more. In
murine schistosomiasis, VIP can inhibit lymphocyte proliferation,
suppress IL-2 production, and stimulate IL-5 secretion in
vitro (10
, 11)
.
VIP acts through VIP-specific receptors on cell surfaces. There are two
distinct VIP receptors called VPAC1 and VPAC2 (12)
. It is
unknown whether they have unique functions. Schistosome granulomas are
composed of ~50% eosinophils, 30% macrophages, 10% T cells, and
5% ß cells. Although the VPAC1 receptors are distributed among at
least several granuloma cell subtypes, VPAC2 expression appears limited
to granuloma T cells (13)
. The resting splenic T cells of
mice infected with S. mansoni make only VPAC1 mRNA as
evidence by a sensitive RT-PCR assay. Here we show that VPAC2 mRNA is
subject to induction and regulation in the splenic and granuloma CD4+ T
lymphocytes. Moreover, VPAC2 mRNA is expressed preferentially in Th1
opposed to Th2 cells, and IL-4 is critically important for restraining
VPAC2 production in Th2 cells. These results suggest that VPAC2 may
have a role in development and functional differentiation of CD4+ T
cell subsets.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. At 8 wk of
infection, mice were killed to obtain splenocytes and liver granuloma
cells.
Isolation and dispersal of splenocytes and granuloma cells
Spleens were dispersed by gently teasing the spleen tissue
through a 100 µm nylon cell strainer (Becton Dickinson, Rutherford,
N.J.) using a rubber policeman and RPMI 1640 medium (Life Technologies,
Grand Island, N.Y.). Splenocytes were spun down and resuspended in 5 ml
of sterile distilled water for a few seconds to lyse RBC by hypotonic
shock. Then, the spleen cells were washed twice in RPMI and resuspended
in RPMI complete medium containing 10% FCS, 10 mM HEPES buffer, 2 mM
L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma,
St. Louis, Mo.).
Livers from infected mice were harvested and homogenized for 30 s at low speed in a blender. The granulomas were collected by centrifugation at 500 g and washed twice in RPMI. The granulomas were dispersed by agitation in a shaking water bath at 37°C for 35 min in RPMI containing 5 mg/ml collagenase (type I from Clostridium histolyticum, Sigma). The granuloma cells then were dispersed further by repeated cycles of suction and expulsion through a 1 ml syringe, and the dispersed cells were passed through sterile gauze to filter out residual particulate matter. The granuloma cells then were washed twice in RPMI and resuspended in 20 ml of RPMI complete medium. Viability of all cells was always >95% as determined using eosin Y exclusion dye. The granuloma cells were composed of ~50% eosinophils, 30% macrophages, 10% CD4+ T cells, 2% CD8+ T cells, and 5% B cells.
Cell lines
D 1.1 and D10.G 4.1, the Th1 and Th2 cell lines, were obtained
from Dr. Abbas (Harvard University, Cambridge, Mass.) and the American
Tissue Culture Collection (ATCC) (Rockville, Md.), respectively. The
other T cell lines were established from the dispersed granuloma cells
of normal CBA/J and C57BL/6 mice, and from IL-4 knockout mice as
described (15)
.
To establish the polyclonal T lymphocyte cell lines, 5 x 107 dispersed granuloma cells were incubated for 10–15 min at 37°C in nylon wool columns pretreated with RPMI. The column nonadherent cells were washed extensively and cultured in T25 flask in 10 ml RPMI containing 10% FCS and 10% supernatant from Con A-stimulated normal spleen cells. These cultures were washed and fed weekly with fresh conditioned medium. After 4 wk, the granuloma cell cultures demonstrated stable growth. At this point, the cultures were split and aliquots frozen in liquid nitrogen. These primary cell lines were composed of CD4+ T cells (>95%) and propagated in culture like normal long-term cell lines.
Induction/modulation of VPAC2 mRNA
Splenocytes or granuloma cells (5x107
cells/flask) in 10 ml RPMI complete medium were incubated at 37°C in
T25 flasks usually for 3 days. Some cultures also contained 0.1 mg
anti-CD3 (2C11, Dr. Bluestone, University of Chicago, Chicago, Ill.)
and/or anti-IL-4 mAb (0.1 mg/ml) (11B11, ATCC), rIL-4 (usually at 200
U/ml)(Biological Response Modifiers Program, National Institutes of
Health, Bethesda, Md. or R&D Systems, Minneapolis, Minn.), or
rat IgG isotype control (ZYMED, San Francisco, Calif.). Also, some
cultures contained soluble egg antigen (SEA) at up to 5 µg/ml. SEA
was made as described previously (14)
. After the
incubation, RNA was extracted from the cells for RT-PCR analysis.
Splenocyte and granuloma cell viability after the 3 day culture were
~70 and 50%, respectively. Anti-IL-4 or rIL-4 treatment did not
appear to affect cell yield or viability.
T cell lines (15–20x107 cells/flask) were cultured in T75 flasks for 72 h at 37°C in 20 ml of RPMI 1640 complete medium supplemented with 100 U/ml of rhIL-2. Some cultures contained rIL4, anti-IL-4, or isotype control mAb as describe above. After incubation, the cells were washed, RNA extracted, and RT-PCR performed.
T cell lysis
Added to 5 x 107 cell suspensions
was 50–100 µl of appropriate cytolytic anti-Thy 1.2 (Accurate
Chemical and Scientific, Westberg, N.Y.), anti-CD4 (GK1.5), or anti-CD8
mAb (TIB211) (hybridoma cell lines from ATCC). After incubation on ice
for 1 h, the cells were spun down, the supernatant was decanted,
and LOW-TOX complement (Accurate Chemical and Scientific) was added at
a 1:10 final dilution. The cells then were suspended gently and
incubated at 37°C for 1 h more. This antibody and complement
lysis was repeated one more cycle to assure thorough deletion of T
cells or T cell subsets. Appropriate selective deletion was confirmed
using flow cytometry.
Cytokine assays
Cytokines were quantified in supernatants using two sandwich
ELISAs. IL-4 was captured with 11B11 and detected with biotinylated
BVD6 (DNAX, Palo Alto, Calif.). The sensitivity of the IL-4
ELISA was 100 pg/ml. IL-5 was captured with TRFK5 and detected with
biotinylated TRFK4 (DNAX). The sensitivity of the IL-5 ELISA was 30
pg/ml. IFN-
was captured with HB170 (ATCC) and detected with a
rabbit polyclonal anti-IFN- antiserum (Dr. Mary Wilson, University
of Iowa, Iowa City, Iowa) followed by application of biotinylated goat
anti-rabbit mAb (Accurate Chemical and Scientific). The sensitivity of
the IFN- ELISA was 30 pg/ml. All ELISAs used streptavidin-peroxidase
conjugate and ABTS substrate (Zymed).
RNA extraction, RT-PCR, and competitive PCR assay for VPAC2
The total cellular RNA was extracted and RT-PCR performed as
mentioned previously (1)
. The spleen or granuloma cells
(5x107) were washed twice in RPMI, and the
pelleted cells were homogenized in guanidinium/acid-phenol to extract
the RNA. The RNA was quantified spectrophotometrically and checked for
intact 18S and 28S bands by gel electrophoresis. RT reactions were
incubated 2 h at 40°C in 40 µl containing 400 units of M-MLV
reverse transcriptase, 0.5 µg of RNA, and 0.5 µg 18-mer oligo (dT)
for random priming. The first strand cDNA was diluted to 250 µl, and
0.1 µl of the product was used in each PCR reaction. PCR was
performed in a total volume of 50 µl using 3 units of Taq
DNA polymerase and primers specific for VPAC2 as mentioned elsewhere
(13)
.
A quantitative RT-PCR assay was performed to measure the amount of
VPAC2 mRNA in total cellular RNA preparations as described
(13)
. The concentration of the unknown mRNA was determined
through competition with known concentration of an engineered plasmid,
containing an appropriate VPAC2 cDNA fragment, in a PCR followed by
localization of bands of equivalence. All RNA preparations were
compared for 18S- and 28S-band expression to assure uniformity of
extraction.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
This again was confirmed using a VPAC2 RT-PCR assay sensitive <0.1 fg
VPAC2 mRNA/µg total RNA. This suggested that T cell VPAC2 mRNA
expression is inducible on T cell subsets.
To address this issue further, splenocytes from schistosome-infected
CBA mice were incubated in vitro with anti-CD3 or various
concentrations of SEA for up to 7 days, but neither stimulus induced
VPAC2 mRNA expression as determined by RT-PCR. Spleen cells from these
infected animals make large amounts of IL-4. It was discovered that
VPAC2 mRNA appeared when splenocytes were cultured for as little as 3
days with anti-CD3 and neutralizing anti-IL-4 mAb (Fig. 1
). Control mAb had no effect suggesting that IL-4 was preventing VPAC2
expression in these cell cultures. Splenocytes cultured alone or with
only anti-IL-4 mAb did not express VPAC2 mRNA (data not shown).
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Next, it was determined if IL-4 also influenced the level of VPAC2 mRNA
expression in granuloma cells. Dispersed granuloma cells constitutively
expressed VPAC2 mRNA. This natural expression decreased substantially
when cells were cultured in vitro for 3 days with or without
anti-CD3 stimulation. However, granuloma cells cultured with both
anti-CD3 and anti-IL-4 mAbs strongly expressed VPAC2 mRNA (Fig. 1)
.
Once more, control mAb had no effect.
Using a competitive RT-PCR assay developed in our laboratory
(Fig. 2
), we measured the content of VPAC2 mRNA in the cell cultures. This
assay showed that both splenocytes and granuloma cells incubated
in vitro with blocking anti-IL-4 mAb expressed much larger
amounts of VPAC2 mRNA than cells maintained without blocking Ab
(Fig. 3
).
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Using RT-PCR, VPAC2 mRNA also was sought in splenocytes from normal
uninfected CBA mice. None was detected even when cells were maintained
in vitro for up to 3 days with either neutralizing IL-4 mAb
or rIL-4 (Fig. 4
). Thus, it appeared that the systemic effects of schistosome infection
were required for the induction of splenic VPAC2 mRNA.
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IL-4 KO mice strongly express VPAC2 mRNA, and this expression is
down-regulated by rIL-4
To further explore the potential importance of IL-4 in limiting
VPAC2 mRNA expression, experiments were performed using C57BL/6 IL-4 KO
mice infected with S. mansoni. These mice form granulomas,
in response to schistosome ova, which are nearly normal in size.
However, the granulomas produce no IL-4, make little IL-5, contain few
eosinophils, and lack other features of a Th2 response
(16)
. In contrast to the CBA mouse strain, freshly
isolated splenocytes from C57BL/6 mice did constitutively express small
amounts of VPAC2 mRNA. Figure 5
shows that both dispersed splenocytes and granuloma cells from IL-4 KO
mice constitutively expressed VPAC2 mRNA much stronger than their
C57BL/6 wild-type controls. The difference in expression within the
granulomas was greater than 30-fold as determined by quantitative
RT-PCR (Fig. 3)
. Unlike granuloma cells from wild-type mice, granuloma
cells from IL-4 mutant mice continued to strongly express VPAC2 mRNA
when maintained in vitro for 3 days with anti-CD3. However,
addition of rIL-4 to the cell cultures resulted in nearly a 20-fold
diminution in VPAC2 mRNA expression (Fig. 6
and Fig. 3
).
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IL-4 regulates VPAC2 mRNA expression in CD4+ T cells
Previous experiments showed that granuloma T cells were the major
source of VPAC2 mRNA within the schistosome granuloma of CBA mice.
Because both CBA mouse splenocytes and granuloma cells maintained
in vitro for 3 days with anti-CD3 and neutralizing anti-IL-4
mAb strongly expressed VPAC2 mRNA, it was determined if T cell deletion
after the incubation affected the level of receptor mRNA detectable in
these dispersed mixed-cell cultures. The granuloma cells were composed
of ~50% eosinophils, 30% macrophages, 10% CD4+ T cells, 2% CD8+ T
cells, and 5% B cells. The splenocytes contained ~70% B cells, 15%
CD4+ T cells, and 8% CD8+ T cells. These experiments used cells from
CBA mice.
After the 3 day incubation with anti-CD3 and anti-IL-4 mAbs,
anti-Thy-1.2 and complement treatment was used to deplete the splenic
and granuloma T cell subpopulation by >99% as determined by flow
cytometry. RNA extracted from the cells depleted of Thy-1.2+
lymphocytes and reverse transcribed contained no detectable VPAC2 cDNA.
However, PCR amplification of reversed transcribed RNA from cells
treated with normal mouse serum and complement yielded VPAC2 cDNA
product (Fig. 7
).
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To further localize VPAC2 mRNA expression after the 3 day incubation,
cells also were depleted of CD4+ or CD8+ T cell subsets using
appropriated cytolytic mAbs and complement. Figure 8
and Table 1
show that RT-PCR could detect little or no VPAC2 cDNA in reverse
transcribed mRNA extracted from CD4-deleted splenocytes. Yet, cells
treated with NMS or anti-CD8 yielded the expected amounts of VPAC2 cDNA
product. Similar experiments using granuloma cells provided equivalent
results. These studies suggested that CD4+ T cells were the source of
VPAC2 mRNA in the granuloma and spleen cell cultures, and that VPAC2
mRNA expression within these cells was subject to IL-4 regulation.
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IL-4 regulates VPAC2 mRNA levels in CD4+ Th2, but not Th1 cell
lines
Three CD4+Th2 cell lines were used to further explore the role of
IL-4 in modulating VPAC2 mRNA expression. These included the D10 Th2
cell line and the C57BL/6 and CBA/J Th2 cell lines produced in our
laboratory from schistosome granulomas. All make large amounts of IL-4
and IL-5, and little or no IFN- (data not shown). Reverse
transcribed mRNA from each line contained small amounts of VPAC2 cDNA
as determined by RT-PCR. Neutralizing anti-IL-4 mAb added to cell
cultures for 3 days substantially enhanced this expression
(Fig. 9
). Although the anti-IL-4 mAb did block IL-4 activity, it did not
significantly alter IL-5 or IFN- release over the 3 day incubation.
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We also examined the effect of rIL-4 on VPAC2 mRNA expression in
CD4+ Th1 cell lines. These included the well-characterized cell line
D1.1 and a Th1 cell line derived from the granuloma of the IL-4 KO
mouse. Relative to Th2 cell lines, RT-PCR amplification detected larger
amounts of VPAC2 mRNA in Th1 cell lines. This high level of expression
did not decrease when the Th1 cells were exposed to even large amounts
of rIL-4 for up to 3 days in vitro (Fig. 9)
. All cell
lines were studied in the absence of feeder cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Because dispersed splenocytes are composed of
~25% T lymphocytes, it was assumed that VPAC2 mRNA would be readily
inducible in splenic T cell subsets. However, we did not incite VPAC2
expression in splenocytes from schistosome-infected mice even after 1
wk of TCR stimulation with SEA or anti-CD3. This suggested that
splenocytes from these schistosome-infected animals contained no cells
capable of VPAC2 mRNA expression or that some factor was preventing
expression.
Schistosomiasis is a parasitic disease in which ova induce a strong
systemic Th2 response associated with the production of large
quantities of IL-4 and IL-5. IL-4 is critically important for
expression of the Th2 phenotype in murine schistosomiasis
(16)
. Thus, we determined if IL-4 was an important factor
in controlling VPAC2 mRNA expression.
We found that cells stimulated through their TCR in the presence of neutralizing anti-IL-4 mAb strongly expressed VPAC2 mRNA. To further address the importance of IL-4 in VPAC2 regulation, we showed that both the granuloma cells and splenocytes of IL-4 KO mice naturally expressed VPAC2 mRNA stronger than cells from the wild-type controls and that rIL-4 significantly inhibited this expression. The composite of these experiments firmly support the hypothesis that IL-4 is a strong down-modulator of both VPAC2 mRNA induction and ongoing expression in T cells of schistosome-infected animals.
Splenocytes of normal uninfected CBA mice did not express VPAC2 mRNA even after TCR activation. These cells released no detectable IL-4 and failed to express VPAC2 mRNA even when cultured with IL-4 neutralizing mAb. In some experiments, splenocytes were maintained in culture for several weeks without evidence of VPAC2 expression (data not shown). Thus, it appears that the process of infection in murine schistosomiasis played an important role in priming VPAC2 mRNA expression in splenocytes of at least the CBA mouse strain.
Schistosome-infected mice have enlarged spleens that contain CD4+ T
cells, CD8+ T cells, and B cells in normal proportions. Yet, a small
fraction of the T cells are activated and secrete IL-4 and IL-5, which
is most evident after soluble schistosome egg antigen or anti-CD3 mAb
stimulation. There are non-T cell elements making IL-4 also
(17)
. The presence of these lymphokines alters the
phenotypes of the accompanying resting B cells shown by elevated CD32,
class II MHC, and IL-2R expression (18)
. Although
schistosome infection modulates splenocyte activation and
differentiation in many ways, the precise factors that prime splenic T
cells for VPAC2 mRNA expression remain unknown.
In this study, we also showed that VPAC2 mRNA localized mostly, if not
exclusively, to splenocyte and granuloma CD4+ T cells from
schistosome-infected mice. This suggested that VPAC2 was subject to
induction and regulation in the splenic and granuloma CD4+ T
lymphocytes. Because the appearance of VPAC2 mRNA in splenocytes
required 3 days of stimulation in vitro, it is possible that
this receptor is a marker of chronically activated primary effector or
memory T cells. Granuloma CD4+ T cells, which contain VPAC2 mRNA, all
display the phenotype of activated T cells (CD44 high, L-selectin low)
(19)
, further supporting this contention.
There are several cell surface markers that can help distinguish Th1
from Th2 cells. For instance, human and murine Th2 cells, as opposed to
Th1, at least transiently express CD30 (20)
and the
eotaxin receptor CCR3 (21)
. They also constitutively and
stably express a receptor-like molecule of uncertain function called
ST2L (22)
. Th1 cells, in contrast to Th2, display the
IL-12R beta2 subunit prominently (23)
. As presented here,
a series of Th1 and Th2 murine cell lines showed that Th1 cells, as
opposed to Th2 lymphocytes, were strong stable producers of VPAC2 mRNA.
Thus, high level expression of VPAC2 mRNA may be an additional marker
of the Th1 cell subset. However, Th2 cells can express VPAC2 mRNA in
large amounts if the cells are cultured with blocking anti-IL-4 mAb.
Hence, IL-4 governs the amount of VPAC2 mRNA in Th2 cells.
It was noteworthy that Th1 cell lines contained large amounts of VPAC2
mRNA, but that rIL-4 failed to down-modulate this expression. While
murine Th1 cells express IL-4R in comparable numbers to those of Th2
cells, they have a major defect in IL-4R signaling through the Stat 6
pathway (24)
. It is tempting to speculate that the failure
of rIL-4 to inhibit VPAC2 mRNA production in our Th1 cell lines was
secondary to this defective IL-4R signaling.
There are two distinct VIP receptors called VPAC1 and VPAC2
(12)
. The receptors share only ~50% homology in amino
acid sequence and display differences in intracytoplasmic tails. Both
are G protein-coupled receptors that activate adenylate cyclase. VPAC1
mRNA is expressed widely throughout the immune system of normal healthy
mice. In schistosomiasis, VPAC1 mRNA is in T cells and other immune
cell types of both the granulomas and spleens. As shown above, the
expression of VPAC2 mRNA is far more restricted than that of VPAC1.
Experiments in vitro suggest that VIP can modulate
lymphocyte proliferation as well as IL-2 and IL-5 production in
schistosomiasis (11
, 25)
. Although the level of VPAC2 mRNA
may help distinguish Th1 from Th2 cells, it remains uncertain if this
receptor preferentially regulates Th1 cell function in murine
schistosomiasis. Studies show that VIP can modulate cytokine production
by murine lymphocytes and macrophages (5
, 26)
. Also, VIP
may stimulate human T cell chemotaxis (27)
and inhibit
human monocyte TNF and IL-12 production (28)
via
VPAC2.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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