Approximately 20% of LILs in PSC expressed CCR9+, whereas <2% were detected in livers of organ donors or patients with other chronic inflammatory liver diseases (). The mean channel fluorescence of CCR9 on LILs in PSC was lower than on PBL, suggesting that CCR9 may have been down-regulated on LILs by ligand interactions. However, CCR9 expression on LILs remained functional and was still able to trigger static adhesion and induce chemotaxis. CCR9+ expression was strongly associated with coexpression of the gut integrin α4β7.
Figure 1. CCR9 expression and phenotyping of peripheral blood and tissue infiltrating lymphocytes. (A) 17% of liver-infiltrating T cells from PSC patients expressed CCR9+ compared with <1% normal PBLs and <2% PBLs from PSC patients (*, P < (more ...)
A functional role for CCR9 in PSC was supported by the strong CCL25 expression detected in the liver in PSC, but not in other chronic inflammatory diseases. CCL25 was detected in portal dendritic cells/macrophages and hepatic sinusoidal endothelium at areas of interface hepatitis in PSC, but was absent from other chronic inflammatory diseases (). The specificity of CCL25 staining for PSC was striking and we confirmed this using Western blotting with which we were unable to detect CCL25 in other liver diseases including primary biliary cirrhosis, another inflammatory biliary disease. Real-time PCR confirmed a 10-fold increase in CCL25 mRNA above control tissue. This is the first report of CCL25 expression outside the thymus or small intestine, and the lack of significant CCL25 up-regulation in other inflammatory liver diseases suggests that this aberrant expression is specific to PSC and likely to be involved in the pathogenesis of the disease.
Figure 2. CCL25 expression in the liver. (A) The livers of patients with PSC demonstrated strong sinusoidal staining with CCL25 ab (A1, brown pigment), staining was particularly intense in periportal areas in association with areas of interface hepatitis (A2), (more ...)
To determine that CCR9 is functionally active on LILs, we used adhesion and migration assays. CCR9+
LILs migrated preferentially to CCL25 rather than to CXCL12 or CCL5 and could also be triggered by CCL25 to bind immobilized MAdCAM-1 via α4β7 integrins (). CCL25-dependent migration and adhesion were both inhibited by pertussis toxin, suggesting that they were mediated by G proteins activated via CCR9. The CCR9+
T cells in the liver are primed/memory cells as demonstrated by their expression of high levels of CD11a (). The majority are also CD45RA+
, suggesting that they belong to the recently described long-lived revertant memory population (25
). We confirmed they are functional memory cells by demonstrating their ability to secrete IFNγ in response to PHA activation. The presence of functional CCR9 and α4β7 on liver-infiltrating memory T lymphocytes is strong evidence that they are of mucosal origin activated in the gut and subsequently recruited to the liver.
Figure 3. CCL25 triggers adhesion to MAdCAM-1 and chemotaxis of PSC LIL. (A) PSC LILs demonstrated a >20-fold dose-dependent increase in adhesion to MAdCAM-1 under the influence of 10 ng/ml CCL25 compared with normal LILs (+, P = 0.002), which was blocked (more ...)
The overlapping expression of endothelial adhesion molecules and now tissue-specific chemokines between the liver and the gut suggests that, under particular circumstances, effector/memory lymphocytes are able to migrate to both sites as part of an entero-hepatic T cell recirculation. Such a mechanism may have evolved to provide surveillance against gut-derived antigens entering the liver via the portal circulation and is likely to be important in the pathogenesis of the immune-mediated hepatic complications of IBD. We propose that T cells activated in the gut during episodes of active IBD differentiate into effector cells that have the ability to bind to both hepatic and mucosal endothelium. Under noninflamed conditions, these cells may be able to enter the liver via interactions with VAP-1, an adhesion molecule that is constitutively expressed on liver endothelium and up-regulated on inflamed mucosal endothelium in IBD. Some of the effector T cells generated in the organized lymphoid tissue of the gut during active IBD (4
) will revert to long-lived memory cells with the ability to recirculate to the liver and subsequently to trigger hepatic inflammation under the right conditions, even in the absence of active gut inflammation. The activation and expansion of these memory cells in the liver may result in the induction of MAdCAM-1 and CCL25 in the liver, promoting the recruitment of CCR9+
mucosal T cells and the development of established inflammation (1
). This model, in which long-lived gut-derived memory T cells recirculate through the liver, could explain why IBD and PSC do not always occur at the same time and how PSC can be present for the first time in patients with IBD whose colons have been removed many years before (1
). The alternative hypothesis, in which the extra-intestinal complications of IBD are driven by activated effector cells that are a consequence of the continuing release of factors by the inflamed colon, can explain the joint and skin complications that run concomitantly with active bowel disease (26
) but not how PSC can develop de novo after colectomy or why the activity of liver disease does not parallel bowel inflammation.
PSC is associated with both ulcerative colitis and Crohn's disease, and usually develops in patients with colitis, suggesting that colonic inflammation is critical. Because CCL25 is largely confined to Peyer's patches and the small bowel mucosa, other gut-specific adhesion molecules might be involved (including CCR10) that may have a role in the recruitment of effector cells to the colon (14
). However, recent papers show significant increases in CCR9+
lymphocytes in the blood during colitis, suggesting that colonic inflammation activates the CCR9 mucosal T cell pool (16
Our results demonstrate for the first time aberrant expression of CCL25 outside the gut or thymus associated with infiltration of CCR9+ T cells into the liver. The ability of CCL25 to activate migration and α4β7-mediated adhesion of LILs to MAdCAM-1 suggests that CCL25 and MAdCAM-1 cooperate in the recruitment of mucosal lymphocytes to the liver in PSC.