IL-4 inhibits vasoactive intestinal peptide production by macrophages

Ahmed Metwali, Arthur M. Blum, David E. Elliott, and Joel V. Weinstock

Division of Gastroenterology-Hepatology, Department of Internal Medicine, University of Iowa, Iowa City, Iowa 52242


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 lambda -DNA 117-bp long, the product of cutting lambda -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).

The concentration of the unknown mRNA was determined through competition with a known concentration of the engineered plasmid in a ppVIP-PCR reaction, followed by localization of the band of equivalence. Data were expressed as total number of ppVIP transcripts in 1 µg total RNA. The assay was sensitive to <100 transcripts.

To further ensure appropriate RNA loading, for most of the RNA preparations a polycompetitor plasmid containing a construct for the housekeeping gene HPTC was used in a quantitative PCR assay to measure HPTC transcripts (24).

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 at -20°C for HPLC.

VIP was identified by elution time by using reverse phase HPLC. HPLC was performed on the cellular extracts reconstituted in 10% acetonitrile by using a µBondpak C18 column (3.9 mm × 30 cm) and a Millipore-Waters HPLC system. Synthetic (Sigma) or splenic VIP was eluted over 30 min by using a linear gradient of 10-45% acetonitrile in 0.05% TFA. Eluent was collected in minute intervals and dried in a Speed-Vac concentrator from Savant Instruments (Hicksville, NY) in preparation for VIP RIA. VIP eluted as a single peak at 23 min.

Dried samples were reconstituted in 5% PBS buffer for RIA. The assay used rabbit VIP antiserum (Peninsula Laboratories, Belmont, CA) and 125I-VIP (NEN, Wilmington, DE).

Statistical analysis. Student's t-test was used to compare the means of two populations for significant difference.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Natural infection with Schistosoma mansoni suppresses vasoactive intestinal peptide (VIP) mRNA expression in splenocytes. RNA was extracted from freshly dispersed splenocytes of healthy C57BL/6 mice (time 0) or from splenocytes of animals colonized with S. mansoni for 4, 6, or 8 wk. Six weeks corresponds with the onset of egg deposition. PreproVIP (ppVIP) mRNA expression was quantified with a competitive PCR assay. Values are means ± SD of duplicate determinations from 2 separate experiments.

The amount of VIP protein in dispersed splenocytes also was measured to further characterize the effect of schistosomiasis on splenic VIP content. VIP protein was extracted from freshly dispersed splenocytes. With HPLC, VIP eluted at the appropriate time as a single peak, as detected by a VIP RIA. There was more than eightfold more VIP in splenocytes from uninfected mice than in splenocytes from 8-wk-infected animals (Fig. 2).


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Fig. 2.   Schistosomiasis inhibits splenocyte VIP protein production. VIP protein was extracted from freshly dispersed splenocytes of healthy C57BL/6 mice (ui SPL) or from splenocytes of animals colonized with S. mansoni for 8 wk (i SPL). VIP protein was isolated by using high-performance liquid chromatography (HPLC) and quantified with an RIA. Values are means ± SD of duplicate determinations from 2 separate experiments.

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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Fig. 3.   Interleukin (IL)-4 inhibits VIP mRNA expression in splenocytes. Unfractionated splenocytes from uninfected mice were cultured 4 h in vitro with or without recombinant (r)IL-4 or rIL-13 (40 U/ml). Following the incubation, cellular RNA was extracted and analyzed for ppVIP mRNA expression by using quantitative PCR. Values are means ± SE of 4 determinations from 2 separate experiments.

IL-13 is a cytokine that shares many functions with IL-4. We also determined if IL-13 could regulate ppVIP mRNA expression. Unlike rIL-4, rIL-13 did not affect the number of ppVIP transcripts in spleen cell cultures (Fig. 3).

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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Fig. 4.   Natural infection with S. mansoni does not inhibit ppVIP mRNA expression in splenocytes of IL-4 knockout (KO) mice. A: RNA was extracted from freshly dispersed splenocytes of healthy IL-4 KO mice (uiSPL) or of IL-4 KO animals infected with S. mansoni for 8 wk (iSPL). B: unfractionated splenocytes of iSPL animals were cultured 4 h in vitro with or without rIL-4 (40 U/ml). Following the incubation, cellular RNA was extracted and analyzed for ppVIP mRNA expression by using PCR. Results are from 2 individual experiments (labeled 1 and 2). RNA samples contained similar amounts of HPTC (bottom). MW, molecular weight.

To address this further, splenocytes from schistosome-infected, IL-4 KO mice were incubated in vitro for 4 h. Spleen cells from these infected animals continued to express ppVIP transcripts. However, addition of rIL-4 to the cultures nearly abolished ppVIP mRNA expression (Fig. 4B).

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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Fig. 5.   Cells expressing F4/80 are the source of ppVIP mRNA in the spleen. T cells, B cells, and macrophages/monocytes were positively selected from dispersed splenocytes from uninfected C57BL/6 mice. The isolation process used paramagnetic beads coupled to anti-Thy1.2 (T cells), anti-B220 (B cells), or anti-F4/80 (macrophages/monocytes). Following cell isolation, RNA was extracted from unfractionated splenocytes and splenic cell subsets. These were analyzed for ppVIP mRNA expression by using PCR. Results are from 2 individual experiments (1 and 2). All samples contained comparable amounts of HPTC transcripts (bottom).

Also examined was the effect of rIL-4 on ppVIP mRNA expression in splenic monocytes/macrophages. PCR amplification detected ppVIP transcripts in F4/80+ splenocytes cultured in vitro for 2 h. However, addition of rIL-4 to these cell cultures completely abrogated ppVIP mRNA expression (Fig. 6). This suggested that VIP production by splenic monocytes/macrophages was subject to IL-4 regulation.


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Fig. 6.   IL-4 constrains ppVIP mRNA expression in F4/80+ splenocytes. Splenic macrophages/monocytes were positively selected by using rat anti-F4/80 monoclonal antibody and anti-rat IgG-coated magnetic beads. Unfractionated splenocytes or F4/80 cells were cultured 2 h in vitro with or without rIL-4 (40 U/ml). Following incubation, cellular RNA was extracted and analyzed for ppVIP mRNA expression by using PCR. Results are from 2 separate experiments (1 and 2). All samples contained comparable amounts of the housekeeping gene HPTC (bottom).

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- cells relative to F4/80- total RNA exceeded that of unfractionated granuloma cells and was >20-fold that of F4/80+ cells. Thus granuloma VIP mRNA derived mostly from cell sources other than macrophages in the IL-4-producing schistosome granulomas.


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Fig. 7.   Macrophages (F4/80+) from schistosome granulomas of wild-type as opposed to those of IL-4 KO animals express little ppVIP mRNA. Granulomas from schistosome-infected wild-type (A) or IL-4 KO (B) animals were isolated from the liver and dispersed with collagenase. Granuloma macrophages were positively selected by using anti-F4/80-coupled paramagnetic beads. RNA was then extracted from unfractionated granuloma cells (whole cells) and from F4/80+ or F4/80- granuloma cells. The RNA was analyzed for ppVIP mRNA expression by using PCR. Values are means ± SD representing duplicate determinations from 2 separate experiments.

Also studied was VIP mRNA expression in the schistosome granulomas of IL-4 KO animals. Dispersed granuloma cells from IL-4 mutant mice expressed VIP mRNA at higher levels than that of granuloma cells from wild-type animals (Fig. 7). Fractionation of these cells into F4/80+ and F4/80- subsets showed that F4/80+ macrophages were a major source of ppVIP mRNA. The relative expression of ppVIP mRNA in F4/80+, as opposed to F4/80-, cells was ~4 to 1. These studies suggested that, in the absence of IL-4, granuloma macrophages produced ppVIP transcripts. However, VIP production switched from macrophages to other granuloma cell types in the IL-4-producing, schistosome granulomas.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -chains but unique beta -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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 283(1):G115-G121
0193-1857/02 $5.00 Copyright © 2002 the American Physiological Society




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