Crucial role of the protein C pathway in governing microvascular inflammation in inflammatory bowel disease

Endothelial protein C receptor (EPCR) and thrombomodulin (TM) are expressed at high levels in the resting microvasculature and convert protein C (PC) into its activated form, which is a potent anticoagulant and antiinflammatory molecule. Here we provide evidence that in Crohn disease (CD) and ulcerative colitis (UC), the 2 major forms of inflammatory bowel disease (IBD), there was loss of expression of endothelial EPCR and TM, which in turns caused impairment of PC activation by the inflamed mucosal microvasculature. In isolated human intestinal endothelial cells, administration of recombinant activated PC had a potent antiinflammatory effect, as demonstrated by downregulated cytokine-dependent cell adhesion molecule expression and chemokine production as well as inhibited leukocyte adhesion. In vivo, administration of activated PC was therapeutically effective in ameliorating experimental colitis as evidenced by reduced weight loss, disease activity index, and histological colitis scores as well as inhibited leukocyte adhesion to the inflamed intestinal vessels. The results suggest that the PC pathway represents a new system crucially involved in governing intestinal homeostasis mediated by the mucosal microvasculature. Restoring the PC pathway may represent a new therapeutic approach to suppress intestinal inflammation in IBD.

The regulatory pathways involved in the pathogenesis of Crohn disease (CD) and ulcerative colitis (UC), the 2 major forms of inflammatory bowel disease (IBD), are not limited to those mediated by immune cells, but also include those mediated by nonimmune cells (1–6). An increasing number of reports have called attention to the significant contribution of epithelial, mesenchymal, nerve, and vascular cells as well as platelets to IBD pathogenesis (3, 7–10). It is now firmly established that the mucosal microvasculature is a nonimmune component crucially involved in intestinal inflammation (3).

Endothelial cells play a key role in mucosal immune homeostasis, acting as gatekeepers that regulate the type and number of leukocytes migrating from the intravascular to the interstitial space. Specialized microvascular cells, like those in high endothelial venules, selectively govern the influx of leukocytes into the gut (11). During intestinal inflammation, the mucosal microvasculature controls the nature and magnitude of such influx through chemokine secretion and expression of cell adhesion molecules (CAMs), which facilitate and amplify communication with leukocytes (12). The mucosal microvasculature is activated in IBD, as shown by aberrant CAM expression and cytokine production as well as the presence of angiogenesis (13). A novel pathway crucially involved in controlling microvascular inflammation is the protein C (PC) pathway (14, 15). Although it was originally described as a major anticoagulant system (16, 17), it is now well established that the PC pathway is a crucial checkpoint for inflammation (14, 15, 18).

Two important molecules in this pathway are thrombomodulin (TM) and endothelial PC receptor (EPCR), both of which are highly expressed on the surface of the microvasculature. An essential step for the pathway to become fully functional involves the capture of PC by the TM/EPCR complex on the endothelial surface and subsequent conversion to activated PC (aPC), a process that is catalyzed by thrombin (16, 17). This assembly process increases the rate of PC activation by more than 1,000-fold and at the same time blocks the ability of thrombin to activate platelets and the endothelium, an additional antiinflammatory step. A unique and critical feature of the PC pathway is its greater relevance to the microcirculation than to the macrocirculation. In fact, because the ratio of endothelial surface to blood volume is much higher in small rather than large vessels, there is a relative increase of TM concentrations in the microvasculature, promoting the conversion of PC to aPC at the local level (14, 16, 17). Each component of the PC pathway, individually or in combination, has been shown to exert antiinflammatory activity and prevent microvascular inflammation (14, 15).

In particular, TM directly inhibits leukocyte adhesion to activated endothelium, and mice expressing a nonfunctional TM mutant show increased leukocyte influx in several models of inflammation (19). TM has also been shown to sequester the high-mobility group–B1 proteins, which are very potent proinflammatory molecules and thus prevent vascular activation (20). EPCR can block the integrin CD11b/CD18 on leukocytes and prevent leukocyte influx into tissues (21). After administration of LPS, mice with a severe deficiency of EPCR exhibit a reduced survival rate compared with wild-type mice, with a marked increase of neutrophilic tissue infiltration and enhanced production of chemokines (22). Finally, aPC inhibits the release of inflammatory cytokines such as TNF-α, IL-6, and IL-8 in experimental models of endotoxin-induced inflammation (23, 24). aPC also limits leukocyte adhesion to the endothelium and extravasation into tissues (25, 26). In endothelial cell–based experimental systems, aPC inhibits a variety of inflammatory events in human umbilical vascular endothelial cells, such as CAM expression, cytokine production, and NF-κB nuclear translocation (27, 28). Nevertheless, each of the antiinflammatory components of the PC pathway is downregulated in inflammation, as observed in various models of experimental inflammation. For instance, expression of TM on lung endothelial cells is suppressed by inflammation secondary to total-body irradiation (29). In the ovalbumin-induced bronchial asthma model, a decrease in aPC concentration was noted in the bronchoalveolar lavage associated with the downregulation of both TM and EPCR, as well as in atherosclerosis. (30). In animal models of LPS-induced sepsis, TM expression diminishes in a dose- and time-dependent manner in lung, liver, and renal peritubular capillaries (31).

Essentially no information exists on the expression and function of the PC pathway in human and experimental IBD, although it is well established that coagulation and inflammation are interdependent processes in IBD (32, 33). To date, no mechanistic study to our knowledge has been performed to investigate the regulatory mechanisms of TM and EPCR in IBD, the expression and function of the components of the PC pathway in the mucosal microvasculature, and their possible involvement in IBD pathogenesis. Therefore, the aim of the present study was to assess the expression and function of the PC pathway in IBD and to explore the therapeutic potential of manipulating such a pathway in experimental intestinal inflammation. In CD and UC, the 2 major forms of IBD, there was loss of expression of endothelial EPCR and TM, which in turns caused impairment of PC activation by the inflamed mucosal microvasculature. In isolated human intestinal endothelial cells, administration of recombinant activated PC had a potent antiinflammatory effect, as demonstrated by downregulated cytokine-dependent cell adhesion molecule expression and chemokine production as well as inhibited leukocyte adhesion. In vivo, administration of activated PC was therapeutically effective in ameliorating experimental colitis as evidenced by reduced weight loss, disease activity index, and histological colitis scores as well as inhibited leukocyte adhesion to the inflamed intestinal vessels. The results suggest that the PC pathway represents a new system crucially involved in governing intestinal homeostasis mediated by the mucosal microvasculature. Restoring the PC pathway may represent a new therapeutic approach to suppress intestinal inflammation in IBD.

Evidence for TM and EPCR downregulation in the IBD microvasculature. In order to investigate the expression levels of TM and EPCR in normal and IBD microvasculature, we initially performed immunohistochemical staining of 12 histologically normal control, 15 active CD-involved, and 14 UC-involved mucosa derived from human surgical specimens. Normal colonic mucosa showed strong vascular immunostaining for both EPCR and TM (Figure 1, A and B). In contrast, microvessels of actively inflamed CD or UC mucosa expressed minimal levels of both proteins, without any apparent difference between the 2 forms of IBD (Figure 1, C and D). To validate this visual assessment, semiquantitative scoring of tissue samples was performed, which confirmed significant decreases of vascular EPCR expression in UC (0.8 ± 0.6 normalized to GAPDH) and CD (0.8 ± 0.8 normalized to GAPDH) compared with normal mucosa (2.5 ± 0.5 normalized to GAPDH; P < 0.01, both comparisons). Similar results were obtained for TM expression, which was significantly decreased in both UC (0.6 ± 0.7 normalized to GAPDH) and CD (0.9 ± 0.7 normalized to GAPDH) vessels compared with those in normal control mucosa (2.6 ± 0.4 normalized to GAPDH; P < 0.01, both comparisons). No differences were found between control mucosa and noninflamed IBD, which showed high expression levels of both EPCR and TM (Figure 1, E and F).

Figure 1

Immunohistochemical staining of EPCR and TM in colons of normal and actively inflamed IBD patients. The panels show brown immunohistochemical staining for EPCR and TM in the microvasculature of colonic mucosa and submucosa from histologically normal control (A and B), active IBD (C and D), and noninflamed IBD (E and F) tissues. Images are representative of 12 control, 14 UC, and 15 CD samples (original magnification, ×10). Red arrows indicate intestinal microvessels.

To confirm the microvascular localization of the staining in the mucosa, we used fluorescence microscopy for detection of EPCR in conjunction with antibodies against von Willebrand factor (Figure 2, A and B). The specificity of EPCR endothelial cell origin was confirmed by the yellow staining of the merged images (Figure 2D) with the DAPI staining for nuclei (Figure 2C). Confocal microscopy was combined with quantitative analysis to confirm the extent of EPCR colocalization with von Willebrand factor (r = 0.85; Figure 2, E and F). Additionally, TM colocalized with von Willebrand factor, demonstrating endothelial specificity (data not shown).

Figure 2

Localization of EPCR in IBD mucosa. Fluorescence micrographs of colonic mucosa microvessels in normal mucosa. (A–C) Staining for EPCR (green, A), von Willebrand factor (red, B), and nuclei (blue, C). (D) Merge (yellow) of panels A–C. (E and F) Binary mask of colocalization (white, E) and colocalization of panels B and E (F). Images are representative of 6 control, 7 CD, and 8 UC samples. Scale bars: 60 μm.

Effect of cytokines on TM and EPCR expression in human intestinal microvascular endothelial cells. The downregulation of TM and EPCR in involved IBD tissue suggests that the local proinflammatory microenvironment could inhibit their endothelial expression. To reproduce this event in vitro, we tested the effect of TNF-α and IL-1β, 2 prototypical proinflammatory cytokines known to be elevated in IBD, on TM and EPCR expression in human intestinal microvascular endothelial cells (HIMECs). Essentially all unstimulated HIMECs expressed EPCR and TM on their surfaces, and stimulation by both TNF-α and IL-1β (50 ng/ml and 100 U/ml, respectively; data not shown) dramatically downregulated these expression levels (Figure 3A). In addition to proinflammatory cytokines, we studied the potential effect of antiinflammatory molecules such as IL-10 (5 ng/ml) and TGF-β on TM and EPCR expression. Notably, IL-10 had no effect on EPCR expression and modestly increased TM expression (Figure 3A). Comparable results were observed with TGF-β (data not shown).

Figure 3

Effect of different cytokines on EPCR and TM expression by HIMECs. (A) HIMEC monolayers were left untreated (Baseline) or stimulated with TNF-α or IL-10 for 24 hours, after which cells were suspended and EPCR and TM expression measured by flow cytometry. Filled curve represents background signal from the isotype control. Values are representative of 6 separate experiments. Numbers represent the net percentage of positive cells. (B) HIMEC monolayers were left untreated or stimulated with TNF-α for 24 hours, after which supernatants were collected for ELISA measurement of soluble EPCR and TM. Cellular mRNA was extracted for real-time PCR of EPCR and TM message levels. Values are representative of 3 separate experiments. *P < 0.05, **P < 0.01 versus baseline.

In order to assess whether the downregulatory effects of TNF-α on HIMECs are the result of shedding or of reduced transcription, we used the above experimental conditions to measure the concentration of soluble EPCR and soluble TM as well as cellular mRNA levels in HIMEC culture supernatants. TNF-α significantly increased soluble EPCR release (P < 0.05), but failed to stimulate TM shedding by HIMECs. Furthermore, TNF-α strongly suppressed both EPCR (P < 0.05) and TM (P < 0.01) at the transcriptional level (Figure 3B).

Inflammatory cytokines impair PC conversion into aPC. Because inflammation downregulates TM and EPCR levels, which in turn leads to impairment of PC conversion to aPC (16), we investigated whether HIMECs are able to generate aPC. We initially added exogenous human recombinant PC to unstimulated HIMECs and measured its conversion into aPC by assaying the generation of proteolytic activity toward a tripeptidyl chromogenic substrate. Under these resting conditions, aPC was readily detected in the HIMEC supernatants (Figure 4). To mimic the inflammatory state of the mucosal microvasculature in IBD, we stimulated HIMECs with TNF-α and measured PC activation. Under these inflammatory conditions, conversion of PC into aPC was drastically impaired (Figure 4), a response consistent with the concomitant downregulation of TM and EPCR (Figure 3A).

Figure 4

TNF-α–inflamed HIMECs exhibit a reduced capacity for PC activation. HIMEC monolayers were incubated 24 hours with or without TNF-α and assayed for their ability to support activation of PC by thrombin. Generation of aPC was measured using the chromogenic tripeptidyl-pNA substrate S-2366; relative capacity for PC activation is presented as change in absorbance at 405 nm over time (mOD/min). Values are representative of 3 separate experiments. *P < 0.05 versus baseline.

aPC is a potent antiinflammatory molecule. Having demonstrated that TNF-α stimulation impairs the generation of aPC and causes the loss of this antiinflammatory molecule, we next investigated whether supplementing HIMECs with aPC would eliminate or ameliorate microvascular inflammation. To test this possibility, HIMECs were either left untreated or stimulated with TNF-α in the absence or presence of 80 nM human recombinant aPC. We investigated whether expression of VCAM-1 and ICAM-1, indicators of the antiinflammatory function of aPC, was inhibited. As previously reported (34), VCAM-1 and ICAM-1 were readily upregulated by TNF-α as detected by flow cytometry (Figure 5). Notably, incubation of HIMECs with aPC both prevented the upregulation of CAMs (data not shown) and inhibited their TNF-α–induced upregulation (Figure 5). Similar results were obtained when IL-1β was used as the inflammatory stimulus (data not shown).

Figure 5

Downregulation of VCAM-1 and ICAM-1 by recombinant aPC in TNF-α–inflamed HIMECs. HIMEC monolayers were left untreated or stimulated with TNF-α in the presence or absence of recombinant human aPC. After 24 hours, cells were suspended and VCAM-1 and ICAM-1 expression was measured by flow cytometry. Filled curve represents background signal from the isotype control. Values are representative of 5 separate experiments. Numbers represent the net percentage of positive cells.

In addition to upregulating CAM, inflamed endothelial cells secrete a large variety of chemokines. Using the same experimental system reported above, we measured the concentration of 3 major chemokines implicated in inflammatory cell recruitment in IBD: epithelial-derived neutrophil-activating peptide 78 (ENA-78), IL-8, and MCP-1. HIMECs produced higher levels of chemokines upon TNF-α stimulation, an effect that was significantly inhibited by the addition of aPC to the cultures, although levels did not return to baseline (Figure 6). Stimulation with IL-1β replicated these results (data not shown).

Figure 6

Downregulation of chemokine production by recombinant aPC in TNF-α–inflamed HIMECs. HIMEC monolayers were left untreated or stimulated with TNF-α in the presence or absence of recombinant human aPC. After 24 hours, supernatants were collected, and ENA-78, IL-8, and MCP-1 were measured by ELISA. Values are representative of 5 separate experiments. *P < 0.05 versus TNF-α without aPC.

aPC inhibits T cell adhesion to inflamed endothelium. It is well known that under inflammatory conditions, endothelium-leukocyte adhesion markedly increases. This aberrant T cell interaction is a key pathogenic event in IBD, and its blockade is currently being tested as a novel therapeutic strategy in both CD and UC (35, 36). Based on our demonstration that aPC inhibited both CAM and chemokine production, it is to be expected that aPC would also inhibit T cell adhesion. Hence, using the same experimental system described above, we quantified the adhesion of T cells to inflamed HIMECs in the presence or absence of exogenous aPC. Unstimulated HIMECs bound few T cells, but adhesion markedly increased after TNF-α stimulation (Figure 7). More importantly, the addition of aPC significantly inhibited T cell adhesion to inflamed HIMECs (P < 0.05; Figure 7).

Figure 7

Inhibition of T cell adhesion to TNF-α–inflamed HIMECs by recombinant aPC. HIMEC monolayers were left untreated or stimulated with TNF-α with or without recombinant human aPC. Calcein-labeled MOLT4 T cells were added to the HIMEC monolayers. Values are mean ± SEM of 4 separate experiments. *P < 0.05 versus TNF-α without aPC.

Impairment of PC activation in dextran sodium sulfate–induced murine colitis. Inflammation downregulates the expression of TM and EPCR in several experimental models of inflammation, resulting in an impairment of PC activation (16, 17). As experimental colitis is characterized by extensive intestinal microvascular inflammation (37, 38), this should be followed by a decrease of PC conversion into aPC in the inflamed mucosa. To verify this, we injected healthy mice and those with dextran sodium sulfate–induced (DSS-induced) colitis with recombinant PC and measured the in vivo activation of PC by a colorimetric reaction, using a standard curve with known quantities of aPC in pooled murine plasma (19). A marked and significant decrease of PC conversion into aPC was observed in colitic mice versus healthy controls (P < 0.05; Figure 8).

Figure 8

Dextran sodium sulfate (DSS) colitic mice exhibit a reduced capacity for PC activation. Healthy and colitic mice (n = 3 per group) were injected i.v. with exogenous human PC. After 30 minutes, blood was collected into sodium citrate anticoagulant supplemented with benzamidine to protect aPC from inhibition by endogenous irreversible inhibitors. Using the immunocapture assay described in Methods, aPC was measured using the chromogenic tripeptidyl-pNA substrate S-2366; relative capacity for PC activation is presented as the change in absorbance at 405 nm over time. *P < 0.05 versus healthy mice.

Effect of aPC administration on DSS-induced murine colitis. Based on the above results, we next investigated whether restoring normal levels of aPC would be beneficial in experimental colitis by assessing the potential therapeutic effect of aPC administration in the DSS colitis model. Mice received either 0.1 or 1 mg/kg murine recombinant aPC or its vehicle, and the colitis disease activity index (DAI) (39–41) and colonic histology were assessed over a period of 10 days. Compared with vehicle-treated control animals, aPC-treated mice displayed less severe colitis, as demonstrated by less severe weight loss (P < 0.05; Figure 9A) and an improved DAI (P < 0.05; Figure 9B). In addition, at the end of the 10-day treatment schedule, histological assessment showed that aPC-treated mice displayed a marked decrease of inflammation compared with control animals (Figure 9, C and D). No significant differences were found between the 0.1 and 1 mg/kg doses (data not shown). Notably, despite the fact that aPC is a natural anticoagulant, administration of aPC failed to induce any bleeding events in the treated animals.

Figure 9

Therapeutic effects of murine recombinant aPC administration. Animals undergoing DSS treatment were given daily i.v. injections of recombinant murine aPC (1 mg/kg; n = 10) or placebo (n = 12). Weight was monitored (A) as well as disease activity index (DAI) (B). After 10 days, mice were sacrificed, and colons were assessed for histological colitis (C and D). *P < 0.05, aPC versus placebo.

Effect of aPC administration on in vivo leukocyte adhesion in the intestinal microcirculation. Having shown that recombinant aPC inhibits leukocyte adhesion to HIMECs in vitro and that aPC administration improves experimental colitis, we next investigated whether the therapeutic effect of recombinant aPC was mediated by the inhibition of CAM and chemokine production in the intestine followed by inhibition of leukocyte adhesion to the microvascular endothelium in vivo. Mice treated with aPC displayed reduced intestinal levels of VCAM-1, ICAM-1, and IL-8 compared with placebo-treated mice (Figure 10, A and B). In order to assess whether such inhibition was functional, we quantified the number of adhering leukocytes in the colonic microcirculation by intravital microscopy (42). In healthy mice, very few leukocytes adhered to colonic venules (Figure 10C). In contrast, a large number of leukocytes adhered to the colonic microvascular endothelium in mice with DSS-induced colitis (P < 0.001). This difference was dependent on a loss of aPC in the microcirculation of the inflamed colonic mucosa, as demonstrated by treatment of DSS colitic mice with recombinant murine aPC, which reduced the number of adhering leukocyte to the colonic venules by 40% compared with untreated DSS colitic mice (P < 0.05; Figure 10).

Figure 10

aPC inhibits CAM expression, IL-8 production, and leukocyte adhesion to the endothelium in the intestine. Animals undergoing DSS treatment were given daily i.v. injections of recombinant murine aPC or placebo. After 10 days mice were sacrificed, and biopsies were collected for Western blot analysis for VCAM-1 and ICAM-1 (A) or organ culture for IL-8 measurement by ELISA (B). (C) Leukocyte–endothelial cell interactions were assessed by intravital microscopy in 3 groups of animals: healthy mice (n = 5); DSS colitic mice treated for 10 days with placebo (n = 8); and DSS colitic mice treated for 10 days with daily i.v. injections of recombinant aPC (n = 6). For each animal, 3–6 unbranched venules were studied and the mean value was calculated. Leukocyte adhesion is expressed as the number of firmly adherent leukocytes to the endothelium per 100 μm of venule. *P < 0.05 versus placebo; **P < 0.001 versus healthy mice.

The results of the present study show that the PC pathway was defective in both CD and UC patients and that this system was crucial for governing microvascular inflammation in the gut. More importantly, restoring the function of the PC pathway may be a new therapeutic approach for translational studies in IBD.

Under physiological circumstances, the tissue microcirculation functions in an anticoagulant and antiinflammatory fashion, both actions being finely tuned by the PC pathway (15, 16, 43–46). When microvascular inflammation occurs, a number of processes are set in motion, including coagulation, angiogenesis, and immune responses (46–48). The coagulation pathway is one of the first to be elicited, and its activation is followed by proinflammatory phenomena, in part because of the loss of the antiinflammatory activity of the PC pathway. In several inflammatory conditions such as lung inflammation, atherosclerosis, and sepsis there is marked downregulation of both TM and EPCR, leading to a functional impairment of PC conversion into aPC (16, 17). Because mucosal microvascular inflammation and coagulation/thrombosis are well-documented events in IBD (32), it would be reasonable to hypothesize that TM and EPCR would be downregulated (3). Indeed, in vivo we found reduced or absent expression of both components of the PC pathway in the microvasculature of CD and UC compared with normal mucosa. Such downregulation appeared to be present in both forms of IBD and occurred in areas of active mucosal inflammation. To complement the results of these descriptive but essential data with a more mechanistic approach, we reproduced in vitro the downregulation of TM and EPCR by stimulating HIMECs with proinflammatory cytokines in order to mimic the IBD inflamed milieu. Both TNF-α and IL-1β triggered prompt surface downregulation of TM and EPCR, an event caused by inhibition of RNA transcription for TM and by both increased shedding of the soluble form of EPCR and inhibition of RNA transcription for EPCR, thus highlighting the crucial role of inflammatory mediators in regulating expression of TM and EPCR. Furthermore, to study the functional consequence of TM and EPCR downregulation in HIMECs, we studied the cells’ capacity to activate PC in resting and inflammatory conditions. As expected, HIMECs activated PC in resting conditions, but this process was strongly inhibited after treatment with proinflammatory cytokines, consistent with the downregulation of TM and EPCR and reinforcing the notion that inflammation impairs PC activation. Taken together, our in vivo and in vitro data suggest that in IBD mucosal microcirculation, expression and function of the PC pathway are impaired.

In order to investigate whether the observed impaired PC pathway activity could have some therapeutic implication, we supplemented HIMECs with exogenous recombinant aPC and assessed its antiinflammatory effects by studying CAM expression, chemokine production, and leukocyte adhesion to endothelium. In vitro, aPC was a potent antiinflammatory agent: it inhibited TNF-α–induced CAM upregulation and secretion of chemokines including MCP-1, which was already produced at high levels by unstimulated HIMECs. Because CAM upregulation and chemokine production by HIMECs are crucial for leukocyte recruitment into inflamed intestine, we assessed whether the inhibition induced by aPC was functional. We performed a series of T cell–HIMEC adhesion assays and consistently observed that aPC exerted its antiinflammatory activities ultimately by inhibiting T cell adhesion.

Based on our results using human cells, we also hypothesized that during the course of experimental colitis, there would be a progressive impairment of the rate of PC conversion. In agreement with our human data, we found that colitic mice exhibited an impaired capacity to convert PC into aPC, thus reinforcing the concept that during gut inflammation the PC pathway is downregulated and cannot exert its physiological antiinflammatory tasks in the microcirculation.

Based on our understanding of the expression and function of PCs in various inflammatory processes, the important therapeutic implications of manipulating the PC system have begun to emerge (49). In several experimental models in which the conversion of PC into aPC is impaired, including asthma, sepsis, and ischemia/reperfusion injury, administration of recombinant aPC has consistently proved to be effective in inhibition of cytokine production, CAM expression, and leukocyte influx into the tissues (26, 30, 50–57). Therefore, we tested the therapeutic efficacy of murine recombinant aPC administration in restoring the functionality of the PC pathway. Importantly, a beneficial therapeutic effect was observed in the DSS model of colitis, resulting in significant reduction in DAI, decreased weight loss, and improvement of the colitis histological score. Our results provide evidence that restoring the PC pathway could be beneficial for IBD treatment. Because our in vitro studies showed that aPC inhibited leukocyte adhesion to endothelium, and because blocking leukocyte-endothelial interactions is a very promising approach for IBD, we used intravital microscopy to investigate whether administration of aPC in colitic mice would affect leukocyte rolling and adhesion to intestinal endothelium. Our results showed that aPC administration inhibited leukocyte adhesion to the inflamed intestinal microcirculation using intravital microscopy, thus supporting its antiinflammatory effects in experimental colitis. Taken together, our observations represent a proof-of-concept for the biological importance of and the antiinflammatory role played by the PC pathway in intestinal inflammation. However, future studies using genetically manipulated mice such as PC^–/–, EPCR^δ/δ, and TM^pro/pro mice will help to address the mechanism whereby defects in PC are associated with amplification of inflammation and whether our therapeutic effects by using recombinant aPC are dependent or independent of EPCR or TM.

The complexity of IBD is well recognized, stemming from limited knowledge about its cause and mechanisms and uncertainty as to whether original pathogenic events persist throughout its course or whether secondary phenomena take over control of inflammation. Until these issues are resolved, it is appropriate to focus on all components whose manipulation may disrupt the cycle of chronic gut inflammation. Given the relevance of immune-nonimmune interactions to IBD, we propose that the endothelial PC pathway is a crucial system in governing microvascular inflammation at the intestinal mucosal level. Our studies complement and strengthen the mounting body of evidence pointing to vascular inflammation as a key pathogenic component of autoimmune and chronic inflammatory disorders. Considering that the treatment of IBD is still a major practical challenge and that the heterogeneity of the patient response to therapy is likely a result of the heterogeneity of mechanisms of inflammation, the significance of our study is further reinforced by the possibility that manipulation of the PC pathway can offer another tool in the therapeutic arsenal against IBD and may be a justifiable new focus for translational studies in both CD and UC.

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This work was supported by grants from the Broad Medical Research Program, by “Premio Giovane Ricercatore” of the Societá Italiana di Medicina Interna, by a grant from the Associazione per le Malattie Infiammatorie Croniche dell’Intestino (A.M.I.C.I. Lombardia) to S. Danese, and by a grant from Ministerio de Educania’n y Ciencia (SAF2005-00280) to M. Sans. The authors thank R. Santangelo and E. Stigliano for technical assistance with mouse experiments and A. Doni for help with confocal imaging and analysis.

Address correspondence to: Silvio Danese, Laboratory of Immunology and Inflammation, Division of Gastroenterology, Istituto Clinico Humanitas—IRCCS in Gastroenterology, Viale Manzoni, Rozzano, Italy. Phone: 011-39-3392318230; Fax: 011-39-0282244590; E-mail: sdanese@hotmail.com.

Nonstandard abbreviations used: aPC, activated PC; CAM, cell adhesion molecule; CD, Crohn disease; ENA-78, epithelial-derived neutrophil-activating peptide 78; EPCR, endothelial PC receptor; HIMEC, human intestinal microvascular endothelial cell; IBD, inflammatory bowel disease; PC, protein C; TM, thrombomodulin; UC, ulcerative colitis.

Conflict of interest: B. Gerlitz and B.W. Grinnell are employees of Lilly Research Laboratories, a division of Eli Lilly & Company Inc., which markets recombinant human activated protein C (Xigris) for treatment of high-risk severe sepsis.

Reference information: J. Clin. Invest.117:1951–1960 (2007). doi:10.1172/JCI31027

Miquel Sans and Stefania Vetrano contributed equally to this work.

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