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Published online before print May 26, 2006
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,1
* Allergy and Inflammation Research Group and
¶ Gene Targeting Group, Division of Biochemistry and Molecular Biology, and
Cancer and Vascular Biology Group, Division of Immunology and Genetics, The John Curtin School of Medical Research, Australian National University, Canberra;
Asthma, Allergy and Inflammation Research Centre, School of Biomedical Sciences, University of Newcastle, Australia;
|| School of Molecular and Biomedical Science, University of Adelaide, Australia;
** Department of Immunology, Murdoch Childrens Research Institute, Royal Children’s Hospital, Victoria, Australia;
Department of Pediatrics, City Hospital of Dortmund, Germany; and
Division of Allergy and Immunology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Ohio
1Correspondence: Division of Allergy and Immunology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45267. E-mail: Simon.Hogan{at}cchmc.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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L, M, and ß2. Using ICAM-1-deficient mice and anti-ICAM-1 neutralizing antibodies, we show that hapten-induced colonic eosinophilic inflammation is critically dependent on ICAM-1. These studies demonstrate that ß2-integrin/ICAM-1-dependent pathways are integral to eosinophil recruitment into the colon during GI inflammation associated with colonic injury.
Key Words: eosinophils • adhesion molecules • gastrointestinal tract
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Numerous inflammatory mediators have been implicated in regulating eosinophil accumulation, including interleukin (IL)-1, -3, -4, -5, and -13 and granulocyte macrophage-colony stimulating factor (GM-CSF) and the chemokines regulated on activation, normal T expressed and secreted (RANTES), monocyte chemoattractant protein (MCP)-3, macrophage inflammatory protein-1, and eotaxin-1, -2, and -3 [5
, 6
]. Of the mediators implicated in modulating eosinophil accumulation, eotaxin-1 appears to be the most important molecule in the regulation of eosinophil trafficking into the GI tract [7
]. Eotaxin-1 is ubiquitously expressed in all segments of the GI tract [8
]. Furthermore, eosinophil levels in the GI tract are reduced significantly in eotaxin-1-deficient mice as compared with wild-type (WT) mice, and overexpression of eotaxin-1 in the GI tract promotes a pronounced eosinophilia in the small intestine [9
, 10
]. Clinical studies have demonstrated increased expression of eotaxin-1 in EGID, including cows milk-associated reflux esophagitis and UC [11
12
13
14
15
].
The transmigration of leukocytes, such as eosinophils, across the vascular epithelium into mucosal tissues, is regulated by coordinated interaction among networks involving chemokine and cytokine signaling, eosinophil adhesion molecules (e.g., selectins and integrins), and integrin receptors [e.g., vascular cell adhesion molecule-1 (VCAM-1), mucosal addressin cell adhesion molecule-1 (MAdCAM-1), and intercellular adhesion molecule 1 (ICAM-1)] expressed on vascular endothelial cells [16
, 17
]. Integrins are heterodimeric surface molecules consisting of an - and ß-chain, and eosinophils express members of the ß1 (4ß1 and 6ß1)-, ß2 (Lß2, Mß2, Xß2, and Dß2)-, and ß7 (4ß7)-integrin families [18
19
20
21
22
]. These various integrin molecules interact selectively with adhesion receptors (VCAM-1, MAdCAM-1, ICAM-1, -2, and -3, and fibrinogen) expressed on the vascular endothelium. 4ß1 selectively binds to VCAM-1 and fibronectin, and 6ß1 is the primary ligand for the extracellular matrix protein laminin. The 4ß7-integrin selectively binds to MAdCAM-1, and lymophocyte function-associated antigen-1 (LFA-1; Lß2) and membrane-activated complex-1 (MAC-1; Mß2) bind to ICAM-1. In addition, LFA-1 (Lß2) can bind ICAM-2 and -3, and Dß2 has been shown to bind VCAM-1 and ICAM-3. Leukocyte integrin/intercellular adhesion molecule interactions, particularly LFA-1/ICAM-1 and very late antigen (VLA)-4/VCAM-1, are regulated by cytokines and chemokines such as IL-1 ( and ß), tumor necrosis fator (TNF; and ß), IL-4, and IL-13 [23
]. IL-1 and TNF stimulate both VCAM-1 and ICAM-1 expression on a variety of cell types. By contrast, IL-4 and IL-13 selectively enhances the expression of VCAM-1. Chemokines also alter the activation state and selectivity of adhesion molecules [24
, 25
]. For example, treatment of eosinophils with MCP-3, RANTES, or eotaxin-2 switches eosinophils from a ß1-integrin/VCAM-1-dominant to a ß2-integrin/ICAM-1-dominant, interacting cell [24
].
The specific interaction of cell surface integrins with adhesion receptors (VCAM-1, MAdCAM-1, ICAM-1, ICAM-2, ICAM-3, and fibrinogen) facilitates eosinophil migration into various tissue compartments during inflammation. For example, eosinophil recruitment to the site of allergic inflammation in the lung and skin is regulated by a VLA-4 (4ß1-integrin)/VCAM-1-dependent processes [26
27
28
29
30
]. Pretreatment of mice with neutralizing monoclonal antibodies (mAb) against 4- or ß1-integrin or genetic deletion of VCAM-1 attenuates eosinophil accumulation in the lung during allergic airways disease [26
27
28
29
30
]. In contrast, eotaxin-1-dependent eosinophil recruitment to the small intestine of the GI tract is MAdCAM-1/4ß7-integrin-dependent [10
].
Recruitment of inflammatory cells into the GI tract is currently believed to occur via an 4ß7/MAdCAM-1-dominant interaction [16
]. The 4ß7-integrin receptor MAdCAM-1 is expressed primarily on GI vascular endothelium and is generally absent on nonintestinal venules and most non-GI sites of inflammation [31
]. Blockade of 4ß7/MAdCAM-1 interactions by neutralizing mAb or genetic deletion inhibits T and B cell and mast cell recruitment into GI compartments including the small intestine, mesenteric lymph nodes, and Peyer’s patches. It is interesting that recent experimental investigations have demonstrated that leukocyte recruitment into the large intestine can occur via a ß7-integrin-independent mechanism, suggesting that leukocytes use different adhesion systems to infiltrate various GI compartments [32
, 33
]. We were therefore interested in identifying the dominant adhesion complex involved in eosinophil trafficking into the large intestine. This is particularly important, as there are numerous diseases characterized by eosinophil accumulation in the colon (e.g., allergic colitis and IBD), yet most studies have concentrated on the upper GI tract (e.g., esophagus and small intestine).
In the present study, we demonstrate that colonic eosinophils express the ICAM-1 ligands [MAC-1 and LFA-1 (ß2, L, and M)]. Furthermore, using in vivo models of colonic eosinophil inflammation, we demonstrate that eosinophil accumulation in the colon is regulated by a ß2-integrin pathway (ICAM-1) and can occur independently of 4- and ß7-integrin-independent pathways. This observation has significant implications for the treatment of disease states characterized by colonic eosinophilic inflammation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fluorescein-activated cell sorter (FACS) analysis
CD2-IL-5Tg mice were killed and spleens were removed, and a single-cell suspension was prepared into single-cell suspensions as described previously [34
]. To analyze integrin expression on splenic and colonic eosinophils, cells were incubated with phycoerythrin (PE)-conjugated anti-4ß7 (Clone DATK32, BD PharMingen, San Diego, CA; 1 µg/106 cells), biotin-conjugated anti-m (Clone M1/70 15.1, Chemicon Europe CBL, UK), biotin-conjugated anti-L (Chemicon Europe CBL), PE-conjugated anti-ß2-integrin (C71/16; BD PharMingen), Alexa Fluor 647 rat anti-mouse CC chemokine receptor 3 (CCR3; 83103; BD PharMingen), or isotype-matched control immunoglobulin (Ig; rat IgG biotin, Southern Biotechnology, Birmingham, AL) in phosphate-buffered saline (PBS)/1% fetal calf serum (FCS) on ice for 30 min and washed twice in PBS/1% FCS. To visualize binding of the anti-integrin antibodies, cells were incubated with streptavidin-fluorescein isothiocyanate (FITC; 1:1600 dilution) in PBS/1% FCS on ice for 30 min and washed twice in PBS/1% FCS. Cells were analyzed by flow cytometry on a FACS advantage SE cell sorter (BD Biosciences, San Jose, CA). Eosinophils were identified by forward-scatter (FSC) versus side-scatter (SSC) and polarizing light as described previously [35
].
Induction of colonic injury
Dextran sulfate sodium (DSS) used for the induction of experimental colitis (ICN Biomedical Inc., Aurora, OH) was supplied as the sodium salt with an average molecular weight of 41 kDA. It was used as a supplement in the drinking water of the mice for 8 days as a 2.5% (w/v) solution.
Disease activity index (DAI)
DAI was derived by scoring three major clinical signs (weight loss, diarrhea, and rectal bleeding) [36
]. The clinical features were scored separately and then correlated with a histological score. DAI = (body weight change) + (diarrhea score) + (rectal bleeding score).
Body weight
Changes in body weight were calculated as the difference between the predicted body weight and the actual weight on a particular day. The formula for predicted body weight was derived by simple regression using the body weight data for the control group. The following formula was used: Y = a + kx, where Y = body weight change (loss or gain), k = daily increase in body weight, x = day, and a = starting body weight.
Diarrhea
The appearance of diarrhea was defined as mucus/fecal material adherent to anal fur. The presence or absence of diarrhea was scored as 1 or 0, respectively. The presence or absence of diarrhea was confirmed by examination of the colon following completion of the experiment [36
]. Mice were killed, and the colon was excised from the animal. Diarrhea was defined by the absence of fecal pellet formation in the colon and the presence of continuous fluid fecal material in the colon.
Rectal bleeding
The appearance of rectal bleeding was defined as diarrhea containing visible blood or gross rectal bleeding and scored as described for diarrhea.
Histopathological examination
Animals were killed on Day 8, and the colon was excised. The length of the colon was measured using digmatic calipers (Mitutoyo, Kawasaji, Japan). Tissue specimens were fixed in 4% paraformaldehyde and stained with hematoxylin and eosin (H/E) and Masson’s trichrome using standard histological techniques. The percentage of colon length with mucosal ulceration was determined by performing morphometric analysis of colon using the ImageProPlus 4.5 software package (Media Cybernetics, Inc., Silver Spring, MD). In brief, digital images of longitudinal sections (1–2 cm in length) of H/E-stained colons were produced. Using the ImageProPlus 4.5 software, the length of ulcerated mucosal lining was divided by the total length of the colonic mucosal surface, and the value was expressed as a percentage of colon length with mucosal ulceration.
Detection and quantification of eosinophils by immunohistochemistry
The colon segment of the GI tract was immunostained with antiserum against mouse major basic protein (MBP) as described previously [8
]. Briefly, 5 µm sections were quenched with H2O2, blocked with normal goat serum, and stained with a rabbit antimurine eosinophil MBP antiserum (kind gift from Nancy and James Lee, Mayo Clinic, Scottsdale, AZ). The slides were then washed and incubated with biotinylated goat anti-rabbit antibody and avidin-peroxidase complex (Vectastain ABC Peroxidase Elite kit, Vector Laboratories, Burlingame, CA). The slides were developed by nickel diaminobenzidine and enhanced cobalt chloride to form a black precipitate and counterstained with Nuclear Fast Red. Quantification of eosinophils was performed by counting the number of immunoreactive cells from 15–25 fields of view (magnification x40) from at least four to five random sections/mouse. Values were expressed as eosinophils per mm2 tissue.
Eosinophil peroxidase (EPO) activity assay
Mice were killed on Day 8, and the colon was excised and flushed with 1 ml PBS solution. The fecal material was vortexed vigorously for 5 min at 4°C and centrifuged at 10,000 g for 10 min at 4°C. The supernatant was collected and placed in a sterile Eppendorf tube and stored at –70°C until analysis. EPO activity was measured in the supernatant of cell-free colon flushes as described previously [37
]. This assay is based on the oxidation of o-phenylenediamine (OPD) by EPO in the presence of H2O2. The EPO substrate solution consisted of 12 mM OPD (Sigma Aldrich, St. Louis, MO), 0.005% H2O2, 10 mM HEPES, and 0.22% cetyltrimethylammonium bromide (CTAB). Substrate solution (75 µl) was added to cell-free supernatants, which were derived from colon flushes (75 µl) in a 96-well microplate and incubated at room temperature for 15 min before stopping the reaction with 50 µl cold 8 N sulfuric acid. Absorbance was measured at 490 nm. Standard EPO activity, 100 U/ml, was determined, as EPO activity produced by 1 x 106-purified eosinophils/µl supernatants purified from the spleen of CD2-IL-5Tg mice as described previously.
mAb treatment
Mice were injected intraperitoneally (i.p.), daily, with rat anti-mouse integrin 4-chain [200 µl 1 mg/ml Clone PS/2 (IgG2b) mAb in saline], anti-mouse ICAM-1 [200 µl 1 mg/ml Clone YN1/1.7.4 (IgG2b) mAb in saline], or rat IgG control antibody (200 µl 1 mg/ml ßGL113 mAb in saline) for 4 days. Three hours following the final i.p. injection, mice were killed, the jejunum and colon were excised and fixed in 4% paraformaldehyde and stained for anti-MBP, and eosinophil levels were quantitiated as described above. In some experiments, mice were injected i.p. daily throughout the 8-day DSS treatment protocol with anti-mouse ICAM-1 [200 µl 1 mg/ml Clone YN1/1.7.4 (IgG2b) mAb in saline] or rat IgG control antibody (200 µl 1 mg/ml ßGL113 mAb in saline).
FACS analysis on colonic eosinophils
Colonic injury was induced with 2.5% DSS as described above. On Day 8, the colon segment of the GI tract was removed and flushed with 20 ml PBS. The colon tissue was cut into 1-cm segments and incubated in digestion buffer containing 48 mg/ml Collagenase A, 24 U/ml Dispase II, and 2.5 mg/ml DNase in RPMI 1640 and incubated for 60 min at 30°C. The tissue was vortexed vigorously every 10 min for 15 s. Following the 60-min incubation, the cell aggregates were dissociated by pipetting and centrifuged at 1200 revolutions per minute for 10 min at 4°C. The supernatant was decanted, and the cell pellet was resuspended in RPMI 1640 + 10% FCS and filtered through a 70-µm filter. The single-cell suspension was prepared by Ficoll density gradient centrifugation and quantitated by trypan blue exclusion analysis. Colonic cells (1x106) were plated in a 96-well round-bottom plate, and flow cytometry analysis staining for CCR3 and ß2-integrin was performed as described above.
Myeloperoxidase (MPO) activity
MPO activity, a marker of polymorphonuclear neutrophil granules, was assessed in colonic luminal contents according to the Bradley method [38
]. Mice were killed on Day 8, and the colon was excised and flushed with 1 ml PBS solution. The fecal material was vortexed vigorously for 5 min at 4°C and centrifuged at 10,000 g for 10 min at 4°C. The supernatant was collected and placed in a sterile Eppendorf tube and stored at –70°C until analysis. MPO activity was measured in the supernatant of cell-free colon flushes as described previously [38
]. This assay is based on the oxidation of o-dianisidine dihydrochloride (oDd) by MPO in the presence of H2O2. The MPO substrate solution consisted of 0.167 mg/ml oDd (Sigma Aldrich), 0.005% H2O2, 10 mM HEPES, and 0.22% CTAB. Substrate solution (75 µl) was added to cell-free supernatants, which were derived from colon flushes (75 µl) in a 96-well microplate and incubated at room temperature for 15 min before stopping the reaction with 50 µl cold 3 M sulfuric acid. Absorbance was measured at 450 nm. Standard MPO (human leukocytes, Sigma Aldrich; 50 U/mg protein), 10 U/ml, was used to generate a standard curve.
Statistical analysis
The significance of differences between the means of experimental groups was analyzed using Student’s unpaired t-test. Values were reported as the mean ± SEM. Differences in mean values were considered significant if P < 0.05. In some experiments, one-way and two-way ANOVA with Bonferroni post-test were performed as indicated. All these calculations were performed using SAS, Version 9.0 (SAS Institute, Cary, NC). To test the hypotheses, that the means were equal, one-factor or two-factor ANOVA was used, and the level of significance was at 0.05. In the one-factor analyses, the comparison groups were the treated groups. If the overall F were statistically significant, Bonferroni’s multiple comparison was applied to determine where the differences were. In the case of two factors, strain of mice and treatment, if the overall F were less than 0.05, Bonferroni’s adjustment was made, dividing the F by the number of comparisons made to adjust the P value.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous investigations have demonstrated that VLA-4 (4ß1) is important in eosinophil recruitment during inflammatory responses [26
27
28
29
30
]. To examine the role of the VLA-4/VCAM-1 pathway in eosinophil recruitment into the colon, ß7-integrin–/– mice were administered anti-4-integrin mAb (PS/2 mAb) or control Ig and exposed to 2.5% DSS in drinking water or drinking water alone for 8 days, and markers of eosinophilic inflammation in the colon were examined. DSS treatment of ß7-integrin–/– mice administered control Ig significantly increased colonic eosinophil numbers and EPO activity as compared with control-treated ß7-integrin–/– mice administered control Ig (Fig. 2a
). Similarily, DSS treatment of ß7-integrin–/– mice administered with anti-4-integrin mAb significantly increased colonic eosinophil levels, as compared with control-treated ß7-integrin–/– mice administered anti-4-integrin mAb (Fig. 2a)
. It is notable that the level of eosinophils and EPO activity in the colon of DSS-treated ß7-integrin–/– mice administered with anti-4-integrin mAb was equivalent to that observed in DSS-treated ß7-integrin–/– mice administered control Ig mAb (Fig. 2b)
. Collectively, these studies suggest that the VLA-4/VCAM-1 and ß7-integrin pathways do not play a major role in eosinophil trafficking into the colon under inflammatory conditions related to colonic injury.
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4- and ß7-integrins, we were next interested in elucidating the involvement of the ß2-integrin members [LFA-1 (Lß2) and MAC-1 (Mß2)]. ß2-Integrin members have previously been shown to be involved in eosinophil recruitment into the lung during allergic pulmonary disease [30
, 39
, 40
]. Initially, we examined the surface expression of ß2-integrin members (L, M, and ß2) on splenic eosinophils from CD2-IL-5Tg mice. Eosinophils were analyzed by flow cytometry based on FSC-H versus SSC-H (Fig. 3a
). To confirm that the identified cell population was eosinophils, the gated cells were sorted by flow cytometry, cytocentrifuged, and stained with chromotrope 2R. The sorted cells (95–99%) were identified as eosinophils by histological analysis (Fig. 3b)
. Splenic eosinophils from CD2-IL-5Tg mice expressed ß2-, 4-, M-, and L-integrins (Fig. 3c
3d
3e
3f)
. Collectively, these studies demonstrate that peripheral eosinophils express the ICAM-1-binding LFA-1 (Lß2) and MAC-1 (Mß2) integrin subunits.
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70% of ß2-integrin+ cells were CCR3+, suggesting that colonic eosinophils expressed ß2-integrin (results not shown). To identify whether CCR3+ eosinophils from the colon of hapten-treated WT mice express L- and M-integrins, we examined L and M expression on ß2-integrin+ CCR3+ eosinophils. We demonstrate that all ß2-integrin+ CCR3+ cells expressed L- and M-integrins (Fig. 4c
and 4d)
. To exclude the possibility that the CCR3+ cells could be mast cells, we performed chloroacetate esterase staining on the colon of control- and DSS-treated WT mice and quantified mast cell levels. We observed no significant increase in mast cell levels in the colon following DSS treatment (results not shown). Collectively, these studies demonstrate that DSS treatment promotes the recruitment of ß2-integrin+ CCR3+ eosinophils into the colon. Furthermore, these studies suggest that colonic eosinophils express the ICAM-1 counter-receptors, LFA-1 (Lß2) and MAC-1 (Mß2) integrins.
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L-, M-, and 2-integrins on eosinophil recruitment into the colon at baselineLß2) interacts with a number of counter-receptors including ICAM-1, ICAM-2, ICAM-3, and fibrinogen, and MAC-1 (Mß2) can bind to ICAM-1 and the iC3b component of complement, the ß2-integrin (LFA-1 and MAC-1) principal receptor is ICAM-1 [46
47
48
]. Overexpression of IL-5 in T cells promotes a pronounced colonic eosinophilic inflammation. Treatment of CD2-IL-5Tg mice with anti-ICAM-1 neutralizing mAb significantly reduced the colonic eosinophilic infiltrate induced by the IL-5Tg [eosinophils/HPF; 176.9±3.8 vs. 101.1±9.3; CD2-IL-5Tg mice+Ig control vs. CD2-IL-5Tg mice+anti-ICAM-1 mAb; mean±SEM (n=3 and 5 mice per group, respectively; P<0.05)], indicating that ICAM-1-binding integrins (ß2-integrins) are involved in the constitutive homing of eosinophils into the colon.
The effect of blockade of the ICAM-1 pathway on eosinophil recruitment into the colon during colonic injury
To examine the role of the ß2-integrin/ICAM-1 pathway in the migration of eosinophils into the colon during colonic injury, we employed WT and ICAM-1–/– mice. Administration of DSS to WT mice induced a colonic eosinophilic infiltrate (Fig. 5a
and 5b
). In contrast, hapten-induced eosinophilic inflammation in ICAM-1–/– DSS-treated mice was significantly attenuated as compared with WT DSS-treated mice (Fig. 5b)
. Consistent with the reduction in eosinophil numbers in the absence of ICAM-1, luminal EPO activity was also reduced significantly in DSS-treated ICAM-1–/– mice as compared with WT mice (Fig. 5c)
. To demonstrate that this was mediated by an ICAM-1-dependent process, we challenged L-selectin–/– mice with DSS (Fig. 5c)
. DSS-induced eosinophil recruitment and EPO activity in L-selectin–/– mice were comparable with that observed in strain-matched WT mice (Fig. 5b
and 5c)
, suggesting that the observed attenuation of eosinophilic inflammation was associated specifically with the ICAM-1 pathway. To exclude the possibility that inherit compensatory mechanisms associated with ICAM-1 gene deficiency can account for the ablation of eosinophil recruitment into the colon in ICAM-1–/– mice, we administered DSS-treated WT mice with Ig control or anti-ICAM-1 neutralizing mAb and examined colonic eosinophil levels. Administration of DSS to control Ig-treated WT mice induced a colonic eosinophilic inflammation (Fig. 5d)
. Eosinophil levels in the colon and the level of EPO in the lumen of the colon of these mice were elevated significantly compared with control-challenged anti-ICAM-1-treated mice (Fig. 5d
5e)
. In contrast, eosinophil and EPO levels in the colon of DSS-administered anti-ICAM-1-treated mice were reduced significantly as compared in DSS-challenged, control Ig-treated WT mice (Fig. 5d
5e)
. These studies confirm that the ß2-integrin/ICAM-1 pathway is critical in the transmigration of eosinophils in the colon during colonic injury. To demonstrate that the observed ablation of eosinophil recruitment into the colon by ICAM-1 blockade is directly a result of inhibition of eosinophil transmigration and not a result of the suppression of the recruitment of other inflammatory cells, we examined neutrophil levels in hapten-treated ICAM-1–/– mice. Neutrophils and not B and T cells have been shown to be important in the inflammatory response observed in hapten-induced colonic injury [49
50
51
]. To quantitate neutrophil levels, we examined MPO activity in colon luminal washes from DSS-treated mice. To block residual EPO activity, we performed the MPO assay in the presence of an EPO-specific inhibitor resorcinol (60 µM) [34
, 37
]. DSS administration to WT mice promoted an increase in luminal MPO activity as compared with control-treated WT mice (Fig. 6
). Similarly, administration of DSS to ICAM-1–/– mice induced a significant and comparable increase in luminal MPO activity, demonstrating no reduction in neutrophil recruitment into the colon in the absence of ICAM-1 (Fig. 6)
. The level of MPO activity is DSS-treated WT, and ICAM-1–/– mice were not reduced significantly in the presence of the EPO inhibitor resorcinol [0, 1, 10 (results not shown) and 60 µM; Fig. 6
]. These studies suggest that the ß2-integrin/ICAM-1 pathway is involved directly in eosinophil but not neutrophil recruitment into the colon during colonic injury.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4ß7-integrin and that the ß7-pathway is critical for eotaxin-1-mediated eosinophil recruitment into the small intestine [10
]. In this study, we have examined the adhesion systems that regulate eosinophil recruitment into the large intestine and have demonstrated that eosinophil recruitment into the colon during colonic injury occurs via an 4- and ß7-integrin-independent pathway; that murine splenic eosinophils express the ligands for ICAM-1 (ß2, L, and M); eosinophils (CCR3+) isolated from colon of hapten-treated mice express the ICAM-1 counter-receptor integrins (ß2, L, and M); and blockade of ICAM-1 activity by gene knockout or by neutralizing mAb attenuated eosinophil recruitment into the colon. Collectively, these studies suggest that the ICAM-1 adhesion pathway and not VCAM-1 and 4ß7/MAdCAM-1 pathways is important for the development of colonic eosinophilic inflammation.
Leukocyte recruitment into the GI tract and gut-associated lymphoid tissue are thought to be critically regulated by ß7-integrin [31
, 32
, 59
, 60
]. Indeed, the expression of MAdCAM-1 and 4ß7 expression on leukocytes in the intestinal mucosa is up-regulated significantly in GI diseases, including cows milk allergy and food allergy [61
, 62
]. Recently, experimental studies have demonstrated that leukocyte recruitment into the large intestine can occur in a ß7-integrin-independent manner [32
, 33
, 60
]. Moreover, no impairment in CD4+ T cell and mast cell infiltration into the colon of ß7-integrin–/– mice was observed following helminth infection [32
]. Furthermore, the concentration of mast cell progenitors in the large intestine of ß7-integrin–/– mice was not reduced as compared with WT mice [60
]. We demonstrate that eosinophil recruitment into colon during inflammatory conditions occurs predominantly via a ß7-integrin-independent mechanism, through a ß2-integrin/ICAM-1 pathway. The contribution of the ß2-integrin pathway in leukocyte and mast cell recruitment into the colon are not yet fully elucidated.
In our previous investigations, we have demonstrated that eosinophil recruitment into the colon during colonic injury is critically regulated by the CCR3 ligand, eotaxin-1 [34
]. It is notable that eotaxin-1 up-regulates the expression of ICAM-1-binding integrins L and m expression on lymphocytes [63
]. Furthermore, we have recently demonstrated that in vitro stimulation of splenic eosinophils with eotaxin-1 in the presence of IL-5 up-regulates the surface expression of L- and M-integrins on eosinophils (results not shown). In light of these findings and our demonstration of a role for the ß2-integrin/ICAM-1 pathway in eosinophil recruitment, it is tempting to speculate that eotaxin-1 preferentially promotes ß2-integrin/ICAM-1 interactions and eosinophil recruitment into the colon. In different disease states, elevated eosinophil numbers are often restricted to specific GI compartments. For example, EE is characterized by elevated level of eosinophils restricted to the esophagus, whereas, eosinophilic colitis and UC are characterized by elevated levels of eosinophils in the colon without increased numbers in other GI compartments. Paradoxically, the recruitment of eosinophils into these various GI compartments (small and large intestine) is thought to be primarily regulated by eotaxin-1 [17
]. How eotaxin-1 selectively orchestrates the trafficking of eosinophils into specific GI compartments remains unclear. It is possible that eotaxin-1 selectively up-regulates L-, M-, and ß2-integrin expression on eosinophils and the ß2-integrin counter-receptor, ICAM-1, on colonic microvascular endothelial cells to promote eosinophil infiltration into the colon. Indeed, eotaxin-1 has been shown to stimulate expression of ICAM-1 on microvascular endothelial cells [64
]. However, as we demonstrate that L-, M-, ß2-integrins are expressed on eosinophils under noninflammatory conditions, the differential expression of integrins cannot fully account for the eotaxin-1-mediated selectivity for the ICAM-1 pathway. An alternate explanation is that chemokines such as eotaxin-1 differentially regulate integrin/adhesion molecule avidity, promoting ß2/ICAM-1-dependent adhesion pathway interaction. Weber and colleagues [65
] have demonstrated that RANTES and MCP-3, eotaxin-1 receptor (CCR3) agonists, can selectively down-regulate eosinophil VLA-4/VCAM-1 adhesion and promote ß2/ICAM-1 adhesion. Furthermore, they showed that the differential regulation of adhesion occurred independently of integrin surface expression and directly involved modulation of integrin avidity [65
]. We postulate that eotaxin-1-mediated recruitment involves a complex step of events involving integrin expression on leukocytes (increased L, M, ß2), adhesion molecule expression on microvascular endothelium (ICAM-1), and increased integrin and integrin receptor avidity (LFA-1/ICAM-1, MAC-1/ICAM-1).
The histological presence of eosinophils in the GI mucosa of patients with IBD has long been recognized; however, the contribution eosinophils make to disease pathogenesis is still not well-understood. Recent clinical investigations of bowel biopsy specimens from UC patients have shown a correlation among the eosinophil numbers in the mucosa, the levels of eosinophil-derived granule proteins (MBP, EPO, eosinophil cationic protein, and eosinophil-derived neurotoxin) in perfusion fluid samples, and disease severity [14 , 56 , 57 , 66 ]. Previously, we have demonstrated that eosinophils mediate disease pathogenesis in experimental colitis through EPO-dependent pathways [34 ]. EPO catalyzes the oxidation of halides and pseudohalides (Cl–, Br–, and SCN–) with the products of respiratory burst (O2 and H2O2) to generate cytotoxic oxidants. Elevated levels of reactive oxygen species (H2O2) have been reported in mucosal tissue samples from patients with UC [67 , 68 ]. Furthermore, H2O2 has been shown to promote an up-regulation of ß2-integrin expression on eosinophils [69 ].
Using L-selectin–/–-deficient mice, we demonstrate that DSS-induced eosinophil recruitment and EPO activity could occur independently of L-selectin. It is notable that eosinophil levels in DSS-treated L-selectin–/–-deficient mice were comparable with WT. However, although EPO activity levels were elevated significantly in DSS-treated L-selectin–/–-deficient mice, the EPO activity levels were reduced by 35%, as compared with DSS-treated WT mice. These studies suggest that eosinophil release of EPO is at least in part dependent on L-selectin, which binds cell-surface carbohydrates including sialylated, fucosylated, and sulfated lactosaminoglycans such as the sialyl LewisX tetrasaccharides. It is notable that liposaccharides of enteric pathogens have been shown to express LewisX antigens. It is possible that during DSS-induced colonic injury, activation of L-selectin via cell-surface carbohydrates on enteric bacteria promotes eosinophil degranulation and release of EPO. Consistent with this notion, eosinophil granule proteins have been shown to possess bactericidal activity [70
, 71
].
In the present study, we also examined the role of ICAM-1 in the recruitment of neutrophils into the colon during hapten-induced colonic injury. To study neutrophil levels, we examined MPO activity, a marker of polymorphonuclear neutrophil granules in the colon of DSS-treated WT and ICAM-1–/– mice. Consistent with previous investigations, we show that hapten treatment enhances colonic neutrophil levels. Furthermore, we show no difference in MPO activity between DSS-treated WT and ICAM-1–/– mice. To confirm that contaminating EPO activity was not contributing to the peroxidase activity, we performed the assay in the presence of an EPO inhibitor, resorcinol (60 µM), which has been shown to selectively inhibit EPO activity without neutralizing MPO [37
, 72
]. These studies suggest that neutrophil recruitment during hapten-induced colonic injury is mediated via ICAM-1-independent mechanisms. Consistent with this observation, Krieglstein and colleagues [49
], using an identical model to what we have described in this investigation, have demonstrated that monocyte and neutrophil (measured by MPO activity) accumulation into the colon is regulated by an 1ß1-integrin-dependent pathway [49
].
It is notable that although we observed a significant attenuation in physical symptoms and histopathological features of disease in DSS-treated ICAM-1–/– mice as compared with matched WT mice, we still observed a significant weight-loss over the course of the experimental regime. We cannot fully account for the observed weight-loss in DSS-treated ICAM-1–/– mice. However, although eosinophils and EPO levels are significantly attenuated, the level of eosinophils and EPO activity is two- to threefold higher than that observed at baseline in ICAM-1–/– control mice. It is possible that this residual level of eosinophils and EPO activity may contribute to the weight-loss, or alternatively, weight-loss may be driven by concurrent pathways, independent of ICAM-1, eosinophils, and EPO.
Clinical and experimental studies have provided evidence for a role for ICAM-1 in UC [73
74
75
76
]. ICAM-1 levels are elevated in sonicated colonic tissue samples from UC patients, as compared with control patients, and in the colon of mice experimental colitis [73
]. Furthermore, blockade of ICAM-1 function by neutralizing mAb or by antisense oligonucleotide against ICAM-1 ameliorates experimental colitis [74
75
76
]. To our knowledge, this is the first demonstration of a critical role for ICAM-1 adhesion pathway in eosinophilic accumulation in the colon during colonic injury. These findings are particularly important, given 1) that activated eosinophils and eosinophil-derived granular proteins have been linked to the pathogenesis of diseases characterized by colonic injury, such as UC in humans and that therapeutic approaches targeting ICAM-1 including as antisense ICAM-1 oligonucleotide (ISIS-2302) therapy, are being examined for the treatment of IBD [77
78
79
]; and 2) that recent clinical trials examining the use of a humanized antibody to 4ß7 as a therapeutic treatment for IBD, particularly UC [80
], suggest the involvement of adhesion pathways, independent of 4ß7 in the regulation of cellular recruitment in UC.
In conclusion, we have shown that eosinophils express ICAM-1-binding integrins (ß2, L, and M) and that recruitment into the colon occurs via an ICAM-1-dependent and not ß7-integrin-dependent pathway. Furthermore, we demonstrate a pathogenic role for eosinophils and ICAM-1-mediated adhesion pathways in the development of experimental colitis. These studies highlight the importance of eosinophils in GI diseases and suggest that antagonism of ICAM-1/eosinophil pathways may be a significant, therapeutic approach for the treatment of UC and allergic colitis.
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ACKNOWLEDGEMENTS |
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Received November 9, 2005; revised March 14, 2006; accepted March 27, 2006.
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REFERENCES |
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ß2 Integrin is expressed on human eosinophils and functions as an alternative ligand for vascular cell adhesion molecule 1 (VCAM-1) J. Exp. Med. 188,2187-2191 6 ß 1, very late activation antigen-6) on human eosinophils Blood 82,2872-2879 4-Integrins mediate antigen-induced late bronchial responses and prolonged airway hyperresponsiveness in sheep J. Clin. Invest. 93,776-787[Medline]
