Gastroenterology
Volume 132, Issue 3 , Pages 955-965, March 2007

CD40–CD40 Ligand Mediates the Recruitment of Leukocytes and Platelets in the Inflamed Murine Colon

  • Thorsten Vowinkel

      Affiliations

    • Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana
    • Department of General Surgery, University Hospital Münster, Münster, Germany
    • T. Vowinkel and C. Anthoni contributed equally to this work.
    • ,
  • Christoph Anthoni

      Affiliations

    • Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana
    • Department of General Surgery, University Hospital Münster, Münster, Germany
    • T. Vowinkel and C. Anthoni contributed equally to this work.
    • ,
  • Katherine C. Wood

      Affiliations

    • Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana
    • ,
  • Karen Y. Stokes

      Affiliations

    • Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana
    • ,
  • Janice Russell

      Affiliations

    • Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana
    • ,
  • Laura Gray

      Affiliations

    • Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana
    • ,
  • Sulaiman Bharwani

      Affiliations

    • Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana
    • ,
  • Norbert Senninger

      Affiliations

    • Department of General Surgery, University Hospital Münster, Münster, Germany
    • ,
  • J. Steven Alexander

      Affiliations

    • Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana
    • ,
  • Christian F. Krieglstein

      Affiliations

    • Department of General Surgery, University Hospital Münster, Münster, Germany
    • ,
  • Matthew B. Grisham

      Affiliations

    • Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana
    • ,
  • D. Neil Granger

      Affiliations

    • Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana
    • Corresponding Author InformationAddress reprint requests to: D. Neil Granger, PhD, Department of Molecular & Cellular Physiology, LSU Health Sciences Center, 1501 Kings Highway, Shreveport, Louisiana 71130-3932. fax: (318) 675-6005.

Received 21 February 2006; accepted 27 November 2006. published online 19 December 2006.

Article Outline

Background & Aims: Although the CD40–CD40 ligand (CD40L) signaling pathway has been implicated in the pathogenesis of a variety of diseases, including inflammatory bowel disease, the nature of its contribution to intestinal inflammation remains poorly understood. The aim of this study was to determine whether CD40–CD40L contributes to the intestinal inflammatory response, tissue injury, and disease activity elicited by dextran sodium sulphate (DSS) through the modulation of leukocyte and platelet recruitment in the colonic microvasculature. Methods: Wild-type (WT), CD40−/−, and CD40L−/− mice were fed DSS drinking water. On day 6, intravital videomicroscopy was performed to monitor leukocyte and platelet recruitment in colonic venules, with measurements obtained for tissue myeloperoxidase and histology. CD40 expression on colonic endothelium was measured using the dual-radiolabeled antibody technique. Results: A comparison of the responses to DSS-induced colitis in CD40−/− and CD40L−/− mice to WT mice revealed a significant attenuation of disease activity and histologic damage, as well as profound reductions in the recruitment of adherent leukocytes and platelets in the mutant mice. Similar down-regulation of the blood cell recruitment responses to DSS was noted in WT mice treated with the CD40–CD40L pathway inhibitor Trapidil. CD40 expression in the colonic vasculature was greatly elevated during DSS-induced inflammation in WT mice, but not in CD40−/− mice. Conclusions: These findings implicate CD40–CD40L in the pathogenesis of DSS-induced intestinal inflammation, and suggest that modulation of leukocyte and platelet recruitment by activated, CD40-positive endothelial cells in colonic venules may represent a major action of this signaling pathway.

Abbreviations used in this paper: CD40−/−, CD40 knockout, CD40L, CD40 ligand, CD40L−/−, CD40L knockout, DAI, disease activity index, DSS, dextran sodium sulphate, HIMEC, human intestinal microvascular endothelial cells, IBD, inflammatory bowel disease, ICAM-1, intercellular adhesion molecule-1, mAb, monoclonal antibody, MPO, myeloperoxidase, VCAM-1, vascular cell adhesion molecule-1, WT, wild type

 

CD40, a member of the tumor necrosis factor receptor family, is widely distributed but primarily found on cells of the vasculature, where it is constitutively expressed on the surface of endothelial cells. In vivo measurements of CD40 expression in the vasculature have revealed significant organ-to-organ differences in the density of CD40 expression on endothelial cells, both under resting conditions and following endotoxin treatment.1 The ligand for CD40 (CD154 or CD40L) is found on cells of the immune system (eg, T-lymphocytes, mast cells) and on activated platelets. The engagement of CD40L with its receptor (CD40) on endothelial cells induces a proinflammatory phenotype that is characterized by the release of proinflammatory cytokines, enhanced secretion of chemokines, and an increased expression of adhesion molecules, that is, E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1).2, 3 Furthermore, both platelet- and T-lymphocyte-associated CD40L appear to play a major role in regulating the density of CD40 expression on vascular endothelial cells in vivo,1 suggesting that CD40–CD40L signaling may contribute to the perpetuation of an inflammatory response by initiating a vicious cycle of CD40L-induced endothelial cell activation and CD40 up-regulation.

CD40–CD40L signaling has been implicated in a variety of physiologic and pathophysiologic processes, including adaptive immune responses, hemostasis/coagulation, and inflammation.4 The importance of CD40–CD40L in inflammation and coagulation is evidenced by the results of a recent report that describes an attenuated recruitment of adherent leukocytes and platelets in the cerebral microvasculature of CD40−/− or CD40L−/− mice subjected to ischemia–reperfusion, a well-characterized model of acute inflammation.5 However, there is also evidence implicating the CD40–CD40L dyad in chronic inflammatory conditions, including inflammatory bowel disease (IBD). IBD patients exhibit an increased expression of CD40L on platelets and increased plasma levels of soluble CD40L, which is largely derived from the activated CD40L+ platelets.2, 6, 7 Studies of biopsy material from IBD patients have revealed increased numbers of CD40+ and CD40L+ cells in the colonic mucosa,8 and the CD40 overexpression on lamina propria cells appears to be positively correlated with clinical disease activity (derived from clinical, endoscopic, and histologic findings).9 Similar support for the involvement of CD40–CD40L in disease progression has been obtained from animal models of IBD. CD40L transgenic mice are known to develop IBD,10 while administration of a CD40L blocking antibody has been shown to reduce disease activity and histologic injury in both the 2,4,6-trinitrobenzene sulfonic acid11 and CD45Rbhigh transfer models12, 13 of experimental colitis.

Although there is growing evidence for the involvement of CD40–CD40L in the pathogenesis of IBD, the mechanisms underlying the contribution of this signaling pathway remain poorly understood. In vitro studies using human intestinal microvascular endothelial cells (HIMEC) have revealed that coculture of CD40L+ platelets derived from IBD patients with HIMEC results in the enhanced production of IL-8 by HIMEC, and an increased surface expression of VCAM-1 and ICAM-1 on the cultured endothelial cells.2 There is additional evidence that activated CD40L+ platelets induce HIMEC to express CD40L,14 although it remains unclear whether CD40 expression is altered in the inflamed intestinal microvasculature. However, it is noteworthy that cultured endothelial cells also assume a pro-thrombogenic phenotype (including tissue factor expression) when exposed to CD40L+ platelets.15 Overall, these in vitro studies suggest that the contribution of the CD40–CD40L dyad to the pathogenesis of intestinal inflammation may relate to phenotypic changes in endothelial cells that are induced by the engagement of platelet CD40L to endothelial CD40, which produce a more adhesive surface that favors the attachment of leukocytes and platelets to endothelial cells. Nonetheless, it remains unclear whether CD40–CD40L contributes to the recruitment and activation of leukocytes and platelets in the microvasculature of the inflamed bowel.

The overall objective of this study was to assess the role of CD40–CD40L in producing the proinflammatory and pro-thrombogenic phenotype that is assumed by the colonic microvasculature and tissue injury that are associated with experimental colitis. The dextran sodium sulfate (DSS) model of experimental colitis was used to determine whether: (1) inflammation is associated with an increased endothelial expression of CD40 in the colonic vasculature, (2) the recruitment of leukocytes and platelets into the inflamed colonic vasculature is altered in mice that are genetically deficient in either CD40 (CD40−/−) or CD40L (CD40L−/−), (3) mucosal injury and disease activity are attenuated in CD40−/− and/or CD40L−/− mice, and (4) a pharmacologic approach (Trapidil) to CD40–CD40L inhibition affords protection similar to genetic ablation against the blood cell recruitment and tissue injury responses to acute inflammation.

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Materials and Methods 

Mice 

The animals used in the experiments were 6–8-week-old male C57BL/6J mice (wild-type [WT] control strain), CD40 knockout mice (CD40−/−), and CD40L−/− mice (all developed on a C57BL/6J background), obtained from Jackson Laboratory (Bar Harbor, ME). All experimental procedures were performed according to the criteria outlined by the National Institutes of Health, and were approved by the LSU Health Sciences Center Institutional Animal Care and Use Committee.

Drugs 

Trapidil (Rocarnal) was obtained from Mochida Pharmaceuticals Co, Ltd, Tokyo, Japan. The agent was dissolved in 0.9% NaCl at a concentration of 1.6 mg/mL.

Induction of Colitis 

DSS model 

Mice were fed 3% DSS (molecular weight, 40 kDa; ICN Biomedicals, Aurora, OH) dissolved in drinking water that was filter-purified (Millipore Corp., Bedford, MA) ad libidum, beginning on day 0 and ending on day 6.16 Control WT mice received the filtered water alone. Previously, we demonstrated that in DSS-induced colitis, the severity of inflammation is DSS load-dependent, and that a critical DSS load ≥30 mg DSS/g body weight is required to reliably induce colitis in the C57BL/6J strain.17 Animals not meeting the DSS load criteria were excluded from the study.

CD 45RBhigh T-cell transfer model 

Chronic colitis was induced in recombinase activating gene-1 knockout (RAG-1 ko) mice using adoptive transfer of naïve (CD4+CD45RBhigh) T cells as previously described.18, 19 Briefly, splenocytes were enriched for CD4+ T cells by negative selection on MACS columns using FITC conjugated antibodies to B220, CD8, and Mac-1 for removal of B cells, CD8+ cells, and myeloid cells, respectively. The flow-through cell suspension was then stained with biotin conjugated anti-CD4 and phycoerythrin (PE) conjugated anti-CD45RB, followed by incubation with streptavidin-red 670. The cells were sorted on a FACS Vantage TurboSort flow cytometer. Lymphocytes that stained brightly with CD4+ and CD45RB mAbs (the 40% brightest in PE staining), as well as those that stained brightly with CD4+ but not CD45RB (the 15% dimmest in PE staining), were collected and termed CD4+CD45RBhigh and CD4+CD45RBlow, respectively. Sorted donor lymphocytes were resuspended in sterile PBS and injected intraperitoneally (IP; 5 × 105 cells per mouse) into male recipient RAG-1 ko mice. The recipient animals were weighed initially, then weekly thereafter. Mice were also observed daily for clinical signs of illness: hunched-over appearance, weight loss, loose stools/diarrhea, and occult blood in the stool.

Assessment of disease activity in DSS-treated mice 

A previously validated clinical disease activity index (DAI)20 ranging from 0 to 4 was calculated using the following parameters: stool consistency (normal, loose, diarrhea), presence or absence of fecal blood (guaiac paper test [ColoScreen; Helena Laboratories Inc., Beaumont, TX]21 and macroscopic evaluation of the anus), and weight loss.

Colon length 

On day 6, intravital microscopy was performed and the mice were sacrificed. Colons were removed, from the cecum to the pelvic floor. Colon length and weight were measured before dividing the colon into samples for measurement of tissue myeloperoxidase (MPO) activity and histologic preparation.

Histology 

Three samples of the distal colon were evaluated histologically for each animal; samples were fixed in 10% formalin and stained with hematoxylin and eosin. Using a previously described scoring system,22 all histology samples were evaluated and quantified in a blinded fashion. Severity of inflammation (0–3: none, slight, moderate, severe), extent of injury (0–3: none, mucosal, mucosal and submucosal, transmural), and crypt damage (0–4: none, basal 1/3 damaged, basal 2/3 damaged, only surface epithelium intact, entire crypt and epithelium lost) were the 3 independent parameters measured. The score of each parameter was multiplied by a factor reflecting the percentage of tissue involvement (×1: 0%–25%, ×2: 26%–50%, ×3: 51%–75%, ×4: 76%–100%), and all numbers were summed. The maximum possible score was 40.

Tissue MPO activity 

Samples of colonic tissue samples were rinsed with cold PBS, blotted dry, frozen in liquid nitrogen, and stored at −80°C until assayed for MPO activity using the o-dianisidine method.23, 24 MPO activity was expressed as the amount of enzyme necessary to produce a change in absorbance of 1.0 unit per minute per gram tissue (wet weight).

Platelet preparation 

Platelets were isolated from untreated donor mice (WT, CD40−/−, or CD40L−/− mice) using a series of centrifugation steps as described previously.25 Platelets were labeled with the fluorochrome carboxyfluorescein diacetate succinimudyl ester (90 mmol/L final concentration; Molecular Probes Inc, Eugene, OR).

Surgical procedures 

Animals were anesthetized using ketamine hydrochloride (150 mg/kg IM) and xylazine (7.5 mg/kg IM). The right carotid artery was cannulated for blood pressure measurements. The right jugular vein was cannulated for infusion of rhodamine-6G (Sigma-Aldrich, St. Louis, MO) for leukocyte labeling and for subsequent infusion of carboxyfluorescein diacetate succinimudyl ester-labeled platelets. On an adjustable acrylic microscope stage, a laparotomy was performed and the mouse was placed on its right side. The proximal large bowel (initial 2–3 cm adjacent to the cecum) was exteriorized with moist cotton swabs, covered with a nonwoven sponge, and continuously superfused at 37°C with bicarbonate-buffered saline solution (pH 7.4).

Intravital fluorescence microscopy 

Platelets and leukocytes in the colonic microcirculation were visualized with an inverted Nikon microscope (Nikon Inc, Tokyo, Japan) equipped with a 75-watt XBO xenon lamp, as described previously. The mice received rhodamine-6G over 5 minutes, which was allowed to circulate for 5 minutes before infusion of carboxyfluorescein diacetate succinimudyl ester-labeled platelets over 5 minutes. After a 5-minute circulation time, the interactions of both leukocytes and platelets in colonic venules were recorded. Five randomly selected postcapillary venules (20–40 μm diameter) in each colon preparation were recorded for 1 minute each.

Off-line video analysis 

Platelets and leukocytes were classified according to their interaction with the venular wall as free flowing, rolling (when cells are slower than centerline blood flow), or adherent (when cells remain stationary for ≥30 seconds). We determined whether a platelet or leukocyte was adherent directly to the endothelium or attached to the vessel wall by binding to another blood cell. Platelet and leukocyte rolling were expressed as number of rolling cells per second per millimeter of vessel diameter, and their adherence was expressed as the number of cells per mm2 of venular surface, calculated from diameter and length and assuming cylindrical vessel shape.26

In vivo measurement of CD40 expression in the colonic vasculature using the dual radiolabeled mAb technique 

The expression of CD40 on vascular endothelial cells was measured in WT mice at 6 days of H2O or DSS, and CD40−/− mice at 6 days of DSS using the dual radiolabeled monoclonal antibody (mAb) technique as described elsewhere in detail.1, 27 Previous studies1 have demonstrated significant expression of CD40 in different vascular beds of WT mice and the absence of CD40 expression in the vasculature of CD40−/− mice using this technique. The mAbs used for in vivo assessment of CD40 expression were 3/23, a purified binding rat immunoglobulin (IgG2a) that is specific for mouse CD40 (20 μg anti-CD40 per mouse) (BD Pharmingen, San Diego, CA), and P-23, a nonspecific, nonbinding murine IgG1 directed against human P-selectin (provided by Dr Donald C. Anderson, Pharmacia-Upjohn, Kalamazoo, MI). The specific binding (3/23) and nonbinding (P23) mAbs were labeled with 125I and 131I, respectively (Du Pont-New England Nuclear, Boston, MA), using the iodogen method as described previously.28, 29 Receptor levels in the proximal and distal portions of the large bowel were expressed as ng mAb/g tissue as described previously.1, 27

Experimental Protocols 

The objective of the first series of experiments was to determine whether CD40 expression is altered in the colonic vasculature of WT mice with DSS-induced inflammation (n = 12), compared to the colon of normal (WT) mice (n = 12). This objective was achieved using the dual radiolabeled mAb technique. To confirm the specificity of our experimental setup a group of CD40−/− mice treated with DSS were tested similarly. A second series of experiments was undertaken to compare different clinical parameters (disease activity index, colon length, histology, and tissue MPO) between control animals receiving water (n = 10) and WT (n = 10), CD40−/− (n = 16), and CD40L−/− (n = 14) mice receiving DSS in their drinking water. In some of these animals, the third objective was achieved using intravital microscopy to quantify leukocyte and platelet adhesion in the colonic microcirculation of control animals (n = 10) and WT (n = 10), CD40−/− (n = 6), and CD40L−/− (n = 6) mice with DSS treatment. Finally, a group of WT mice (n = 6) were treated twice (on day 0 and on day 3 of the induction of colitis with DSS) with 40 mg/kg IP Trapidil and both clinical parameters (disease activity index, colon length, histology, and tissue MPO) and leukocyte/platelet endothelial adhesion were monitored and compared to separate WT groups receiving water (n = 6) or receiving DSS in their drinking water (n = 6) set up in parallel to the Trapidil-treated mice. All experiments/data collection/tissue sample collection, without exception, were performed on the 6th day of DSS feeding counting the starting day as day 0.

In the CD45RBhigh transfer model animals were allowed to recover for 14 days after transplantation and then divided between 2 groups: untreated and Trapidil-treated. The latter received 40 mg/kg IP Trapidil every 3 days until the time of the experiment.

Data analysis 

Statistical analyses were performed with StatView 4.5 software (Abacus Concepts Inc, Berkeley, CA) using 1-way ANOVA with Fisher’s (post hoc) test. All values are reported as means ± SE. Statistical significance was set at P < .05.

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Results 

Expression of CD40 in the Colonic Vasculature During DSS-Induced Inflammation 

The expression of CD40 on colonic endothelial cells was measured in the proximal and distal colon of control (H2O) and 3% DSS-treated WT mice. CD40 expression was elevated (compared to H2O controls) in the vasculature of the proximal and distal colonic segments of mice treated with DSS, although this increase reached statistical significance (P < .05) only in the distal colon (Figure 1). Although the mesenteric vasculature in DSS-treated mice also exhibited a significant increase in CD40 expression (data not shown), all other organs studied, including lung, liver, brain, kidneys, heart, small intestine, stomach, and pancreas did not exhibit significant changes compared to H2O control mice. CD40−/− mice treated with DSS did not exhibit detectable expression of CD40 in the colonic and mesenteric vasculatures (data not shown).

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  • Figure 1. 

    Effects of DSS (vs H2O) treatment (day 0–day 6) on the expression of CD40 in the vasculature of the proximal and distal colon in WT mice at day 6. Zero denotes no detectable expression. Data are presented as the mean ± SE. *Denotes statistical significance (P < .05) compared with corresponding control (H2O).

Clinical Indices of Disease Activity 

All mice placed on 3% DSS survived the entire treatment period. Control mice drinking Millipore water showed no clinical signs of intestinal inflammation (diarrhea, occult, or perianal bleeding, weight loss; Figure 2A and B). WT mice treated with DSS exhibited a gradual time-dependent increase in DAI. In addition, DSS induced a reduction in body weight that was significant beginning on day 4. The changes in DAI and body weight were most prominent on day 6. Another sign of disease activity noted in DSS-treated WT mice was colonic shortening (Figure 3). In CD40−/− and CD40L−/− mice treated with DSS, the changes in DAI, body weight, and colonic shortening were significantly blunted when compared with their WT counterparts.

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  • Figure 2. 

    Changes in the disease activity index (A) and body weight (%) (B) over the course of DSS (or H2O) treatment in WT, CD40-deficient (CD40−/−), and CD40L-deficient (CD40L−/−) mice. Data are presented as the mean ± SE. *P < .05 vs H2O, #P < .05 vs DSS.

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  • Figure 3. 

    Length of the resected colon (cm) at day 6 of DSS (or H2O) treatment in WT, CD40-deficient (CD40−/−), and CD40L-deficient (CD40L−/−) mice. Data are presented as the mean ± SE. *P < .05 vs H2O, #P < .05 vs DSS.

Histology 

Control mice showed no signs of intestinal inflammation (Figure 4) and had normal morphologic features (Figure 5A). In contrast, at day 6 of DSS treatment, WT mice exhibited evidence of severe colitis as assessed by the overall score as well as significant changes in the specific parameters examined (inflammation, extent, crypt damage). Severe inflammation was evident in the mucosa with dense infiltrates of inflammatory cells, loss of epithelial integrity, and loss of goblet cells, accompanied by submucosal edema and perivascular accumulation of inflammatory cells (Figure 5B). However, DSS-treated CD40−/− and CD40L−/− mice exhibited significantly attenuated histologic scores, as reflected by the reduction of total scores and of all 3 component variables (inflammation, extent, and crypt damage) (Figure 4). In both CD40−/− and CD40L−/− mice, the infiltration of inflammatory cells and submucosal edema were reduced and the mucosal architecture remained intact (Figure 5C and D).

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  • Figure 4. 

    Blinded histologic assessment of colonic mucosa at day 6 of DSS (or H2O) treatment in WT, CD40-deficient (CD40−/−), and CD40L-deficient (CD40L−/−) mice. Zero denotes no detectable response in H2O-treated WT mice. The total histologic score was derived from the severity and extent of inflammation and crypt damage. For each mouse, 3 sections were analyzed (mean ± SE). * and # denote statistical significance (P < .05) compared with control and WT DSS-treated animals, respectively.

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  • Figure 5. 

    Histology (H&E staining; magnification ×100) of colonic samples taken at day 6 from WT animals receiving H2O (control) or from WT, CD40−/−, and CD40L−/− mice receiving DSS in drinking water. Compared with the colon of control animals (A), the colon of WT DSS-treated mice (B) showed destruction of the bowel wall architecture with loss of the epithelial lining (arrowheads), vanished crypts, submucosal edema (asterisk), and dense cellular inflammation in all layers (arrows). Histologic specimens of CD40−/− (C) and CD40L−/− (D) mice exhibited an attenuated morphologic damage with only mild cellular infiltration (arrow).

Tissue Myeloperoxidase 

Figure 6 illustrates the significant increase in colonic myeloperoxidase (MPO) activity observed at day 6 of DSS treatment in WT mice, compared to WT mice receiving water. A significant reduction in the MPO response to DSS was noted in CD40−/− mice, whereas the small attenuation observed in the CD40L−/− mice did not reach significance (P = .12).

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  • Figure 6. 

    Colonic myeloperoxidase (MPO) activity at day 6 in H2O-treated WT mice and WT, CD40−/−, and CD40L−/− mice receiving DSS in drinking water from day 0–day 6. *P < .05 vs water, #P < .05 vs DSS.

Intravital Microscopic Evaluation of Platelet and Leukocyte Adhesion 

DSS treatment of WT mice elicited profound increases in rolling and firmly adherent platelets and leukocytes in colonic venules (Figure 7). The majority of the adhesive interactions between platelets (rolling and adherent) and the venular wall in the DSS-treated WT mice involved binding to leukocytes, with a small proportion (14% for rolling and 5% for adherent) of the platelets directly binding to venular endothelium (Figure 7A and C). In DSS-treated WT mice, about 1/3 of the rolling leukocytes and approximately 1/2 of the adherent leukocytes in colonic venules were directly attached to the endothelium without bound platelets, and the remaining leukocytes were bound to the endothelium as leukocyte-platelet aggregates (Figure 7B and D).

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  • Figure 7. 

    Rolling platelets (A) and leukocytes (B) and firmly adherent platelets (C) and leukocytes (D) in postcapillary venules on day 6 of treatment with H2O (WT control) or DSS in WT, CD40−/−, and CD40L−/− mice. The number of platelets bound to leukocytes (platelet–leukocyte interactions) and those bound directly to endothelium (platelet–endothelial interactions) are shown in A and C. The number of platelet-bearing and platelet-free rolling and adherent leukocytes are presented in B and D. *P < .05 relative to H2O, #P < .05 relative to WT mice treated with DSS &P < .05 relative to CD40−/−.

In both CD40- and CD40L-deficient mice, the increment in numbers of rolling platelets and leukocytes during DSS-induced inflammation did not differ from the responses noted in WT mice treated with DSS. However, both CD40−/− and CD40L−/− mice exhibited profound reductions in DSS-induced firm adherence of platelets (Figure 7C) and leukocytes (Figure 7D). The protective effect of CD40 or CD40L deficiency on platelet adherence resulted entirely from an attenuated binding of platelet–leukocyte aggregates to the venular wall. However, the adherence of both platelet-free and platelet-bearing leukocytes was attenuated in the DSS-treated CD40−/− and CD40L−/− mice.

Effects of the CD40–CD40L Pathway Inhibitor, Triazolopyrimidine (Trapidil), on DSS-Induced Colonic Inflammation 

Table 1 summarizes the effects of Trapidil treatment on the responses of WT mice to DSS colitis. Disease activity index and colon weight/length ratio were significantly reduced in the drug-treated DSS mice compared to their untreated counterparts. Although the recruitment of rolling platelets and leukocytes induced by DSS was not affected by drug treatment, the numbers of firmly adherent platelets and leukocytes were significantly attenuated and reflected actions on both the binding of leukocytes to venular endothelium and to platelets.

Table 1. Effects of Trapidil on the Colonic Inflammatory Responses to DSS (on Day 6)
H2ODSS 3%DSS 3% + trapidil
Disease activity index0.056±0.063.55±0.15a2.47±0.23ab
Body weight change (%)102.5±0.6488.28±2.15a89.79±0.73a
Colon length (cm)8.11±0.185.64±0.19a7.64±0.51b
Histology score0.18±0.1318.84±2.02a3.89±0.96b
MPO (U/g tissue)1.48±0.4815.5±3.25a17.62±4.52a
Rolling platelets (#/sec/mm)
Plt–endo. interaction0.34±0.130.55±0.170.25±0.06
Plt–leuk. interaction0.66±0.175.1±0.63a4.19±0.44a
Total0.99±0.295.64±0.66a4.45±0.46a
Rolling leukocytes (#/sec/mm)
Plt-bearing leuk.0.65±0.174.59±0.6a3.9±0.38a
Plt-free leuk.1.09±0.177.14±1.36a4.76±0.62
Total1.74±0.3911.73±1.88a8.66±0.57a
Adherent platelets (#/mm2)
Plt–endo. interaction1.42±1.4149.17±8.45a10.63±6.52b
Plt–leuk. interaction10.7±8.38225.2±37.39a31.41±19.52b
Total12.11±9.75274.37±35.96a42.04±24.8b
Adherent leukocytes (#/mm2)
Plt-bearing leuk.10.69±8.38201.77±29.99a26.01±14.53b
Plt-free leuk.28.29±22.71188.32±22.02a37.09±23.25b
Total38.99±25.16390.09±41.13a63.19±29.66b

Endo, endothelial; leuk, leukocytes; plt, platelet.

aP < .05 vs H2O.

bP < .05 vs DSS 3%.

We also assessed the actions of Trapidil in a T-cell-dependent model of experimental IBD, that is, the CD45RBhigh T-cell transfer model. This model produces a very pronounced colitis at 6–8 weeks following adoptive transfer of donor T cells. For all variables measured in this model, including colitis score, colonic weight/length ratio, histologic injury, and the adhesion of platelets and leukocytes in colonic venules, no significant differences were noted between untreated mice and those receiving Trapidil (data not shown).

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Discussion 

There are several lines of evidence in the literature that implicate the CD40–CD40L signaling pathway in the pathogenesis of IBD. This evidence has been derived from studies of mucosal biopsy material from IBD patients,8, 9 from experiments on mouse models of experimental colitis,11, 12, 13 and from cultured monolayers of HIMEC that are allowed to interact with CD40L-positive platelets obtained from IBD patients.2 Although these studies support a role for multiple cell types in the CD40–CD40L-mediated responses during IBD, the latter in vitro studies suggest that a critical component of the involvement of this signaling pathway is endothelial cell activation and the consequent production of inflammatory mediators and expression of adhesion molecules that serve to sustain the inflammatory response. The results of the present study are consistent with such a mechanism, and provide the first evidence for: (1) a large (10-fold) increase in CD40 expression in the colonic vasculature during DSS-induced inflammation, (2) a major contribution of the CD40–CD40L dyad in promoting the recruitment of adherent leukocytes and platelets in colonic venules during experimental colitis, and (3) a pharmacologic approach (Trapidil) to inhibition of CD40–CD40L signaling that effectively mimics the beneficial effects of genetic ablation of either CD40 or CD40L in a widely used animal model of intestinal inflammation.

We have recently used the dual radiolabeled mAb technique to obtain quantitative in vivo measurements of constitutive and induced expression of CD40 in different regional vascular beds, and demonstrated a tissue-specific, time-dependent up-regulation of endothelial CD40 in mice challenged with bacterial endotoxin.1 In the present study, the same method was used to determine if DSS-induced colonic inflammation is associated with an altered expression of CD40 in the colonic vasculature. The findings of these experiments reveal a large (10-fold) increase in the density of CD40 in the inflamed colonic vasculature of WT (but not CD40−/−) mice treated with DSS, compared to their control counterparts. This response was stronger than the 3.5- to 4.0-fold increase in intestinal vascular CD40 expression that is elicited 4–48 hours after intraperitoneal administration of 5 μg of bacterial endotoxin into WT mice. The CD40 up-regulation noted in DSS-induced intestinal inflammation appears to reflect an intense activation of endothelial cells that is not likely mediated by bacterial endotoxin.

Our finding of increased CD40 expression in the colonic vasculature of DSS-treated mice is consistent with reports describing strong immunohistochemical staining of CD40 on endothelial cells in the mucosa and submucosa of biopsy samples derived from IBD patients,30, 31 which contrasts with the weak or absent CD40 immunostaining seen in the normal colonic mucosa. Although the increased CD40 expression in mucosal tissue of patients with ulcerative colitis was found to be directly proportional to disease activity based on clinical, endoscopic, and histologic criteria,9 we did not detect such a correlation in our DSS model. This apparent discrepancy may result from the detection of CD40 expression in other cell types (eg, monocytes, platelets) within mucosal biopsy specimens of human IBD.

It has been previously shown that engagement of CD40L on activated, adherent platelets with constitutively expressed CD40 on endothelial cells results in phenotypic changes in the endothelial cells that are similar to that induced by tumor necrosis factor alpha (a cytokine that is known to contribute to the pathogenesis of IBD), including an increased expression of E-selectin, ICAM-1, and VCAM-1.3, 32 It has also been shown that adherent activated platelets induce the expression of tissue factor, which initiates the extrinsic pathway of blood coagulation, on endothelial cells in a CD40/CD40L-dependent manner.15 These observations indicate that the engagement of platelet CD40L with endothelial cell CD40 may represent a component of a juxtacrine signaling system wherein CAM-dependent adhesion of platelets to vascular endothelial cells brings together a ligand–receptor pair to mediate cell–cell signaling that amplifies the inflammatory response by promoting the recruitment of leukocytes and more platelets. Our comparison of the platelet and leukocyte adhesion responses in colonic venules of DSS-treated wild-type mice to those observed in DSS-treated CD40−/− and CD40L−/− mice clearly indicates that CD40–CD40L signaling plays a major role in promoting the recruitment of adherent leukocytes and platelets in colonic venules during experimental colitis. Interestingly, intravascular leukocyte adhesion as measured by intravital microscopy was almost completely abrogated, whereas a relatively small reduction in MPO levels was observed in the mutant mice. One potential explanation is that MPO reflects the total number of adherent and emigrated neutrophils, suggesting that CD40/CD40L may have a more important role in modulating adhesion than emigration, at least in the proximal colon. Interestingly, histologic evaluation revealed significant reductions in neutrophil infiltration in the distal portion of the colon of CD40 and CD40L knockouts when compared to WT mice. The discrepancy between the MPO data and the histologic findings may reflect the fact that MPO measurements are normalized to the wet weight of the tissue, which would lead to an underestimation of MPO in the DSS group (where edema is present), but not in the knockout mice (where edema is reduced), thereby obscuring a reduction in MPO that results from CD40 or CD40L deficiency. Our adhesion data also indicates that CD40–CD40L signaling is critical not only for the adhesion of platelets and leukocytes to endothelial cells lining the venular wall, but also for the heterotypic adhesive interactions between these blood cells. The number of platelet–leukocyte aggregates is increased in patients with IBD, and it has been proposed that the aggregates may be pathogenic in IBD.33, 34 Our results indicate that the aggregation of platelets and leukocytes that is elicited by intestinal inflammation may be largely dependent on CD40–CD40L signaling. Because soluble CD40L has been shown to enhance P-selectin expression on platelets,35 and platelet P-selectin avidly binds to PSGL-1 that is constitutively expressed on circulating leukocytes,36, 37, 38 it is possible that activation of the CD40–CD40L pathway during intestinal inflammation induces platelet–leukocyte aggregation by promoting adhesive interactions between platelet-associated P-selectin and its natural ligand (PSGL-1) on leukocytes. Furthermore, our finding that ablation of CD40–CD40L interferes with the recruitment of both platelets and leukocytes is consistent with the view that this signaling pathway is an important link between the inflammatory and hemostatic systems in different pathologic conditions.39, 40, 41

The beneficial effect of disruption of the CD40–CD40L signaling pathway in different models of experimental colitis12, 13 raises the possibility of targeting this mechanism for the treatment of IBD. One drug that has been shown to exert an inhibitory effect on the CD40–CD40L pathway is the triazolopyrimidine Trapidil. This compound, which was developed for the prevention of restenosis after vascular injury, has been shown to inhibit the responses of monocytes to CD40 ligation.42 We provide evidence in this study that treatment with Trapidil significantly attenuates the colonic inflammatory responses to DSS, including disease activity and the number of adherent platelets and leukocytes recruited into inflamed venules. The protection afforded by Trapidil in the DSS model was similar to that noted in CD40−/− and CD40L−/− mice. Although it is tempting to attribute the beneficial effects of Trapidil to inhibition of the CD40–CD40L pathway, the contribution of the antiplatelet action of this drug cannot be excluded, particularly because clinical2, 43 and experimental animal studies44 have recently implicated platelets in the pathogenesis of IBD.

Although Trapidil was effective in blunting the inflammatory and injury responses in the DSS model of colitis, no such benefit was noted in the T-cell transfer model of colitis. Although a definitive explanation for the differential effectiveness of Trapidil in the 2 models is not readily available, it may reflect the nature of the underlying cause of inflammation in the 2 models, that is, mucosal injury-induced inflammation in the DSS model vs T-cell–induced inflammation in the T-cell transfer model. Furthermore, this observation suggests that Trapidil may prove more effective in promoting mucosal repair following the epithelial injury induced by DSS than on the T-cell–dependent mechanisms responsible for promoting the chronic inflammatory response as observed in the CD45RBhigh T-cell transfer model. Also, the findings may relate to differences in the quality and severity of the inflammatory response between the 2 models, with the latter model producing a more chronic and robust response. Additional work is needed to determine whether a different treatment regimen (eg, higher dose) would yield a beneficial response in the T-cell model. Because intra- and extraintestinal thrombosis are now recognized pathologic features of IBD,43, 45, 46 the drug may prove useful in reducing the thromboembolic events associated with this disease.45 In view of the potential therapeutic benefits of Trapidil in the DSS model of gut inflammation, additional work is needed to more precisely define the nature and relative importance of its anti-inflammatory and antithrombotic actions in gut inflammation.

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 Supported by grants from the National Institute of Health (DK065649 to D.N.G.) and the Deutsche Forschungsgemeinschaft (VO998/1-1 to T.V.).

PII: S0016-5085(06)02677-1

doi:10.1053/j.gastro.2006.12.027

Gastroenterology
Volume 132, Issue 3 , Pages 955-965, March 2007