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*
Mucosal Inflammation Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada;
Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109; and
Serono Pharmaceutical Institute, Geneva, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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7 days after induction). Colonic
RANTES mRNA expression was also significantly elevated during the
chronic phase of colitis. The numbers of macrophages and monocytes in
the colonic mucosa increased substantially during the chronic phase, as
did expression of two of the receptors (CCR1 and CCR5) to which RANTES
is known to bind. Administration on days 7 through 14 after
trinitrobenzene sulfonic acid administration of a CCR1/CCR5 receptor
antagonist, Met-RANTES, resulted in a significant reduction of both
macroscopic and microscopic colonic damage, as well as reducing the
recruitment into the colon of monocytes, mast cells, and neutrophils.
In some rats, treatment with Met-RANTES resulted in a near-complete
resolution of colonic damage and inflammation. These results suggest a
crucial role of RANTES in the progression from acute to chronic
inflammation in a rat model of colitis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RANTES is a C-C chemokine that promotes the recruitment and activation of inflammatory cells such as monocytes (6), lymphocytes (7), mast cells (8), and eosinophils (9). RANTES is a ligand for the chemokine receptors CCR1, CCR3, and CCR5 (10), and increased expression of RANTES has been observed in vivo in inflammatory diseases such as glomerulonephritis (11, 12), adjuvant-induced arthritis (13), and granulomatous inflammation (14). Of particular relevance to this study is a report of increased expression of RANTES in colonic biopsies from patients with active inflammatory bowel disease (IBD)3 (15). In that study, the expression of RANTES mRNA was found to be increased in biopsies from patients with Crohn’s disease or ulcerative colitis, particularly in intraepithelial lymphocytes and in the subepithelial lamina propria. RANTES expression in animal models of colitis has not been described. Moreover, no interventional studies have been performed to determine whether RANTES makes an important contribution to inflammation and/or injury in human IBD or in experimental colitis.
The purpose of this study was to assess the contribution of RANTES to the pathogenesis of colitis induced in the rat by trinitrobenzene sulfonic acid (TNBS). In particular, we were interested in the possibility that RANTES, through its ability to recruit monocytes and mast cells, may play a role specifically in the chronic phase of colonic inflammation following TNBS administration. Previous studies using this model have documented important roles for a number of inflammatory mediators (e.g., leukotrienes, tachykinins, prostaglandins, IL-1, TNF) in the inflammation and injury occurring during the first week after TNBS administration. Indeed, inflammation during the acute phase could be markedly reduced by a number of drugs that targeted these inflammatory mediators (16, 17, 18, 19, 20, 21). However, modifying the inflammatory response once it has progressed to a more chronic phase has proven to be much more difficult.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male Wistar rats (175–200 g) were purchased from Charles River Breeding Farms Limited (Montreal, Canada). The rats were fed a standard chow pellet diet, had free access to water, and were maintained on a 12-h light/dark cycle. All procedures in this study were approved by the Animal Care Committee of the University of Calgary and conformed to the guidelines established by the Canadian Council on Animal Care.
Induction of colitis
Rats were lightly anesthetized with halothane, and the hapten
TNBS (60 mg/ml in 0.5 ml of 50% ethanol) was administered into the
distal colon via a cannula (22). At selected times
thereafter (2 h to 14 days), the rats were killed by cervical
dislocation. The colon was excised and examined for macroscopic damage
by an observer unaware of the treatments. The colonic damage was scored
using the criteria outlined in Table I
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After examination, tissue samples were taken for determination of
tissue chemokine levels and for measurement of RANTES mRNA expression.
Age-matched, untreated rats served as controls.
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Samples of distal colonic tissues were homogenized in 1.5 ml of lysis buffer containing 0.1% Triton X-100, 500 mM NaCl, 50 mM HEPES, 0.1 mg/ml leupeptin, and 10 mg/ml PMSF. The homogenates were incubated on ice for 30 min and then centrifuged (400 x g for 10 min). The supernatants were collected and stored at -80°C until used for determination of chemokine protein levels by ELISA (23) and total protein concentration by colorimetric protein assay (Bio-Rad, Richmond, CA). The chemokines that were measured were RANTES, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-2, and cytokine-induced neutrophil chemoattractant (CINC).
mRNA expression
RNA was extracted from colonic tissue using Trizol reagent according to the manufacturer’s instructions (Life Technologies, Burlington, Canada). The yield and purity of the RNA was determined spectrophotometrically at 260 and 280 nm. Isolated RNA (2 µg in diethyl pyrocarbonate-treated water) was reverse transcribed to cDNA and amplified by PCR as previously detailed (24). Briefly, DNA amplification was performed under the following conditions: denaturation at 94°C for 1 min, annealing at 55°C for 30 s, and extension at 72°C for 1 min. The RANTES/GAPDH genes were coamplified for 30 and 22 cycles, respectively. The sequences for the RANTES and GAPDH primers used have been described elsewhere (9, 24, 25). To ensure that the amplified PCR product was measured only during the exponential phase of the amplification reaction, the optimum number of PCR cycles was determined for each primer pair. In addition, to exclude the presence of contamination, amplification was always performed in which cDNA was omitted from the PCR mixture. Amplified products were visualized on a 2% agarose gel stained with ethidium bromide, and the size of the PCR product was confirmed with m.w. markers (Life Technologies). The relative quantity of mRNA was estimated by densitometric analysis using Quantity One software (Bio-Rad), normalized to the respective GAPDH, and expressed as a ratio. Data are presented as the mean normalized ratio ± SE.
Expression of mRNA for the chemokine receptors CCR1 and CCR5 was also examined by RT-PCR at various times after induction of colitis, as described above. The primers used for this purpose have been described previously (26).
Effects of Met-RANTES
Met-RANTES is a modified chemokine that binds with high affinity, but does not activate, the chemokine receptors CCR1 and CCR5 (27, 28). Rats were treated with Met-RANTES (40 or 200 µg/rat; i.v.) on day 7 after the administration of TNBS and every 24 h thereafter for 1 wk. Control rats received the vehicle (sterile saline) at the same times. All rats were killed 14 days after TNBS administration for assessment of colonic damage. Additional tissues were taken for analysis of myeloperoxidase (MPO) activity as an index of granulocyte infiltration (17) and for histological examination. Slides were coded to prevent observer bias, and histological sections were then evaluated and scored by an observer unaware of the treatments. The 200-µg dose of Met-RANTES used in this study has been shown to be effective in reducing inflammation in a rodent model of glomerulonephritis (12).
Morphological studies
Colonic tissues were fixed overnight in 10% neutral buffered formalin, dehydrated in graded concentrations of ethanol, embedded in paraffin, and sectioned. Sections (5 µm thick) were stained with hematoxylin and eosin according to standard protocols for histological examination. RANTES has been shown to play a role in the recruitment of mast cells (8) and monocytes (12) into tissues; thus, the effect of treatment with Met-RANTES on the recruitment of these cells into the colon after TNBS administration was determined. For monocyte enumeration, sections were pretreated with 3% hydrogen peroxide in methanol for 10 min to inactivate endogenous peroxidases, followed by permeabilization in PBS containing normal horse serum and Triton X-100 at room temperature for 60 min. Specific labeling of monocytes was performed by incubation of sections overnight at 4°C with anti-ED1 Ab (clone 1C7, 1:10 dilution; PharMingen, Mississauga, Ontario, Canada) to detect mononuclear phagocytes. Sections were then treated with HRP-conjugated secondary Ab (rat anti-mouse IgG; Jackson ImmunoResearch, West Grove, PA) for 90 min at room temperature. The slides were treated with diaminobenzidine for color development and counterstained with hematoxylin. As a negative control, mouse IgG was used.
For mast cell staining, colonic tissues were fixed for 48 h in Carnoy’s fixative (ethanol/chloroform/acetic acid, 6:3:1), processed, and then stained with Alcian blue/safranine (29). Slides were coded to prevent observer bias, and histological sections were then evaluated and scored by an observer unaware of the treatments the rats had received.
Materials
TNBS was obtained from Fluka Chimica (Buchs, Switzerland). RT-PCR reagents were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Met-RANTES was synthesized by Serono Pharmaceutical Institute (Geneva, Switzerland). All other chemicals were obtained from Sigma (St. Louis, MO) or VWR Scientific (Edmonton, Canada).
Statistical analysis
All data are shown as mean ± SE. Comparisons between two experimental groups of data were performed using Student’s unpaired t test, except in the case of the colonic damage scores, in which a Mann-Whitney U test was used. Comparisons among three or more experimental groups were performed using a one-way ANOVA followed by a Dunnett’s multiple comparison test. Probability values of <5% (p < 0.05) were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Over the course of the 2-wk study, there was a gradual decline in the
colonic damage score. MPO activity reached a peak at 72 h
post-TNBS, and gradually declined thereafter. RANTES expression was not
significantly different from controls (i.e., rats without colitis)
during the acute phase of colitis (2–72 h post-TNBS). However, RANTES
expression was significantly elevated during the period 7–14 days
after administration of TNBS, a time when the expression of the other
chemokines did not differ significantly from controls. In addition to
the changes in chemokine protein expression shown in Fig. 1, we
observed that expression of CINC was markedly up-regulated during the
acute phase of colitis (2–72 h post-TNBS; peak increase of 20-fold at
4 h), but was not significantly different from controls during the
7- to 14-day post-TNBS period.
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).
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As summarized in Fig. 3, significant
increases in colonic monocyte and mast cell numbers occurred during the
period 7–14 days after TNBS administration (i.e., the numbers were
significantly elevated at day 14, but not at day 7).
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The expression of mRNA for the chemokine receptors CCR1 and CCR5 increased significantly following induction of colitis. The ratio of CCR1 to GAPDH mRNA increased from 0.23 ± 0.14 in healthy controls to 1.55 ± 0.19, 1.98 ± 0.07, and 2.32 ± 0.20 at 3, 7, and 14 days after TNBS administration, respectively (p < 0.01 at each of these times). In the case of CCR5, the expression of mRNA as a ratio of GAPDH mRNA expression was not significantly changed at day 3 post-TNBS (1.78 ± 0.09) vs the healthy controls (1.25 ± 0.04). However, at both day 7 and day 14 there was a significant (p < 0.01) increase in expression (2.28 ± 0.10 and 2.81 ± 0.27, respectively).
Effects of Met-RANTES on macroscopic severity of colitis
Treatment with Met-RANTES (200 µg) after the onset of colitis
significantly reduced the macroscopic scores for colonic damage (Fig. 4). All rats in the vehicle-treated group
exhibited severe damage. In the group treated with Met-RANTES, four of
the six rats exhibited little or no colonic damage, whereas the
remaining two rats exhibited damage of similar severity to that seen in
the controls (Fig. 4).
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Effects of Met-RANTES on histological severity of colitis
Histological assessment confirmed the significant reduction of
tissue injury in rats treated with the higher dose of Met-RANTES (Fig. 4
). This histological evaluation confirmed the very low level of damage
or inflammation in four of the six rats treated with Met-RANTES,
whereas all six rats receiving the vehicle had widespread tissue
destruction and massive, transmural inflammation. Examples of the
histological appearance of the colonic tissues from rats treated with
Met-RANTES or vehicle are shown in Fig. 5. In the vehicle-treated rats, there was
widespread destruction of the mucosa with extensive, transmural
infiltration of neutrophils, monocytes, and lymphocytes (mastocytosis
was also evident in sections stained with Alcian blue/saffranine). In
contrast, in the rats treated with Met-RANTES in which a macroscopic
response was evident, there was minimal damage to the surface
epithelium, and there was mild inflammation of the mucosa (mainly
neutrophils), but no transmural inflammation.
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A significant reduction of the numbers of mast cell and monocytes
in the colon was seen in rats treated with Met-RANTES (Fig. 6). In addition, a significant reduction
in the number of granulocytes, as measured by tissue MPO activity, was
observed (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Met-RANTES is one of a number of chemokine analogs that act as RANTES
antagonists (27, 28, 34). The ability of Met-RANTES to
reduce tissue injury in this study is consistent with its effects in
models of arthritis (35) and renal inflammation
(12). Moreover, a role for RANTES in the progression from
acute to chronic colitis, which our results strongly suggest, is
consistent with the conclusions of Lloyd et al. (36) with
respect to the role of RANTES in murine crescentic nephritis. RANTES
could contribute to such a conversion of the inflammatory state by
virtue of its chemotactic effects on cells that are involved in chronic
inflammation such as monocytes, lymphocytes, and mast cells, in
addition to its ability to activate T cells and monocytes (6, 7, 8, 37). RANTES has also been suggested to stimulate neutrophil
activation (37), which could contribute to the
prolongation of an established inflammatory response, as well as to
fibrosis. Moreover, granulocytes have been shown to contribute
significantly to acute tissue injury in experimental colitis in rats
and rabbits (38, 39, 40). Treatment with Met-RANTES produced a
substantial (
63%) reduction in tissue MPO activity, an index of
tissue granulocyte numbers.
The effectiveness of Met-RANTES in abrogating colitis suggests that the CCR1 and/or CCR5 are key receptors in mediating the effects of RANTES in this model. Therefore, it is noteworthy that CCR1 has been implicated in chronic inflammation in other tissues. For example, CCR1 was reported to mediate the gross pulmonary fibrotic response to bleomycin treatment (41). The role of CCR5 in colitis has been examined in a mouse model. The colitis induced by oral administration of dextran sodium sulfate was found to be significantly reduced in mice lacking the gene for CCR5 than in wild-type controls, although there was not any evidence of a reduction in macrophage infiltration into the colon (42). It is also important to note that CCR1 and CCR5 are expressed to differing degrees by various leukocyte subsets. CCR1 has been reported to be expressed on activated T cells, monocytes, eosinophils, dendritic cells, and neutrophils (4, 41, 43). In contrast, CCR5 has been shown to be expressed on activated T cells, monocytes, macrophages, and dendritic cells, but not on neutrophils (41, 43, 44). The expression of CCR1 on neutrophils may account for the earlier increase in expression of this receptor than of CCR5 after induction of colitis because neutrophil recruitment is an early event in the TNBS model.
Although Met-RANTES at a dose of 200 µg/day produced a statistically significant reduction of colonic damage and MPO activity as compared with vehicle-treated controls, it was apparent that this antagonist did not reduce the severity of tissue injury or inflammation in all of the rats that received this agent. The reasons for the apparent failure of response in two of the six rats are not clear. It is possible that these rats had more severe colonic injury at the onset of treatment than the other rats treated with Met-RANTES. Alternatively, it is possible that in some rats, other mediators of inflammation play a more predominant role than RANTES in mediating the progression from acute to chronic colitis. It is noteworthy that heterogeneous responses to therapies are very common in patients with IBD, with 20–40% of patients failing to show a response to the most widely used therapeutic agents (44, 45, 46, 47). Of course, one cannot rule out that possibility that treatment with higher doses of Met-RANTES or treatment over a longer period of time may produce beneficial effects in a higher percentage of rats.
In summary, the results presented herein demonstrate that RANTES expression is markedly elevated in the chronic phase of TNBS-induced colitis in the rat, a time when the expression of a number of other chemokines (MIP-1, MCP-2, CINC) was not significantly elevated above control levels. Expression of two of the key receptors for RANTES was also elevated during the chronic phase of colitis. Moreover, treatment with a CCR1/CCR5 receptor antagonist significantly accelerated the healing of colonic injury, most likely by antagonizing the actions of RANTES. Our results provide novel evidence of a crucial role for RANTES in the progression from acute to chronic colitis in this model. The beneficial effects of RANTES antagonism in experimental colitis suggest that chemokine receptor antagonists are potentially useful therapeutic approaches to the treatment of IBD.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John L. Wallace, Department of Pharmacology and Therapeutics, University of Calgary, 3330 Hospital Drive Northwest, Calgary, Alberta, T2N 4N1, Canada.
3 Abbreviations used in this paper: IBD, inflammatory bowel disease; TNBS, trinitrobenzene sulfonic acid; MCP, monocyte chemoattractant protein; CINC, cytokine-induced neutrophil chemoattractant; MPO, myeloperoxidase; MIP, macrophage inflammatory protein.
Received for publication June 15, 2000. Accepted for publication October 4, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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