Published online before print July 26, 2007
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* Department of Preclinical Pharmacology, Dompé pha.r.ma s.p.a., L’Aquila, Italy;
Department of Oncology and Neurosciences, Surgical Pathology Section, "G. d'Annunzio" University, Chieti, Italy;
Ce. S.I. Aging Research Center, "G. d'Annunzio" University Foundation, Chieti, Italy;
Experimental Medicine Department, University of L’Aquila, L’Aquila, Italy;
¶ Operative Unit of Pathological Anatomy, S. Salvatore Regional Hospital, L’Aquila, Italy;
|| Biometry Unit, Dompé pha.r.ma s.p.a., Milan, Italy; and
# Nerviano Medical Sciences, Milan, Italy
1 Correspondence: Department of Preclinical Pharmacology, Dompé pha.r.ma s.p.a., Via Campo di Pile 67100 L’Aquila, Italy. E-mail: bertini{at}dompe.it
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: rodent • neutrophils • inflammation
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Interleukin-8 (IL-8/CXCL8), a member of CXC chemokines, is a chemoattractant of PMN. Two high-affinity human CXCL8 receptors are known, CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2). Although a mouse ortholog of CXCL8 has not been identified, the two corresponding receptors have been identified in the mouse [7 ,8 ]. By recruiting and activating PMN, CXCL8 has been implicated in a wide range of disease states characterized by PMN infiltration in different organs [9 ], including UC [10 ]. CXCL8 is expressed in the colonic mucosa of patients affected by UC [11 ,12 ] and IBD [13 ,14 ] and a correlation between CXCL8 levels, PMN number in mucosal tissue, and the severity of UC has also been described [15 ,16 ].
To investigate the pathophysiological role played by CXCR2 and its specific ligands in experimental UC, we induced chronic relapsing DSS-mediated colitis in WT and CXCR2–/– mice. Among animal models of UC, the chronic relapsing DSS-induced colitis resembles human UC under several features, including clinical symptoms and histopathological changes [17
]. CXCR2–/– mice were reported to bear and develop normally, but their PMN were unable to migrate in response to its specific ligands CXCL1 (GRO
/KC) and CXCL2 (GROβ/MIP-2) when compared with their WT littermates [18
]. The results reported hereafter point out that the functional lacking of CXCR2 produced an amelioration of experimental UC, as shown by a significant improvement of clinical conditions paralleled by a reduction of the histopathological hallmarks of this disease model. These data are in keeping with a key role of CXCR2 in mediating PMN recruitment and tissue damage in experimental UC, suggesting that CXCR2 activation may represent a relevant pharmacological target for the design of novel pharmacological treatments in human UC.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All the procedures were performed in the animal operating rooms, according to ethical guidelines for the conduct of animal research (Authorization Italian Ministry of Health No. 50/2001-B; Italian Legislative Decree 116/92, Gazzetta Ufficiale della Repubblica Italiana No. 40, February 18, 1992; EEC Council Directive 86/609, OJL 358, 1, December 12, 1987; National Institutes of Health Guide for the Care and Use of Laboratory Animals, NIH Pub. No. 85-23, 1985).
Chronic DSS colitis model
Chronic colitis was induced by 4% DSS (36–50 kDa, ICN Biochemicals, Milan, Italy) in drinking water in two consecutive cycles, as previously reported [20
,21
]. The first DSS cycle was from day 0 to day 6. This cycle was followed by 14 days of simple ultrafiltered water (1st washout phase), from day 6 to day 20. The second DSS cycle (from day 20 to day 24) induced the relapse of the disease and was followed by three days of ultrafiltered water (2nd washout phase), from day 24 to day 27. Control group received water during all the experimental sessions. During the setup of the model, colitis was induced in Balb/c mice obtained from Charles River Laboratories and WT mice (CXCR2+/+) derived from founders by Jackson Laboratories. No differences were seen in terms of clinical and histopathological response between the two inbred Balb/c strains. In all of the experiments reported hereafter, WT mice derived from founders by Jackson Laboratories were used.
Clinical evaluation of DSS-induced colitis
Disease progression was determined at crucial time points by assessment of disease activity index (DAI), which ranged from 0 to 7. DAI score was calculated accordingly to previous reports [17
,22
], adding two separate scores: Stool score (0=Normal; 1=>30% pellets with smooth consistency or <30% pellets with diarrheic consistency; 2=30–70% pellets with diarrheic consistency; 3=>70% pellets with diarrheic consistency) and Emo score (0= negative; 1=occult blood positive; 2=small blood drops on the pellet; 3= gross anal bleeding; 4=blood drops at the bottom of the cage). Occult blood in feces was determined by Hemo-fec (Roche Diagnostics, Mannheim, Germany).
Histopathological, ultrastructural, immunohistochemical, and immunofluorescent analysis
Colon from the ileocecal valve to the anus was removed, washed in ice-cold saline, fixed in 10% neutral buffered formalin, embedded in paraffin and stained with hematoxylin and eosin. Histopathological score was assigned by two blinded pathologists. According to grading scales of histological assessment in human UC [23
,24
], histopathological score was calculated as sum of single scores (ranged from 0 to 15), taking into account crypt distortion, loss of muco-secretive capability, percentage of severe ulcers, amount of inflammatory infiltrate in the severe ulcers expressed as granulocytes/severe ulcer thickness ratio (G/U ratio), and extension of tissue damage. The amount of inflammatory infiltrate in severe ulcers (expressed as G/U ratio) was singularly considered and defined as inflammatory score ranging from 0 to 5.
For electron microscopy, specimens were fixed in cacodylate buffered 2.5% glutaraldehyde, postfixed in osmium tetroxide and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate-lead citrate.
For immunohistochemistry, large intestines were embedded in optimum cutting temperature compound (Miles), snap-frozen in liquid nitrogen and stored at –80°C. Then, frozen samples were sectioned, air dried overnight, fixed with acetone, and immunostained with anti-CXCL1 (Peprotech, Rocky Hill, NJ), anti-CD11c (Chemicon International, Temecula, CA, USA), anti-CD11b (Mac-1; Sera-Lab), anti-cytokeratins (CK) 8/18 (Progen Biotechnick, Heidelberg, Germany), anti-CD8 (Ly/T2; Sera-Lab), and anti-CD4 (LT34; Sera-Lab), anti-GR-1 (American Type Culture Collection) Abs. After washing, sections were overlaid with biotinylated goat anti-rat, anti-hamster, anti-rabbit and horse anti-goat Ig (Vector Laboratories, Burlingame, CA, USA). Unbound Ig was removed by washing, and slides were incubated with ABC (avidin/biotin complex)/alkaline phosphatase (DakoCytomation, Glostrup, Denmark).
For double immunofluorescent staining and confocal analysis, acetone-fixed frozen sections were incubated with the first primary antibodies and then with biotinylated secondary antibodies. After washing, sections were incubated with the second primary antibodies and, after washing, with biotinylated secondary antibodies and then with Alexa Fluor 594 conjugated StreptAvidin. Cross-reactions between the first secondary antibodies and Alexa Fluor 594 was prevented by saturation of all of its binding sites with Alexa Fluor 488. Slides were mounted with Vectashield medium (Vector Laboratories) and examined with a LSM 510 Meta laser scanning confocal microscope (Zeiss, Jena, Germany).
Myeloperoxidase activity
Three centimeters of colon from anus were ice-homogenized (Ultra–Turrax, Ika-Werk) in a tube containing 1 ml/100 mg of tissue of 50 mM PBS, pH 6.0 and 0.5% HTAB (Sigma-Aldrich, St. Louis, MO, USA). Then samples were ice-sonicated (Ultrasonic processor XL) and ultracentrifuged (Sorvall RC5C) at 40,000 g at 4°C [25
]. The supernatants were assayed for MPO activity [26
]. Protein assay was performed by bicinchoninic acid method (Pierce Biotechnology, Rockford, IL, USA).
CXCL1 and CXCL2 production
Three centimeters of colon from the anus were removed, ice-homogenized with protease inhibitors (5 µg/ml leupeptin, pepstatin, chimostatin, aprotinin and 1 mM PMSF; Sigma-Aldrich) and centrifuged at 20,800 g at 4°C (Centrifuge 5417 R, Eppendorf). Measurement of CXCL1 and CXCL2 was performed in the supernatants by using mouse CXCL1 and CXCL2 ELISA kits (R&D Systems, Minneapolis, MN, USA), according to manufacturer’s instructions.
Statistical analysis
Data are expressed as means ± SEM. All the analyses were conducted by using SAS 9,1 TS Level 1M3 in Windows XP professional environment.
DAI score was analyzed by Cochran-Mantel-Haenszel test (CMH) [27] and mixed linear models of analysis of variance (ANOVA) for repeated measures on data transformed by ranks [28
]. Histopathological and inflammatory scores were processed by applying both Exact 
2-test or CMH and mixed linear models of ANOVA for a model completely random on data transformed by ranks. ANOVA followed by Dunnett’s multiple comparison test was performed for CXCL1 and CXCL2 production data.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To induce relapsing colitis, a second cycle of DSS administration was started at day 20 until day 24, as described previously [20 ]. DAI score increased in a time-dependent manner, reaching the maximum value at day 24 and persisting until day 27.
To assess the role of CXCR2 in the chronic relapsing disease, DSS colitis was induced in CXCR2–/– mice. As shown in Fig. 1 , the first cycle of DSS administration induced in CXCR2–/– mice a clinical outcome similar to WT mice (2.00 ± 0.51 and 2.11 ± 0.50 in CXCR2–/– and WT mice at day 6, respectively). DSS-second cycle caused a time-dependent increase in DSS-treated WT mice, reaching the maximum value at day 24. A significant reduction (P<0.001) of clinical signs of colitis (from 30% up to 48% of inhibition at day 24 and day 27, respectively) was observed in CXCR2–/– mice in comparison with WT. At day 27, the time of sacrifice, the DAI score of CXCR2–/– group was 42% lower than that of WT group (1.44 ± 0.22 and 2.47 ± 0.19 in CXCR2–/– and WT mice, respectively) (Fig. 1) . The clinical score improvement observed in CXCR2–/– mice was evident both as stool consistency and as blood in feces.
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Figure 1. Clinical evaluation of DSS-induced colitis in wild-type (WT) and CXC chemokine receptor 2 (CXCR2–/–) mice. The clinical progression of the colitis was determined by disease activity index (DAI), calculated by adding two separate scores (ranging from 0 to 7): Stool score (0=normal; 1=>30% pellets with smooth consistency or <30% pellets with diarrheic consistency; 2=30–70% pellets with diarrheic consistency; 3=>70% pellets with diarrheic consistency) and Emo score (0=negative; 1=occult blood positive; 2=small blood drops on the pellet; 3=gross anal bleeding; 4=blood drops at the bottom of the cage). Colitis was induced in WT and CXCR2–/– mice by giving 4% DSS in drinking water in two cycles. Control group received water. Data are reported as means ± SEM (24–35 animals for each time point from one experiment of three). **, P< 0.01 vs. DSS-treated WT group by ANOVA.
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According to previous reports [17 ,22 ], the first cycle of DSS administration induced a pathological status with a low involvement of inflammatory cells and reduced tissue damage. Only few PMN, lymphocytes, and plasma cells, incoming from submucosa, were detected between crypts and into crypt epithelium. Initial erosions were also observed but without PMN stratifications (data not shown).
During the second DSS cycle, clinical symptoms (see above) were related by progressive histopathological changes, characterized by a dramatic infiltration of PMN from the lamina propria into the colonic mucosa causing severe ulcers (Fig. 2C 2D 2E) and microabscesses formation (Fig. 2F) . No histopathological change was reported in control group (Fig. 2A 2B) . As shown in Fig. 3 , massive PMN recruitment into colonic segment was further confirmed by increased tissue MPO, a well-known specific marker of PMN (5.81 ± 0.11 and 171.25 ± 12.16 mU/mg protein in water and DSS-treated WT mice at day 27, respectively). Large tracts of mucosa lost the epithelium and was replaced by granulation tissue (Fig. 2E) . The overall histopathological and inflammatory scores peaked at day 27 (Fig. 4A B ).
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Figure 2. Histopathological analysis of DSS-induced colitis in WT and CXCR2–/– mice. Colon from the ileocecal valve to the anus was removed and stained with hematoxylin and eosin. (A) and (B) control mice. At day 27, DSS induced massive infiltration of PMN into the crypt epithelial layer with necrosis and ulcerative lesions of colonic mucosa (C), (D), large tracts of mucosa lost crypt epithelium appeared to be replaced by granulation tissue (E) associated with a dramatic infiltration of PMN, causing microabscesses formation (F) in WT mice. The overall aspect of colonic mucosa was less severe in CXCR2–/– mice. A significant decrease of PMN aggregates was associated with a reduction of severe ulcers formation (G), (H). The inflammatory cells (indicated by arrow) were mainly confined into microvascular space, confined around the crypts (I). Muco-secretive ability preserved in CXCR2–/– mice (G), (H), (L). Original magnification, x100 (A), (C), (D),(G); x200 (B), (H); x400 (E), (I), (L); x1000 (F).
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Figure 3. MPO activity in the colon of DSS-treated WT and CXCR2–/– mice. WT and CXCR2–/– mice were killed at day 27 after DSS administration, and colonic mucosa were processed for determination of MPO activity. Control group received normal water. (**, P<0.01 vs. DSS-treated WT group by ANOVA). Data are expressed as mean ± SEM (14 animals for each experimental group from one experiment of three).
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Figure 4. Histopathological and inflammatory scores in DSS-treated WT and CXCR2–/– mice. Animals were killed during the chronic phase of DSS-induced colitis, and histopathological analysis was performed in the tract of the colon mainly involved by disease. (A) Histopathological score in CXCR2–/– and WT mice (**, P< 0.01 vs. DSS-treated WT group by ANOVA). (B) Inflammatory score in CXCR2–/– and WT mice (**, P<0.01 vs. DSS-treated WT group by ANOVA). Data are expressed as means ± SEM (14–25 animals for each time point from one experiment of three).
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Figure 5. Immunohistochemical analysis of colon section from WT and CXCR2–/– mice. Immunohistochemistry on intestinal sections from mice exposed to normal water or two cycles of DSS and killed at day 27. (A) Control (water-treated) WT animals. (B) DSS-treated WT mice. PMN infiltrated (arrows) and destroyed the crypt epithelial layer. (C) DSS-treated CXCR2–/– mice. PMN were mostly confined among the crypts (arrows). (D) DSS-treated WT mice. Magnification of details indicated by arrows in (B). (E) DSS-treated CXCR2–/– mice. Magnification of details indicated by arrows in (C). Original magnification, x400.
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Figure 6. Ultrastructural features of colonic lamina propria from DSS-induced chronic colitis in WT and CXCR2–/– mice. Colon sections from WT and CXCR2–/– mice killed at day 27 after DSS administration. (A, B) WT mice. In the colonic lamina propria, both eosinophils (E) and neutrophils (N) largely extravasated. Endothelial cells are indicated by arrow; vessel lumen indicated by asterisk. Eosinophils and neutrophils spread to the stroma, reaching tight contact with epithelial crypts (indicated by arrows). (C, D) CXCR2–/– mice. PMN extravasated in the colonic lamina propria but did not migrate, remaining in close proximity to microvessel walls (indicated by arrows). PMN stopped and filled microvessels. Bars in A and B = 100 µm. Bars in C and D = 50 µm.
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Time-dependent increase of production of CXCR2 ligands in DSS-treated WT
To further evaluate the different clinical and histopathological course of colitis in WT vs. CXCR2–/– mice, we examined the expression of CXCR2 ligands into colonic mucosa after DSS administration in both WT and CXCR2–/– mice. As shown in Fig. 7A
, CXCL1 expression was almost absent in water-treated WT mice. During the chronic colitis, CXCL1 was consistently expressed in WT mice, peaking at day 27 (13.96±1.73 pg/mg of tissue). CXCL1 mucosal production was further confirmed by confocal microscopy (Fig. 8
). CXCL1 was found to be expressed in the stroma of the lamina propria, especially close to the areas of PMN recruitment (Fig. 8A)
, mainly by (CD11c+) dendritic cells (Fig. 8C)
and (CD11b+) macrophages to a lesser extent (Fig. 8D)
, whereas epithelial cells of the intestinal crypts were mostly negative (Fig. 8B)
. Also CXCL2 was consistently expressed in WT mice, with CXCL2 levels peaking again at day 27 (0.03±0.00 and 6.89±2.16 pg/mg of tissue in water-treated and DSS-treated animals, respectively (Fig. 7B)
.
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Figure 7. Expression of CXCL1 and CXCL2 in the colon of DSS-treated WT mice. WT mice were killed after different times of DSS administration, and colonic mucosa was processed for CXCL1 and CXCL2 detection. Control group received normal water. (A) Colonic production of CXCL1 (**, P<0.01 vs. control group by ANOVA). (B) Colonic production of CXCL2 (**, P<0.01 vs. control group by ANOVA). Data are expressed as means ± SEM (14 animals for each time point).
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Figure 8. Confocal analysis of CXCL1 expression in colonic mucosa of DSS-treated WT mice. Colon sections from WT mice exposed to two cycles of DSS and killed at day 27. (A) Massive influx of PMN (red stained) close to the mucosal areas rich in CXCL1 expression (green stained) without aspects of colocalization, as shown by the merge image. (B) Expression of CXCL1 (green stained) was mainly among the intestinal epithelial (red stained) crypts. (C) Most of dendritic cells (CD11c, red stained) infiltrating the lamina propria produce CXCL1 (green stained), as revealed by the yellow color in the merge image. (D) Macrophages (CD11b, red stained) infiltrating the lamina propria cooperate to CXCL1 production (green stained), as revealed by the yellow color in the merge image. Original magnification, x400.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ligands of CXCR2 were expressed at high levels in experimental colitis of both WT and CXCR2–/– mice, and their production was strongly related to the increase of PMN recruitment into colonic mucosa of WT mice.
To assess the role of CXCR2, we used a model of DSS-induced colitis that closely resembles human UC under several features [30
,31
]. In this model, repeated DSS administration induced epithelial atrophy, extensive loss of mucus production, widespread hyperplasia in large areas of the colonic mucosa, complete loss of crypt epithelium in large tract of mucosa, crypt abscesses, severe ulcers and, most notably, a massive infiltration of PMN into colonic mucosa. Also, from the clinical point of view, morphological changes observed after chronic administration of DSS were paralleled by clinical symptoms induced by repeated DSS administration that closely resemble severe human UC, including diarrhea and gross rectal bleeding [30
]. Moreover, selected drugs currently used in the therapy of UC are also efficacious in this model of repeated DSS administration, including cyclosporin A [32
] and infliximab [21
,33
], a monoclonal antibody anti-TNF- [34
35
36
], further suggesting that the DSS model we used closely resembles the human condition.
In the DSS-induced colitis, both high levels of CXCR2 ligands and a massive recruitment of PMN were evident in WT mice during the chronic phase of disease.
The main histopathological changes and clinical symptoms in the DSS-induced experimental colitis were strongly reduced in CXCR2–/– mice. Lack of functional CXCR2 prevented PMN infiltration in the mucosa, suggesting that PMN recruitment into the gut mucosa is mainly attributable to CXCR2 activation. Inhibition of PMN recruitment was associated with a strong reduction in all histopathological changes associated with experimental colitis, including necrotic and ulcerative lesions in the colonic mucosa. Large areas of mucosa with intact muco-secretive properties were also observed in CXCR2–/– mice, whereas in WT mice, there was a strong reduction of mucus production, and large tracts of mucosa lost all crypt epithelium, leaving in its place granulation tissue. These data are further in keeping with the concept that PMN recruitment and activation in UC are key events in inducing mucosal damage and clinical symptoms. This evidence was apparently in contrast to a previous report [37
], in which the abrogation of PMN has a deleterious effect on DSS-induced colitis. However, these results are referred to a single cycle of DSS administration, an experimental condition that does not resemble UC condition. Indeed, a single cycle of DSS administration induces a pathological state, with a low involvement of inflammatory cells and reduced tissue damage, mainly caused by the toxic effect of DSS on colonic epithelial cells [17
,22
]. In addition, the efficacy of treatment with a monoclonal antibody anti-TNF- and corticosteroids clinically proved in UC was not experimentally observed after a single cycle of DSS [38
].
Whereas intraepithelial infiltration of PMN seems to be mediated mainly by CXCR2, we found that lack of CXCR2 has no effect on intravascular accumulation of leukocytes. These findings suggest that intravascular leukocyte recruitment might be under the control of other leukocyte-activating factors, such as TNF- [20
] and products of the complement system [39
]. On the other hand, the impairment of PMN to cross endothelial surface and subsequently infiltration into the crypt epithelial barrier strongly suggest that PMN recruitment into the mucosa could be mainly dependent upon CXCR2 activity on endothelial and epithelial cells. In keeping with this hypothesis, several reports support the crucial role played by CXCR1 and CXCR2 activity on endothelial and epithelial cell layer in chemokine-mediated PMN arrest and subsequent transmigration into inflamed tissue [40
41
42
43
]. Indeed, PMN of CXCR2–/– mice were unable to cross the epithelial barrier and accumulate in the tissue in an experimental model of infection of the urinary tract [42
], and immunoneutralization of CXCL1 and CXCL2 did not reduce endotoxin-induced leukocyte intravascular accumulation, whereas PMN transmigration and extravascular tissue accumulation was abolished [40
]. Similarly, CXCR1 and CXCR2 overexpression was detected in infected human uroepithelial cell lines, and neutralization of CXCR1 strongly inhibited CXCL8-dependent PMN migration across the epithelial cell layer [42
]. Finally, the crucial role of CXCR2 for transendothelial and transepithelial migration of PMN, but not for their accumulation in the vasculature, was also reported in a murine model of acute lung injury in which PMN of CXCR2–/– mice did not migrate into the lung interstitium following endotoxin aerosolization, remaining into the pulmonary vasculature without inducing lung damage [44
].
Although PMN infiltration is believed to be a key event in DSS-induced colitis, information as to the soluble factors involved in the regulation of PMN recruitment in this model are scanty. Here, we demonstrate that CXCR2 ligands (CXCL1 and CXCL2) are highly expressed in the stroma of the lamina propria, close to the areas of PMN recruitment. Production of these PMN attractants was mainly attributable to mononuclear and dendritic cells in the colonic mucosa. High levels of both CXCR1/R2 expression and CXCR1/2 ligands have also been found in the mucosa of UC patients [45 ].
Taken together, these findings demonstrate that CXCR2 plays a key, nonredundant role in mediating PMN infiltration into the mucosa in an animal model of colitis that closely resembles human UC. Prevention of PMN infiltration was associated with a strong reduction in both histopathological signs of the disease and clinical symptoms. These data may provide the rationale for testing CXCL8 receptors inhibitors as a new therapeutic approach to UC in the clinic.
Received February 19, 2007; revised March 28, 2007; accepted June 21, 2007.
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inhibitor, is effective in the dextran sulphate mouse model of chronic colitis Inflamm. Res. 51,522-531[CrossRef][Medline]
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