Division of Gastroenterology-Hepatology, Department of Internal Medicine, University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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In
schistosomiasis, eggs induce granulomas that have a vasoactive
intestinal peptide (VIP) immunoregulatory circuit. This study explored
the regulation of VIP production at sites of inflammation. Splenocytes
from uninfected C57BL/6 mice expressed VIP mRNA and protein, which
stopped following egg deposition. Eggs induce a Th2 response,
suggesting that Th2 cytokines like interleukin (IL)-4 can regulate VIP.
To address this issue, splenocytes from uninfected mice were incubated
for 4 h with or without recombinant IL-4. IL-4 inhibited
VIP mRNA expression. F4/80+ macrophages were the source of
constitutively expressed VIP, subject to IL-4 regulation. In IL-4
knockout mice, splenic VIP production did not downmodulate during
schistosome infection, suggesting that IL-4 is a critical cytokine
regulating VIP production in wild-type mouse spleen. IL-4-producing
granulomas in schistosomiasis made VIP. Experiments showed that
granuloma VIP derived from F4/80 (nonmacrophage) cell
populations, explaining this paradox. Granuloma F4/80+
cells from IL-4 knockout mice expressed VIP. Thus macrophages can make
VIP, which is subject to IL-4 regulation. However, in the Th2
granulomas, other cell types produce VIP, which compensates for loss of
macrophages as a source of this molecule.
granulomas; interleukin-4; Th1; Th2
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INTRODUCTION |
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MANY STUDIES HAVE SHOWN THAT the nervous and immune systems use similar mediators like neuropeptides and cytokines to govern biological processes. One such molecule is vasoactive intestinal peptide (VIP). It is a 28-amino acid peptide that is both a neurotransmitter and immunomodulator. Both nerves and leukocytes can make VIP (14). VIP functions through interaction with its two receptor subtypes, called VIP adenylate cyclase-activating types 1 and 2 (VPAC1, VPAC2) (25). Lymphocytes and macrophages can express one or both of these VIP receptors. VIP can modulate various aspects of T cell and macrophage function (23).
Schistosomiasis is a parasitic disease (caused by Schistosoma mansoni) in which granulomas form in the liver and intestines in response to eggs that deposit in tissue. There is a functional VIP immunoregulatory circuit readily evident in the splenocytes and liver inflammation of this experimental model of chronic inflammation. These granulomas make VIP (26). Granuloma T lymphocytes express VPAC1 and VPAC2 (21). Other granuloma cell subtypes express mostly VPAC1. Interleukin (IL)-4 inhibits VPAC2 expression on T cells (19). VIP can modulate granuloma lymphocyte proliferation as well as IL-2 and IL-5 production (16, 18). This suggests that VIP plays a role in this inflammatory process.
Splenocytes can make VIP (13). In murine schistosomiasis, it was observed that splenocytes cease expression of preproVIP (ppVIP) mRNA and contain little VIP protein at the time of egg deposition. The eggs induce a strong Th2 response.
The aim of this study was to characterize mechanisms that regulate the release of VIP from immunocytes. IL-4 is an important cytokine that drives development of the Th2 response. Our data suggest that macrophages can be a major source of VIP and that IL-4 is an important regulator of macrophage VIP expression in inflammation. IL-13 has no effect. Thus IL-4 appears to play a critical role in modulating various aspects of the VIP immunoregulatory circuit in inflammation.
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MATERIALS AND METHODS |
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Mice and infection. C57BL/6J IL-4 knockout and C57BL/6J littermate control mice (Jackson Laboratory, Bar Harbor, ME) were used throughout this study. The mice were bred and maintained at the University of Iowa. Some mice were infected subcutaneously with 35 cercariae of the Puerto Rican strain of the parasite S. mansoni (12). 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) by using a rubber policeman and RPMI 1640 medium (Life Technologies, Grand Island, NY). Splenocytes were spun down and resuspended in 5 ml sterile distilled water for a few seconds to lyse red blood cells by hypotonic shock. Then the spleen cells were washed twice in RPMI and resuspended in RPMI medium containing 10% FCS, 10 mM HEPES buffer, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (complete medium; Sigma, St. Louis, MO). The cells were counted, and viability was determined by using Trypan blue exclusion dye.
Livers from infected mice (usually 3) were harvested and homogenized for 30 s at low speed in a Warring 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 the nondispersed tissue fragments. The granuloma cells then were washed twice in RPMI and resuspended in 20 ml RPMI complete medium. Viability of these cells was also determined by using trypan blue exclusion dye.Isolation of T cells, B cells, or macrophages/monocytes. T and B cell populations were isolated from dispersed splenocytes by using magnetic beads (Dynabeads M-450; Dynal, Lake Success, NY) coated with Thy 1.2 and B220, respectively. Spleen cells from C57BL/6 mice were exposed directly to these beads, and the T and B cell populations were separated as suggested by the manufacturer.
Macrophage/monocyte populations from dispersed splenic or granuloma cells were also isolated by using the Dynabead method. Dispersed cells were exposed to a rat IgG anti-F4/80 monoclonal antibody (MCAP 497; Serotec, Raleigh, NC) at 1 µg/106 cells for 1 h on ice. The cells then were washed three times in complete medium, and magnetic beads coated with sheep anti-rat IgG were added at four times that of the targeted cells. Cells were incubated at 4°C with slow rocking for 20 min. At the end of this incubation, F4/80+ cells were separated by using a magnet, as suggested by the manufacturer. Flow analysis confirmed the adequacy of all separations, which always was >95%.Cell culture. In some experiments, dispersed splenocytes (5 × 107 cells/flask) or isolated F4/80+ splenocytes were incubated for 4 h in 10 ml RPMI complete medium at 37°C in T25 flasks. Some cultures contained recombinant murine (r-m)IL-4 (40 U/ml; PeproTech, Rocky Hill, NJ) or r-mIL-13 (500 U/ml; R&D Systems, Minneapolis, MN). After the incubation, RNA was extracted from the cells for PCR analysis.
RNA extraction, RT-PCR, and competitive PCR assay for ppVIP. The total cellular RNA was extracted as described previously (3). Briefly, the spleen or granuloma cells (~5 × 107) 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 performed for 2 h at 42°C by using 5 µg of RNA, 400 units of Moloney murine leukemia virus RT, and 0.5 µg of 18-mer oligo(dT) for random priming, all in a total volume of 40 µl. The first-strand cDNA was diluted to 250 µl, and 25 µl of the product was used in each PCR reaction. PCR was performed by using a Robocycler 40 (Stratagene, Menasha, WI) in a total volume of 50 µl by using 3 units of Taq DNA polymerase and a primer pair specific for mouse ppVIP mRNA. The primers spanned a 412-bp fragment of the full-length ppVIP transcript. The sequences of the primers were 5'-AGTCTGCAGAATCTCCCTCACT and 5'-CCTGGCATTCCTGATACTCTTC. Each tube contained 5 µl each of 2 mM dNTP, 1.4 mM Mg2+, 1.5 units Taq DNA polymerase, and 10 pM of both primers. The PCR sequence was 93°C for 1.0 min to melt, 65°C for 1.25 min to anneal, and 72°C for 1.12 min to extend. The PCR was repeated for 40 cycles.Quantitative PCR for ppVIP.
A quantitative PCR assay measured the amount of ppVIP mRNA in total
cellular RNA preparations. To develop the assay, the ppVIP cDNA
fragment generated via the ppVIP PCR described above was ligated into
pGEM-T plasmid (Promega, Madison, WI). The recombinant plasmid then was
cut with BstEII restriction endonuclease (New England
Biolabs, Beverly, MA). A fragment of -DNA 117-bp long, the product
of cutting
-DNA with the same restriction endonuclease and
electroeluting the fragment, was inserted into the cut recombinant plasmid by a ligation reaction. The engineered plasmid then was transformed into JM109-competent cells (Promega).
High-performance liquid chromatography and RIA for quantification of VIP. Dispersed splenocyte cells in RPMI from infected or uninfected C57BL/6 mice (~5 × 108 cells) were pelleted at 1,500 rpm for 10 min and resuspended in 1 ml 1 N acetic acid. The cells were placed in a boiling water bath for 2 min and then chilled on ice for another 2 min. Next, they were sonicated (Heat Systems-Ultrasonics) on ice for three 5-s bursts. The crude cellular extracts were centrifuged to remove particulate matter.
The homogenates were passed through a C18 SEP-PAK cartridge (Millipore-Waters Associates) to prepare the sample for high-performance liquid chromatography (HPLC). The cartridges were moistened with 100% acetonitrile followed by 0.05% trifluoroacetic acid (TFA; Sigma) in water. The homogenates were loaded onto the cartridge, washed with 5 ml of 0.05% TFA, and eluted with 5 ml of 50% acetonitrile in 0.05% TFA. The samples then were vacuum dried and stored atStatistical analysis. Student's t-test was used to compare the means of two populations for significant difference.
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RESULTS |
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Schistosomiasis affects splenic VIP mRNA and protein expression. Splenocytes isolated from normal, healthy C57BL/6 mice express VIP. In murine schistosomiasis, ova deposit in the liver and intestines beginning at about the sixth week of infection. These ova induce a Th2-type granulomatous response. It was determined whether the immunologic response to schistosome ova affected VIP production in the spleen. RNA was extracted from freshly dispersed splenocytes from uninfected mice or from animals colonized with S. mansoni for 4, 6, or 8 wk. A sensitive PCR assay readily detected ppVIP transcripts in splenocytes from uninfected mice, but none were evident in splenocytes from infected animals at or beyond the sixth week of infection.
Using a competitive RT-PCR assay developed in our laboratory, we measured the content of ppVIP mRNA in various spleen cell RNA preparations. Dispersed splenocytes from uninfected mice contained >3 × 105 ppVIP transcripts/µg total splenic RNA. Beginning at the sixth week of infection, this fell to <100 transcripts/µg total RNA, which was the lower limit of sensitivity for this PCR assay (Fig. 1).
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IL-4 inhibits VIP mRNA expression in splenocytes. Schistosome ova induce IL-4 production, which is the cytokine most critical for driving the Th2 response in murine schistosomiasis. It also regulates the expression of VPAC2, one of the VIP receptor subtypes on murine T cells (19). Therefore, we determined whether IL-4 regulated ppVIP mRNA expression in splenocytes.
Splenocytes from uninfected mice cultured in vitro continued to express VIP mRNA. However, cells cultured for 4 h with rIL-4 displayed a >80% reduction in ppVIP transcripts (Fig. 3).
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Splenic ppVIP mRNA in IL-4 knockout mice.
To further study the importance of IL-4 in limiting ppVIP mRNA
expression, additional experiments used C57BL/6 IL-4 knockout (KO) mice
infected with S. mansoni. These mice form granulomas in
response to schistosome ova that make no IL-4 and lack the features of
a Th2 response (20). Figure
4A shows that freshly dispersed splenocytes from uninfected IL-4 KO mice and from
8-wk-infected KO animals expressed ppVIP mRNA comparably, unlike that
of wild-type controls. These studies suggested that IL-4 was important
in vivo for controlling splenic VIP production.
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Macrophages produce ppVIP mRNA in the spleen.
Experiments explored the origin of ppVIP transcripts within the
dispersed splenocytes of uninfected wild-type mice. The splenocyte preparations comprised ~70% B cells (B220+,
Thy1.2), 23% T cells
(Thy1.2+/CD4+ and
Thy1.2+/CD8+), and 5% monocytes/macrophages
(F4/80+, Thy1.2
, B220
).
Paramagnetic beads coated with anti-Thy1.2, -B220, or -F4/80 monoclonal
antibody were used to positively select splenic T cell, B cell, or
monocyte/macrophage subsets, respectively. Flow analysis confirmed the
adequacy of the enrichment process, which was always >95%. Figure
5 shows that ppVIP mRNA localized
exclusively to the splenic monocyte/macrophage subset. T lymphocytes
and B cells were devoid of ppVIP transcripts as determined by RT-PCR
amplification.
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IL-4 regulates VIP production in schistosome granulomas. Schistosome granulomas in wild-type mice are composed of ~50% eosinophils, 30% macrophages, 12% T cells, and 5% B cells. They generate large amounts of IL-4 but also produce VIP. Thus we determined if F4/80+ granuloma macrophages contained ppVIP transcripts.
As expected, RNA extracted from the unfractionated, dispersed granuloma cells contained ppVIP mRNA. However, F4/80+ granuloma macrophages expressed nearly none (Fig. 7). The ppVIP mRNA content of F4/80
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DISCUSSION |
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Leukocytes (e.g., eosinophils) within the granulomas of murine schistosomiasis make VIP (26). The objective of this study was to determine if cytokines regulated the expression of VIP within the inflammation.
The early experiments showed that dispersed splenocytes from healthy, uninfected mice contained ppVIP mRNA and VIP protein. Dispersed splenocytes are composed of ~25% T cells, 65% B cells, and 5-10% monocytes. Positive-selection experiments using splenocytes from C57BL/6 mice showed that splenic ppVIP mRNA localized exclusively to the F4/80+ monocyte subpopulation. Purified T cells and B cells tested negative for ppVIP mRNA using PCR.
In contrast, dispersed splenocytes isolated from schistosome-infected mice contained nearly no ppVIP mRNA and ~10% the amount of VIP protein compared with uninfected controls. This suggested that these spleens had fewer leukocytes capable of VIP expression.
It takes ~6 wk for worms to mature and to begin egg production in mice infected with schistosomes. It was noted that the loss of VIP in the spleens of schistosome-infected mice began at the sixth week of infection, which corresponded with the onset of egg deposition in the liver and intestines.
These ova induce a Th2-type granulomatous response. This is associated with the production of IL-4, IL-5, and IL-13 in the granulomas and spleens of infected animals. These cytokines are critically important for induction of the Th2 phenotype characteristic of schistosome granulomas (1, 2, 20). Thus we addressed the issue of whether IL-4 or IL-13 could regulate ppVIP mRNA expression.
It was found that IL-4 could readily inhibit the ppVIP mRNA that was expressed constitutively by dispersed splenocytes from uninfected mice. Also studied were IL-4 KO mice. Splenocytes from these animals infected for 8 wk expressed ppVIP mRNA at levels similar to both uninfected, wild-type control animals and uninfected IL-4 KO mice. Also, IL-4 added to IL-4 KO spleen cell cultures quickly suppressed VIP mRNA expression. These studies taken in total showed that IL-4 was a strong inhibitor of VIP synthesis by splenic monocytes.
Although IL-4 had a marked inhibitory effect on VIP expression, IL-13
had no effect. IL-13 is a Th2 cytokine that has some functional
similarities to IL-4. Also, IL-4 and IL-13 signal via signal transducer
and activator of transcription 6 (17) through dimeric receptors containing the same -chains but unique
-chains. Although not yet explored, it is possible that VIP-producing
macrophages fail to express the complete IL-13 receptor or perhaps that
VIP inhibition requires intracellular signaling through more than the
Stat6 pathway.
Schistosome granulomas, which are a rich source of IL-4, do make VIP (26). The granulomas are composed of ~50% eosinophils, 30% macrophages, and 15% lymphocytes. This suggested that either granuloma macrophages overcome IL-4 to produce ppVIP mRNA or that other granuloma cell types were the source of ppVIP transcripts. Our experiments showed that granuloma F4/80+ macrophages were a weak source of ppVIP transcripts in schistosome granulomas of wild-type mice. Since the cellular composition of the granuloma in wild-type mice is ~30% macrophages, it is reasonable to assume that 30% of RNA obtained from unfractionated granuloma cells derived from F4/80+ cells. This implies that <5% of the total granuloma ppVIP mRNA transcripts were of granuloma macrophage origin.
If IL-4 was important for VIP regulation in vivo, conceivably
F4/80+ macrophages would be more important for VIP
expression in the granulomas of IL-4 KO animals. This was indeed the
case. IL-4 KO granulomas are ~50% macrophages. Fractionation of
these granuloma cells into F4/80+ and F4/80
subsets showed that F4/80+ macrophages were the major
source of ppVIP mRNA, accounting for >80% of the total VIP
transcripts. This represented a true increase in macrophage ppVIP mRNA
synthesis in IL-4 KO granulomas, since the relative number of ppVIP
transcripts expressed in granulomas was similar for IL-4 KO and
wild-type animals.
Previous studies have suggested that VIP can be produced in many lymphoid organs by various leukocyte subsets. VIP is in rat and mouse thymus (11), spleen, and lymph nodes (13). VIP has been detected in mast cells (4), T and B lymphocytes (14), and eosinophils (26). Human peripheral blood mononuclear cells have VIP (15). This suggests that VIP can come from many cellular origins, akin to other immunomodulatory molecules like IL-4 and IL-10, which derive from various inflammatory sources.
In the schistosome granuloma, IL-4 inhibits VIP production by macrophages, but eosinophils (26), and perhaps other cell types, assume this function. CD4+ and CD8+ T cells can make VIP after engagement of their T cell receptor (7). Th2 cells preferentially produce it. It is tempting to speculate that Th2 cells in schistosome granulomas are also a source for this molecule. Immune responses use various overlapping and independent immunoregulatory processes to control cytokine production. Thus there likely are several potential sources of VIP under diverse mechanisms of regulation that make it difficult to completely downregulate VIP production in the granuloma.
VIP acts through two receptor subtypes called VPAC1 and VPAC2. In schistosomiasis, VPAC1 is widely distributed among granuloma leukocytes, whereas VPAC2 is expressed predominantly on granuloma T cells (22). IL-4 inhibits T cell VPAC2 expression (19). Thus VPAC2 is most prominently expressed on Th1 cells. It remains unknown if IL-4 also affects VPAC1 production.
VIP inhibits Th1 while stimulating Th2 cell differentiation in vivo (9). This is perhaps through enhancing B7.2 expression (10) and via inhibition of IL-12 synthesis (5). Studies using the murine T cell hybridoma 2B4.11 showed that VIP acting through VPAC2 can inhibit Fas ligand expression (6, 8). This may favor the survival of activated T cells, allowing them to differentiate into appropriate memory subsets. The counterregulatory effects of IL-4 on VIP and its receptor VPAC2 and the effect of VIP on T cells suggest that VIP and IL-4 are part of an interactive circuit active in the immune response to eggs in schistosomiasis and in other inflammatory states.
In summary, this study is the first to show that splenic and granuloma macrophages in C57BL/6 mice are a source of VIP, whose production is downregulated by IL-4. Moreover, the source of leukocyte VIP can shift from macrophages to other cellular sources at the site of inflammation under the influence of IL-4, an immunoregulatory cytokine.
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ACKNOWLEDGEMENTS |
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Grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-38327, DK-02428, and DK-25295), the Crohn's and Colitis Foundation of America, and the Veterans Administration supported this research.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. Metwali or J. V. Weinstock, Dept. of Internal Medicine, 4607 JCP, Univ. of Iowa, 200 Hawkins Drive, Iowa City, Iowa 52242 (E-mail: joel-weinstock{at}uiowa.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published March 6, 2002;10.1152/ajpgi.00491.2001
Received 16 November 2001; accepted in final form 11 February 2002.
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