TGF-beta and IL-10 regulation of IFN-gamma produced in Th2-type schistosome granulomas requires IL-12

Khurram Qadir, Ahmed Metwali, Arthur M. Blum, Jie Li, David E. Elliott, and Joel V. Weinstock

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta ) regulate CD4+ T cell interferon-gamma (IFN-gamma ) secretion in schistosome granulomas. The role of IL-12 was determined using C57BL/6 and CBA mice. C57BL/6 IL-4 -/- granuloma cells were stimulated to produce IFN-gamma when cultured with IL-10 or TGF-beta neutralizing monoclonal antibody. In comparison, C57BL/6 wild-type (WT) control granuloma cells produced less IFN-gamma . IL-12, IL-18, and soluble egg antigen stimulated IFN-gamma release from C57BL/6 IL-4 -/- and WT mice. IFN-gamma production in C57 IL-4 -/- and WT granulomas was IL-12 dependent, because IL-12 blockade partly abrogated IFN-gamma secretion after stimulation. All granuloma cells released IL-12 (p70 and p40), and IL-12 production remained constant after anti-TGF-beta , anti-IL-10, recombinant IL-18, or antigen stimulation. C57 WT and IL-4 -/- mouse granuloma cells expressed IL-12 receptor (IL-12R) beta 1-subunit mRNA but little beta 2 mRNA. TGF-beta or IL-10 blockade did not influence beta 1 or beta 2 mRNA expression. CBA mouse dispersed granuloma cells released no measurable IFN-gamma , produced IL-12 p70 and little p40, and expressed IL-12R beta 2 and little beta 1 mRNA. In T helper 2 (Th2) granulomas of C57BL/6 WT and IL-4 -/- mice, cells produce IL-12 (for IFN-gamma production) and IL-10 and TGF-beta modulate IFN-gamma secretion via mechanisms independent of IL-12 and IL-12R mRNA regulation. We found substantial differences in control of granuloma IFN-gamma production and IL-12 circuitry in C57BL/6 and CBA mice.

T helper 1 cell; interleukin-12 receptor


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN MURINE SCHISTOSOMIASIS mansoni, eggs from the parasite settle in the liver and intestines during the course of natural infection. The eggs evoke a strong granulomatous response that surrounds the ova. The granulomas in the liver of mice with schistosomiasis make large amounts of interleukin-4 (IL-4) and IL-5 and display the phenotypic characteristics of a T helper 2 (Th2)-type response. However, granulomas from mice with a C57BL/6 genetic background contain T cells that produce interferon-gamma (IFN-gamma ) (30). The various immunoregulatory circuits that control IFN-gamma production within these strong Th2-type granulomas are not well defined.

Various cytokines have a role in Th1/Th2 cell differentiation in murine schistosomiasis. IL-4 is important for phenotypic expression of most aspects of the Th2 response (8, 26). In contrast to wild-type controls, the liver granulomas of IL-4 knockout mice produce no IgE and little IgG1, IL-5, and IL-13. Also, there is a substantial reduction in eosinophil and mast cell content. Regional lymph node cells, splenocytes, and granuloma T cells from IL-4 -/- mice produce more IFN-gamma than cells from their wild-type counterparts. However, this shift toward Th1 is modest (8, 15, 26, 26). Although deletion of IL-13 by itself has little effect, the cytokine profile in schistosomiasis does shift toward Th1 in the IL-4/IL-13 double-mutant mouse (15). This suggests that IL-4 and IL-13 have a synergistic inhibitory effect on development of the Th1 phenotype. Some other factors that influence Th1/Th2 development include IL-10, transforming growth factor-beta (TGF-beta ), IL-12, and B7 expression (18, 19).

IFN-gamma production by granuloma cells increases in response to recombinant IL-12 (rIL-12), suggesting that some granuloma T cells display the IL-12 receptor (IL-12R). Also, the granulomas make large amounts of IL-10 and TGF-beta that help limit this IFN-gamma expression. IL-12, IL-10, and TGF-beta all appear to act directly or indirectly on the granuloma Thy 1.2+ T cells that make IFN-gamma (30).

IL-12 is a powerful inducer of IFN-gamma production and Th1 cell development (16). Pulmonary and natural models of granulomatous inflammation show that rIL-12 administration before the onset of egg deposition can enhance IFN-gamma production and Th1 cell development in response to schistosome eggs (19). However, largely unexplored is the role of intralesional IL-12 and IL-12R in the regulation of ongoing IFN-gamma production within granulomas of murine schistosomiasis, which was the subject of this study.

In C57BL/6 and IL-4-deficient mice, we showed that Th2 granulomas of murine schistosomiasis produce IL-12 that is required for the ongoing IFN-gamma response. Although granuloma IFN-gamma is subject to regulation by IL-10, TGF-beta , and antigen stimulation, none of these factors affected IL-12 production or IL-12R mRNA expression. Similar studies using CBA mice revealed substantial stain differences regarding control of granuloma IFN-gamma production and IL-12 circuitry.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Mice and schistosome infection. This study used normal C57BL/6 and CBA/J mice obtained from the National Cancer Institute (Bethesda, MD). Also used were C57BL/6 IL-4 -/- and C57BL/6 IL-12 p40 knockout mice. Breeding colonies for the mutant animals were maintained at the University of Iowa. At 7-8 wk of age, mice were infected subcutaneously with 50 cercariae of the Puerto Rican strain of Schistosoma mansoni.

Granuloma cell and splenocyte dispersal and cell culture. Livers of mice killed during the eighth week of infection were homogenized for 30 s at low speed in a Waring blender. Granulomas were collected by 1 g sedimentation and washed three times in RPMI 1640 medium (RPMI). To prepare a single cell suspension from these granulomas, the intact granulomas were incubated in a shaking water bath at 37°C for 30 min in RPMI containing 0.5% collagenase (type 1 from Clostridium histolyticum, Sigma Chemical, St. Louis, MO). Repeated suction and expulsion through a 1-ml syringe disrupted the softened granulomas further. The dispersed granuloma cell suspensions were passed through sterile gauze to exclude nondispersed fragments. The cells were collected by centrifugation, washed three times in RPMI, and counted. Cell viability was determined by eosin Y exclusion.

Single cell suspensions of splenocytes were prepared from individual spleens from 8-wk infected mice by gentle teasing in RPMI. The cells were briefly resuspended in distilled water to lyse red blood cells. The splenocytes were then washed three times in a large volume of RPMI.

For cytokine measurements in supernatants, cells were cultured in microtiter plates at 106 cells/well in medium (200 µl) for 18 h at 37°C in 5% CO2 atmosphere with or without anti-IL-10 (1 µg/ml; SXC1, DNAX, Palo Alto, CA), anti-TGF-beta (1 µg/ml; MAB 240, R&D Systems, Minneapolis, MN), soluble egg antigen (SEA; 5 µg/ml), rIL-12 (5 ng/ml; PeproTech, Rocky Hill, NJ), or rIL18 (5 ng/ml; PeproTech). Some cultures also contained anti-IL-12 (C17-15, a gift from Dr. G. Trinchieri, Wistar Institute, Philadelphia, PA). The culture medium was RPMI containing 10% FCS, 10 mM HEPES buffer, 2 mM L-glutamine, 100 U/ml penicillin, 5 mg/ml gentamycin, and 100 mg/ml streptomycin (all from Sigma Chemical). Soluble egg antigen (SEA) was made from ova isolated from the liver of hamsters infected for ~7 wk with 1,000 cercariae as previously described (14).

For IL-12R beta 1 and beta 2 mRNA analysis, granuloma cells were cultured for 5 h at 4 × 107 cells/well with or without anti-IL-10 or anti-TGF-beta monoclonal antibody (MAb) each at 1 µg/ml.

T cell line. The D1.1 T cell line was maintained in T25 flasks in complete RPMI medium. For boosting, 6 ml RPMI complete medium containing rabbit IgG (100 µg/ml) was added to T25 flasks containing adherent D1.1 cells. Also added were irradiated (3,000 rad) splenocytes (106/ml). The cultures were split every 2-3 days and maintained in complete medium. Two weeks after boosting, cellular RNA was extracted for mRNA analysis.

RNA extraction and RNase protection assay. Each experiment used RNA from splenocytes or granuloma cells pooled from three to four mice. Total cellular RNA was extracted from cell suspensions by homogenization in guanidinium-acid phenol as previously described (9). Total RNA preparations contained equivalent 18S and 28S RNA bands. RNA extracts were quantified spectrophotometrically.

RPA was used to quantify IL-12R beta 1 and beta 2 mRNA in samples of total RNA extracted from schistosome granuloma cell suspensions. RNA was extracted from ~4 × 107 granuloma cells. RPA was performed using the mCR-3 cytokine receptor multiprobe template set as suggested by the manufacturer (PharMingen, San Diego, CA). Protected RNA fragments were electrophoresed in a 5% denaturing polyacrylamide gel. The radioactive gels were dried and exposed to a phosphorimaging screen. The images on the screen were scanned and quantified using the Cyclone detection system (Packard BioScience, Meriden, CT). IL-12R beta 1 and beta 2 mRNA expression were compared relative to that of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

ELISAs. We used ELISAs to measure cytokine concentrations in supernatants. To measure IFN-gamma , plates were coated with a MAb to IFN-gamma (HB170, American Type Culture Collection) and incubated with supernatant. IFN-gamma was detected with polyclonal rabbit anti-IFN-gamma (a gift from Dr. Mary Wilson, Department of Medicine, University of Iowa) followed by biotinylated goat anti-rabbit IgG (Accurate Chemical, Westbury, NY), streptavidin-horseradish peroxidase (HRP), and 2,2'-azino-di(3-ethylbenzthiazoline) sulfonic acid substrate (Zymed, San Francisco, CA).

IL-12 p70 was captured with anti-IL-12 MAb (MM-120, Endogen, Woburn, MA), and IL-12 p40 was captured with anti-IL-12 MAb (C15-6, a gift from Dr. G. Trinchieri). C15-6 captured both free p40 and p40 bound to p35 (p70). Both p40 and p70 were detected with biotinylated anti-IL-12 MAb (MM-121-B, Endogen) and HRP-conjugated streptavidin (Zymed).

MAbs to IFN-gamma (HB170) and IL-12 (C15-6) derived from cell lines maintained in our laboratory. These MAbs were purified from culture supernatants by ammonium sulfate precipitation. Sensitivities of the ELISAs were <30 pg/ml for IFN-gamma , IL-12 p70, and IL-12 total p40. IL-12 p70 and p40 were expressed in femtometers per milliliter to allow comparison of their concentrations. Free p40 was calculated by subtracting the femtometers of p70 from that of total p40.

Statistical analysis. Data are means ± SE or SD of multiple determinations. Differences between two groups were compared using Student's t-test. ANOVA and Dunnett's t-test were used for multiple comparison data. P < 0.05 was considered significant.


    RESULTS
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IFN-gamma production within granulomas is subject to regulation. Dispersed granuloma cells from the livers of C57BL/6 or C57BL/6 IL-4 -/- mice secrete IFN-gamma constitutively when cultured in vitro (30). Table 1 shows that granuloma cells cultured with blocking anti-IL-10 or anti-TGF-beta MAb produce substantially more IFN-gamma , suggesting that IL-10 and TGF-beta made by the dispersed cells function to restrain IFN-gamma synthesis. Isotype control antibody had no effect. Also, cells incubated with schistosome SEA, rIL-12, or rIL-18 secreted more IFN-gamma . As expected, cells from schistosome granulomas that developed in C57BL/6 IL-4 -/- mice made more IFN-gamma than granuloma cells from IL-4-producing wild-type control animals (Table 1).

                              
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Table 1.   Production of IFN-gamma by dispersed granuloma cells

Inducible IFN-gamma production requires IL-12. IL-12 induces T lymphocytes to secrete IFN-gamma and promotes development of the Th1 CD4+ T cell phenotype. It was determined if IL-12 was necessary for ongoing IFN-gamma production within schistosome granulomas. Dispersed granuloma cells cultured with neutralizing anti-IL-12 MAb produced less IFN-gamma than cells grown without blocking anti-IL-12. In the C57BL/6 IL-4 -/- mouse, constitutive production of IFN-gamma by the dispersed granuloma cells decreased by ~40%. Also, IFN-gamma secreted by these granuloma cells in response to anti-IL-10 MAb, anti-TGF-beta MAb, SEA, or rIL-18 decreased by 70-97% (Fig. 1A). Granuloma cells from C57BL/6 wild-type control mice yielded similar results (Fig. 1B). The amount of blocking anti-IL-12 MAb added to each culture was sufficient to completely neutralize the biological activity of rIL-12 used at 500 pg/ml in a bioassay.


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Fig. 1.   Granuloma cells required interleukin-12 (IL-12) to make interferon-gamma (IFN-gamma ) optimally. Dispersed granuloma cells from C57BL/6 IL-4 -/- (A) or C57BL/6 mice (B) were cultured in vitro in microtiter plates at 106 cells/well for 18 h with or without anti-IL-10 (1 µg/ml), anti-transforming growth factor-beta (TGF-beta ; 1 µg/ml), soluble egg antigen (SEA; 5 µg/ml), recombinant IL-12 (rIL-12; 5 ng/ml), or rIL-18 (5 ng/ml). Some cultures also received neutralizing anti-IL-12 monoclonal antibody (MAb; 1 µg/ml). IFN-gamma was measured in the culture supernatants after the incubations. Data are means ± SE of 6 separate experiments performed in triplicate. Anti-IL-12 MAb decreased IFN-gamma production in all cultures (P < 0.01).

Granuloma cells from C57BL/6 IL-12 -/- mice make no measurable IFN-gamma . C57BL/6 IL-12 -/- mice were infected with S. mansoni to further analyze the role of IL-12 in intragranuloma IFN-gamma production. In three separate experiments, the dispersed granuloma cells from the IL-12 -/- animals cultured in vitro failed to secrete IFN-gamma (<30 pg/ml) even when stimulated with rIL-12, rIL-18, SEA, anti-IL-10, or anti-TGF-beta . Also, granuloma cells cultured with rIL-12 (5 ng/ml), anti-IL-10, and/or anti-TGF-beta MAb produced no measurable IFN-gamma .

Granuloma cells secrete IL-12. Because culturing granuloma cells with neutralizing anti-IL-12 MAb substantially decreased inducible IFN-gamma production, we determined whether the granuloma cells released ample amounts of this cytokine. Bioactive IL-12 (p70) is a heterodimeric cytokine consisting of p35 and p40 subunits. The p40 subunit can be produced independently of the p35 subunit. Free p40 and p40 dimers can function as IL-12 inhibitors (29). Thus we used two separate ELISAs to measure both p70 (active IL-12) and total p40 (p40 + p40/p35 and p40/p40 complexes).

Granuloma cells were cultured in vitro for 18 h. After the incubation, the cell-free supernatants were assayed for IL-12. Figure 2 shows that dispersed granuloma cells from C57BL/6 mice constitutively secreted the bioactive form of IL-12 (p70). Because the concentration of immunoreactive p40 exceeded that of p70, this suggested that the granuloma cells secreted free p40 at about twice the rate of bioactive p70. Granuloma cells from C57BL/6 IL-4 -/- mice, which make more IFN-gamma , produced free p40 and bioactive p70 at about twice the rate of granuloma cells from wild-type control animals (Fig. 2).


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Fig. 2.   Dispersed granuloma cells cultured in vitro secrete IL-12 constitutively. Dispersed granuloma cells from C57BL/6 or C57BL/6 IL-4 -/- mice were cultured in vitro in microtiter plates at 106 cells/well. They were maintained for 18 h in 200 µl of RPMI complete medium. IL-12 p70 or total p40 was measured in the culture supernatants after the incubations using ELISAs. Data are means ± SE (in fm/ml) of 3 separate experiments performed in triplicate. For either C57BL/6 or C57BL/6 IL-4 -/-, the granuloma cells made p40 in excess to that of p70 (p70 vs. p40, P < 0.01). Also, C57BL/6 IL-4 -/- granuloma cells made more IL-12 p70 and p40 than granuloma cells from C57BL/6 wild-type controls (P < 0.01). KO, knockout.

Manipulation of IFN-gamma production does not change rates of IL-12 p70 or total p40 secretion from granuloma cells cultured in vitro. As shown above, granuloma IFN-gamma production is partly dependent on IL-12, and rIL-12 can strongly stimulate IFN-gamma secretion in granuloma cells from C57BL/6 mice. Thus we tested the hypothesis that IL-10, TGF-beta , IL-18, and antigen modulated IFN-gamma synthesis via regulation of IL-12 production. Dispersed granuloma cells were cultured in vitro with or without anti-IL-10 MAb, anti-TGF-beta MAb, rIL-18, SEA, or anti-CD3 MAb. After the incubation, the cell-free supernatants were assayed for IL-12 p70 and total p40. Table 2 shows that these manipulations had no affect on IL-12 release.

                              
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Table 2.   Manipulation of IFN-gamma production does not change amount of IL-12 secreted by granuloma cells cultured in vitro

IL-12R beta 1 and beta 2 mRNA expression in dispersed granuloma cells. IL-12 functions through a receptor with a binding beta 1-subunit and a signaling beta 2-subunit. Using an RNase protection assay, we examined the expression and regulation of these subunits within the granulomas of murine schistosomiasis. Mice on the C57BL/6 genetic background strongly expressed the beta 1-subunit. Compared with the GAPDH housekeeping gene, granuloma cells from C57BL/6 IL-4 -/- mice had amounts of IL-12 beta 1 mRNA similar to those of cells from C57BL/6 wild-type control animals. In both mouse strains, beta 2 mRNA was detected only at exceedingly low levels (Table 3).

                              
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Table 3.   IL-12R beta 1 and beta 2 mRNA expression in inflammatory cells from dispersed granulomas

Experiments examined the effect of IL-10 or TGF-beta neutralization on IL-12R beta 1 and beta 2 mRNA expression in dispersed granuloma cells cultured in vitro. Table 3 shows that neither IL-10 nor TGF-beta blockade modulated IL-12R beta 1 or beta 2 mRNA expression in dispersed granuloma cells from C57BL/6 IL-4 -/- or wild-type control animals. SEA stimulation also failed to modulate granuloma IL-12R mRNA expression.

IL-10 and/or TGF-beta blockade enhance granuloma cell IFN-gamma production even with rIL-12 added to cultures. rIL-12 stimulates dispersed granuloma cells from C57 BL/6 wild-type and IL-4 -/- mice to produce IFN-gamma . We also determined whether anti-IL-10 and anti-TGF-beta MAbs could enhance IFN-gamma secretion even with rIL-12 added to the cultures. IL-10 and TGF-beta neutralization enhanced IFN-gamma production beyond that induced by rIL-12 used at 0.01, 0.1, 0.5, or 5 µg/ml. rIL-12 at 0.5 and 5 µg/ml were both at the top of the IL-12 dose-response curve (see Table 1 for results using rIL-12 at 0.5 µg/ml with anti-IL-10/TGF-beta ).

IFN-gamma production in CBA mice is regulated differently. Experiments evaluated IFN-gamma production from dispersed granuloma cells of 8-wk infected CBA mice. These cells made no detectable IFN-gamma constitutively or after stimulation with rIL-12, rIL18, anti-TGF-beta , anti-IL-10, or SEA (Table 1). Table 2 shows that dispersed granuloma cells from CBA mice constitutively secreted the bioactive form of IL-12 (p70) at similar rates to that of C57BL/6 granuloma cells. However, in contrast, granuloma cells from CBA mice released little free p40. Also, analysis of IL-12R mRNA expression yielded an unexpected result. These granuloma cells expressed IL-12R beta 2 mRNA in excess to that of IL-12R beta 2 (Table 3). Similar to the C57BL/6 granuloma cells, neither IL-10 nor TGF-beta blockade affected IL-12 secretion (Table 2) or IL-12R mRNA expression (Table 3) during short-term cultures.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
REFERENCES

Granulomas in the liver of mice with schistosomiasis make large amounts of IL-4 and IL-5 and display the phenotypic characteristics of a Th2-type response. However, the granulomas from mice on a C57BL/6 genetic background produce some IFN-gamma that appears to derive from granuloma T cells (30).

IL-12 is a component of the innate immune response important for induction of Th1 cells. Its early release at the onset of inflammation promotes IFN-gamma production and Th1 cell development and inhibits Th2 cell differentiation (36). Macrophages and dendritic cells probably are the major sources of IL-12 (21). There are T cell-dependent and -independent mechanisms for induction of IL-12 (13). The T cell-dependent pathway involves CD40-CD40L interaction (13, 31). Lipopolysaccharide, endotoxin, and various microorganisms can induce IL-12 expression independently of T cells (3). IL-12 production can be upregulated by IFN-gamma (24) and PGE2 and downregulated by IL-10 (2, 11), TGF-beta , and other factors (1, 10, 37). Also, IL-4 and IL-13 can suppress IL-12 production, helping to prevent emergence of the Th1 response (12, 23).

Thus it was surprising to discover that schistosome Th2-type granulomas from mice on the C57BL/6 genetic background produced IL-12 as well as IL-10, TGF-beta , IL-4, and IL-13. This constitutive IL-12 secretion appeared to be independent of IL-10 and TGF-beta regulation, because IL-10 and TGF-beta blockade had no effect on IL-12 release (p40 or p70). Granulomas in IL-4 -/- mice make no IL-4 and little IL-13 (8). IL-12 secretion from granulomas of IL-4-deficient mice was only modestly higher, suggesting that IL-4 and IL-13 were not major determinants of IL-12 production within schistosome granulomas.

IL-12 is a heterodimeric molecule composed of a light (p35) and heavy (p40) chain. Together, they constitute the active form of the molecule (p70). Cells that make IL-12 can produce the p40 chain in great excess over that of the biologically active p70 heterodimer. The free p40 chain can bind to IL-12R beta 1 and may act as a physiological antagonist of IL-12 (29). We therefore determined if changes in IFN-gamma secretion by granuloma cells after antigen stimulation or cytokine manipulation also changed the relative production of p40 to p70.

C57BL/6 mice produced twice as much free p40 as p70. However, SEA, anti-IL-10 MAb, anti-TGF-beta MAb, rIL18, or anti-CD3 MAb stimulation did not affect the secretory rate of either the free p40 or p70 component. Thus it is unlikely that an alteration in the relative production of free p40 to p70 is the mechanism underlying IL-10, TGF-beta , SEA, or anti-CD3 modulation of IFN-gamma secretion.

Although TGF-beta IL-10 and SEA did not appear to govern the ongoing granuloma IL-12 secretion, IL-12 blocking experiments did suggest that IFN-gamma produced in response to these agents required the presence of IL-12. Also notable was the capacity of anti-IL-12 MAb to block rIL18-induced IFN-gamma secretion. IFN-gamma released constitutively from the granuloma cells was only moderately decreased by IL-12 blockade. Thus IL-12 produced locally within the granuloma is necessary for complete expression of IFN-gamma by granuloma cells in murine schistosomiasis.

To further evaluate the relevance of IL-12 in granuloma IFN-gamma synthesis, we also infected C57BL/6 IL-12 p40 -/- mice with schistosomes. These IL-12 -/- animals formed granulomas that made no detectable IFN-gamma either constitutively or after stimulation with rIL-12, rIL18, or other biological agents. This confirms the importance of IL-12 for the development of IFN-gamma -producing T cells within these granulomas. Experiments using lethally schistosome-infected IL-4/IL-12 double knockout mice suggest that at least splenocytes can overcome the IL-12 deficiency to produce IFN-gamma (28).

The IL-12R consists of two covalently linked components: IL-12R beta 1 and beta 2, each encoded by separate genes. Either unit alone binds IL-12 at low affinity, whereas coexpression of both units can result in high-affinity binding sites. The IL-12R beta 2-subunit likely is most involved in intracellular signaling (16).

The components of the IL-12R are subject to regulation. Activation of T and natural killer cells results in upregulation of the IL-12R. Some of the factors that induce IL-12R display include T cell receptor activation, B7 costimulation (20), IFN-gamma , and IL-12 (7, 34). IL-4, TGF-beta , and IL-10 are reported to downregulate this expression (16). Under various circumstances, either beta 1- or beta 2-subunits are susceptible to immunoregulation in mice and humans (5, 38). Naive T cells stimulated with IL-4 selectively lose the beta 2-subunit, suggesting that this subunit is a critical regulator of Th1/Th2 differentiation (32). It is proposed that IL-12 responsiveness is controlled predominantly through regulation of the beta 2-subunit (16).

TGF-beta has been shown to attenuate IL-12R expression in mice (17). Thus it was possible that TGF-beta and IL-10 blockade stimulated IFN-gamma secretion through modulation of IL-12R beta 1- or beta 2-transcription. Therefore, we examined the expression of IL-12R beta 1 and beta 2 mRNA in the Th2 granulomas of murine schistosomiasis.

Granulomas from mice on the C57BL/6 genetic background strongly expressed IL-12R beta 1 mRNA, but little beta 2. The level of expression was similar in granuloma cells from C57BL/6 IL-4 -/- and wild-type controls. This suggested that neither IL-4 nor IL-13 produced within the granuloma were critically important for regulation of the IL-12R, at least at the mRNA level. Similarly, anti-IL-10 MAb, anti-TGF-beta MAb, and SEA did not alter either IL-12R beta 1 or beta 2 mRNA expression. Thus these factors do not regulate granuloma IFN-gamma production by altering transcription of the IL-12R. It remains possible that IL-10 and TGF-beta affect IL-12R protein expression posttranscriptionally or modulate IL-12 intracellular signaling (6). The stimulation of IFN-gamma production in the presence of blocking IL-12 MAb (Fig. 1) provides some evidence that IL-10 and TGF-beta may work at least in part through mechanisms that do not affect IL-12 signaling.

Our IL-10 and TGF-beta blockade experiments suggest that IL-10 and TGF-beta are factors that help limit IFN-gamma production within the ongoing schistosome granulomas. Although we showed no effect on IL-12 secretion, IL-10 can inhibit macrophage secretion of various cytokines and macrophage expression of major histocompatibility complex class II and B7 (33). B7 interactions can influence IFN-gamma responses in murine schistosomiasis (18). The multiple direct effects of IL-10 on antigen-presenting cells and perhaps T cells could account partly for IL-10 suppression of IFN-gamma synthesis in granulomas. Similarly, TGF-beta has multiple suppressive actions on T and B cells, macrophages, and other cell types (22, 25). TGF-beta can disrupt early IL-12 signaling events in T cells (6, 17, 27), which could be one point of regulation.

The granulomas in the liver of CBA and C57BL/6 mice have a similar morphological appearance. A surprising observation was that IFN-gamma and IL-12 circuitry was substantially different in the granulomas of CBA and C57BL/6 mice. As opposed to the granuloma cells of C57BL/6 mice, CBA granuloma cells expressed IL-12R beta 2 mRNA and little IL-12R beta 1 mRNA. They also secreted much less IL-12 p40, but similar amounts of the bioactive form of IL-12 (p70). The dispersed granuloma cells from CBA mice also produced no detectable IFN-gamma even after stimulation. rIL-12, rIL18, anti-IL-10, anti-TGF-beta , and SEA used alone or in combination had no effect on IFN-gamma secretion. However, CBA granuloma T cells separated from the other granuloma cell types do make IFN-gamma when cultured in vitro (4). In fact, granuloma T cells highly enriched using magnetic beads can produce IFN-gamma (in ng/ml quantities) on stimulation with adherent anti-CD3 MAb (unpublished observation). This suggests that these T cells retain the ability to make IFN-gamma and that perhaps there are unique regulatory circuits within the CBA granuloma environment limiting T cell IFN-gamma expression.

Our findings are in agreement with Todt et al. (35), who showed with CBA mice that the granuloma-bearing livers contained mRNA for IFN-gamma , IL-12, and IL-12R. Also, similar to our results, their data (35) revealed that dispersed granuloma cells cultured even with rIL-12 failed to secrete IFN-gamma . However, Todt et al. (35) failed to detect IL-12 secretion from the dispersed granuloma cells. This latter apparent contradiction to our observations may have resulted from their (35) use of an IFN-gamma bioassay to indirectly measure IL-12 production rather than a direct measurement of IL-12 protein using sensitive and highly specific ELISA assays. Todt et al. (35) also reported IL-12R beta 1 and no beta 2 in intact granuloma-bearing livers of 8-wk-infected CBA mice, which is contrary to our findings using isolated granulomas. This could be a consequence of liver tissue contamination of their (35) RNA preparations. While we showed that blocking IL-10 had no immediate effect on IFN-gamma production, IL-12 secretion, or IL-12R mRNA expression in dispersed CBA granuloma cells cultured in vitro, the findings of Todt et al. (35) suggest that IL-10 neutralization in vivo during early granuloma development can lead to liver granulomas expressing stronger Th1 features.

In summary, this study showed that the Th2 granulomas of murine schistosomiasis in the C57BL/6 mouse make IL-12, which is a necessary cofactor for maintenance of the ongoing IFN-gamma response within the granuloma of natural infection. Granuloma IFN-gamma is subject to regulation by IL-10, TGF-beta , and antigen stimulation. However, none of these factors affect IL-12 production or IL-12R mRNA expression. We also found substantial differences between CBA and C57BL/6 mice in granuloma IFN-gamma production, IL-12 p40 secretion, and IL-12R beta 1 and beta 2 mRNA expression. This study clearly reveals that there are many complex interactions involved in the regulation of IFN-gamma production in schistosome Th2-type granulomas.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-38327, DK-02428, and DK-25295, the Crohn's and Colitis Foundation of America, and the Veterans Administration.


    FOOTNOTES

Address for reprint requests and other correspondence: K. Qadir, Dept. of Internal Medicine, 4607 JCP, Univ. of Iowa, Iowa City, Iowa 52242.

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.

Received 12 January 2001; accepted in final form 21 June 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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