1Intestinal Diseases Research Program, and 2 Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
Submitted 12 January 2004 ; accepted in final form 28 June 2004
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ABSTRACT |
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colitis; adenovirus; cytokine; gene transfer
IL-4 is a pleiotropic cytokine with regulatory effects on B cell growth, T cell growth and function, immunoglobulin switching, hematopoietic cells, and tumor cells (21). There is evidence to support both anti- and counterinflammatory roles for IL-4 in experimental models of intestinal inflammation. IL-4 downregulates Th1-associated injury in T cell receptor -chain-deficient mice (15), but ameliorates colitis induced by hapten (14) or dextran sulfate sodium (23). Dohi et al. (7, 8) found that hapten-induced colitis shifts from Th1 to a Th2 response during the resolution phase, again suggesting the counterinflammatory properties of Th2 cytokines. The number of studies that support IL-4 as a proinflammatory mediator of injury in the intestine are quite limited. In contrast to extensive literature on Th1-based models of colitis, Th2-driven models of colitis are few and include colitis induced by oxazalone (2) and that seen in T cell receptor (TCR)-
/ mice (18). IL-4 has also been shown to enhance inflammation in a Th1 cell-mediated transfer model of colitis (11).
Studies examining the effects of IL-4 in the intestine have used several established models of experimental colitis and have shown that the effects of IL-4 are dependent on the particular model of colitis. Thus the aims of this study were to determine the potential for IL-4 to induce intestinal inflammation in normal naive mice and to examine underlying mechanisms. We also determined the extent to which IL-4-induced colitis resembles that induced by a classic proinflammatory cytokine such as IL-12. This was determined by the use of a replication-deficient adenovirus vector (Ad5) to overexpress either IL-4 or IL-12 directly in the colonic mucosa. We showed that the overexpression of IL-4 in the colonic mucosal microenvironment of healthy mice induces a potentially fatal colitis but that this does not occur when Ad5IL-4 is administered systemically. In contrast to colitis induced by IL-12, the damage induced by IL-4 is less severe, of shorter duration, and is T cell independent. These results provide support for the proinflammatory properties of a Th2 cytokine in the intestinal compartment.
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MATERIALS AND METHODS |
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Mast cell-deficient mice (WWv/) and littermate controls (WWv+/+) were purchased from Jackson Laboratories. All mice were kept in sterilized filter-topped cages and had ad libitum access to autoclaved food and water during the study. Each experimental group of animals, controls, and test subjects, including all wild-type and knockout strains, contained 610 animals. The protocols used in this study were in accordance with guidelines drafted by McMaster Animal Care Committee and Canadian Council on the Use of Laboratory Animals.
Reagents.
Human Ad5 lacking the E1 replication region of its genome were transfected with murine IL-12 cDNA (Ad5IL-12) or murine IL-4 cDNA (Ad5IL-4) according to established procedures (3, 25). Cytokine ELISA kits for detection of TNF-, IFN-
, and IL-4 were purchased from R&D Systems (Minneapolis, MN), and for IL-12, they were purchased from Biosource International (Medicorp, Montreal, PQ, Canada). All reagents for RT-PCR procedures were purchased from GIBCO-BRL and Sigma (St. Louis, MO). Primers for cytokine mRNA analysis were synthesized by MOBIX (McMaster University).
Study protocol.
Mice were anesthetized with enflurane (Abbott Laboratories, St. Laurent, PQ, Canada). Three hours before administering the Ad5 vectors, 150 µl of 50% ethanol in PBS were introduced into the colon at 4 cm proximal to the rectum, using a polyethelene 90 catheter, attached to a 20-gauge needle and 1-ml syringe. Ad5 vectors were suspended in PBS at concentrations of 106-109 plaque-forming units (pfu)/mouse and administered intrarectally in 100-µl volumes. Some mice received only the vector intraperitoneally at 109 pfu/mouse. Control mice were given 50% ethanol only. There were 610 mice in each experimental group, and each experiment was repeated at least twice.
On days 1, 3, 7, and 14, after Ad5 administration, full-thickness samples were removed from an area 6 cm proximal to the anus in control mice or from the upper margin of the macroscopically damaged area in the inflamed distal colons. Samples for histology were fixed in 10% formalin, and samples for cytokine mRNA and protein measurements were snap frozen in liquid nitrogen and stored at 70°C until needed. Assessment of macroscopic and microscopic damage was based on a modification of a previously published scoring system (Table 1). All tabular data, graphs, and micrographs were generated from results using the 108 pfu of Ad5IL-4 and Ad5IL-12.
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Concentrations of IFN-, IL-4, IL-12, and TNF-
were measured by enzyme-linked immunosorbent assay technique using commercially available kits purchased from R&D Systems or Biosource International.
Measurement of cytokine mRNA expression by RT-PCR.
Total cellular RNA was isolated from each tissue sample using a previously described (4) guanidium isothiocyanate method. The concentration of RNA was determined by measuring absorbance at 260 nm, and purity was assessed by the ratio of absorbency of 260/280 nm. During the RT-PCR, the mRNA was reverse transcribed to yield cDNA and was then amplified by PCR using gene-specific primers. The housekeeping gene GAPDH was used as an internal control and detected by using upstream 5'-CCA TGG AGA AGG CTG GGG-3' and downstream primers 5'-CAA AGT TGT CAT GGA TGA CC-3' (9). Primer sequences for the cytokines of interest were as follows: for IL-4 (12), upstream, 5'-TGA TGG GTC TCA GCC CCC ACC TTG C-3' and downstream, 5'-CTT TCA GTG TTG TGA GCG TGG ACT C-3'; for IFN-, upstream 5'-AAC GCT ACA CAC TGC ATC TTG G-3' and downstream, 5'-GAC TTC AAA GAG TCT GAG G-3' (24); and for TNF-
, upstream 5'-GCC CTT GCT GTT CTT CTC TGT-3', and downstream 5'-GGC AAT CAG TTC CAG GTC AGT-3' (17). PCR was performed in 50-µl volumes containing dNTP (200 µM), MgCl2 (1.5 mM), 2.5 units Taq polymerase (GIBCO-BRL) and 50 µg aliquots of cDNA (0.1 µg) mixed with 20 pmol of each primer, along with buffer and distilled water. Messages for IFN-
, IL-4, TNF-
, and GAPDH were amplified by using the following parameters: denaturation at 94°C for 30 s, followed by annealing at 55°C for 30 s and extension at 72°C for 60 s. The number of cycles varied for each set of primers with 42 cycles for IFN-
, 48 cycles for IL-4, 40 cycles for TNF-
, and 27 cycles for GAPDH. PCR products were loaded onto a 2.5% agarose gel and visualized under ultraviolet light after ethidium bromide staining.
Statistical analysis. Data were analyzed by using either Students unpaired t-test or Mann-Whitney U-test with 95% confidence intervals being considered significant. All data were normally distributed and are expressed as means ± SD.
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RESULTS |
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Gross examination of the colon within 3 days after gene transfer revealed severe ulceration and hemorrhage in NIH Swiss and Balb/c mice that received Ad5IL-4 (Fig. 2, C and D). Damage was significantly less by 3 days and reached baseline by day 7. Severity of damage was less at the lower doses but with the same time course as the high doses (Fig. 3).
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Effect of IL-4 overexpression in knockout mice.
Animals lacking either the STAT6 gene, which regulates the Th2 pathway of cytokine production, or the TNF- receptor did not develop colitis after administration of Ad5IL-4 (Table 2). Mast cell-deficient mice (WWv/) and the wild-type strain (WWv+/+), however, showed comparable damage to the colon after overexpression of IL-4. Overexpression of IL-4 in the colon of RAG-2/ animals caused the same amount of damage as in the wild-type strain (Fig. 4, A and C).
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Ad5IL-4 overexpression in IFN- knockout mice induced comparable amounts of IL-4 protein and mRNA expression as wild-type mice at 24 h; however, the amount of IL-4 protein and mRNA expression in IFN-
R knockout mice was significantly less (Table 3). Minimal IL-4 mRNA and protein production was found in STAT6 knockout mice at days 1 and 3 (Table 3).
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Macroscopic damage was severe within 3 days after IL-12 gene transfer with extensive ulceration and hemorrhage in all strains of mice. Damage scores were significantly less after 3 days and reached baseline by day 7 (Fig. 1). Lower doses of the Ad5IL-12 construct caused less damage (Fig. 2), but with a similar time course to the higher doses (data not shown).
Histological examination of hematoxylin and eosin-stained sections from Ad5IL-12 mice showed complete destruction of the mucosal architecture within 24 h accompanied by inflammatory and mononuclear infiltrate (Fig. 2). Mucosal damage lessened by 3 days with some evidence of repair (Fig. 2B); however, disruption of the mucosal layer persisted until 14 days, after which the colon looked structurally normal.
Effect of IL-12 overexpression in knockout mice.
There was minimal damage and no lethality in mice deficient in the IFN-R gene in response to overexpression of IL-12 in the colon (Table 2). Histological damage was completely absent by 3 days. There was, however, considerable damage in the colons of IFN-
-deficient mice given Ad5IL-12 (Fig. 5B) and that was comparable to the Balb/c wild-type controls (Fig. 5B).
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Cytokine mRNA expression and protein production in colonic tissue after administration of Ad5IL-12.
IFN- mRNA and protein (0.96 ± 0.2 IFN-
/G3, and 205 ± 63 pg/ml of IFN-
) was detected in animals that overexpressed IL-12 but not IL-4 in the colon. Peak expression occurred at 24 h. There was no measurable IL-4 mRNA or protein in the colons of NIH Swiss, Balb/c, or C57BL/6 mice that received the Ad5IL-12 virus, or the control virus. TNF-
mRNA (1.1 ± 0.04 TNF-
/G3) and protein (654 ± 101 pg/ml) expression occurred within 24 h of IL-12 overexpression and persisted at elevated levels until day 14.
Expression of IFN- mRNA and protein was present at very low levels in the IFN-
knockout (0.005 IFN-
/G3 ratio, and 0 pg/ml), IFN-
R knockout (0.005 IFN-
/G3 and 0 pg/ml), and in the CD4 or RAG-2 knockout (0.015 or 0.18 IFN-
/G3 ratio, and 1.5 or 2.1 pg/ml) mice after Ad5IL-12 administration. High amounts of TNF-
mRNA and protein were present in the IFN-
knockout mice (1.31 TNF-
/G3 and 526 pg/ml) and were comparable to amounts in wild-type mice (1.1 TNF-
/G3 and 654 pg/ml). TNF-
mRNA and protein were found at much lower concentrations in the IFN-
R knockout and the CD4 and RAG-2 knockout mice (e.g., 0.23 TNF-
/G3 and 25 pg/ml).
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DISCUSSION |
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In the Th1-mediated hapten-induced model of colitis in mice, a Th2 cytokine profile emerges during the later phases of the inflammatory process (7, 8) raising the possibility that Th2 cytokines contribute to recovery. This is supported by preliminary data showing that hapten-induced colitis is aggravated and often fatal in STAT-6/ mice (D. El-Ashry, unpublished observation), implying a counterinflammatory action for IL-4 and IL-13. This again emphasizes the importance of the micro environment in determining the action of a cytokine.
The proinflammatory effect of IL-4 documented in this study is in keeping with a role for Th2 cytokines in models of colitis induced by oxazolone (2), in that seen in TCR--deficient mice (18), and in intestinal inflammation associated with helminth infection (13). Colitis induced by IL-4 overexpression was evident in CD4 and RAG-2-deficient mice, indicating a T cell-independent mechanism. This is supported by a previous study (23) in which colitis induced by DSS, which is generally accepted to be a T cell-independent model, was ameliorated in IL-4-deficient mice. The ability of IL-4 to exacerbate a Th-1-driven model of colitis suggests that IL-4 may also exert proinflammatory effects in a context of T cell-mediated injury (11).
The results of this study implicate TNF- as an important mediator of colitis induced by IL-4 and by IL-12. This cytokine is, of course, well known as a mediator of intestinal inflammation. The higher and more sustained levels of this cytokine are associated with the more severe colitis seen after overexpression of IL-12. TNF-
has also been implicated in intestinal inflammation that accompanies helminth infection, in which IL-4 plays a critical role (1). However, in contrast to the models of parasitic infection in which mast cells are prominent, the proinflammatory effect of IL-4 seen here is mast cell independent. The identity of the cells involved in this response remain to be identified. Weaver and colleagues (16), have shown that Th1 and Th2 cells can mediate intestinal inflammation, but each in a qualitatively distinct manner. An examination and quantification of specific cell types that infiltrate the site would be a useful comparison between IL-12- and IL-4-induced mechanisms of action. Because colitis was seen in RAG-2 as well as mast cell-deficient mice, a role for innate immune cells seems likely and should be examined further.
As expected, overexpression of IL-12 induced a severe colitis which, in contrast to that induced by IL-4, was T cell dependent. The effect of IL-12 was dose dependent, and similar to findings from other studies (6, 10, 19). Davidson et al. (6) reported that IL-12 of a particular dose played a major role in sustaining the chronic phase of colitis in the IL-10-deficient spontaneous model of colitis. Increasing the dose of IL-12 used in vitro to stimulate fetal gut explants led to mucosal degradation and an increase in stromelysin-1 concentrations (19). In the present study, IFN- did not appear critical for IL-12-induced injury. Colitis was attenuated in IFN-
R-deficient mice but not in IFN-
-deficient mice. Perhaps IFN-
R is stimulated by some other molecule that contributes to inflammation. This is a possible focus of future work in this area. Whereas IFN-
was absent in both knockouts, it was the marked reduction in TNF-
in the IFN-
R-deficient mouse that accompanied the attenuation of colitis. These results suggest that TNF, rather than IFN, is critical for the proinflammatory effects of IL-12 under these conditions. Our finding of an IFN-independent action of IL-12 is consistent with conclusions drawn by others in different experimental contexts (5, 22).
In conclusion, this study provides direct evidence of the potential for a counterinflammatory Th2 cytokine to cause acute and lethal damage in a healthy mouse colon, through IFN-- and T cell independent mechanisms. These results establish the basis for more in-depth examination of the effects of Th2 cytokine overexpression, and its implications for the design of drugs and therapies that steer the immune response toward a Th2 response.
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GRANTS |
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FOOTNOTES |
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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.
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REFERENCES |
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