Hepatic endothelial CCL25 mediates the recruitment of CCR9+ gut-homing lymphocytes to the liver in primary sclerosing cholangitis - PubMed (original) (raw)

Hepatic endothelial CCL25 mediates the recruitment of CCR9+ gut-homing lymphocytes to the liver in primary sclerosing cholangitis

Bertus Eksteen et al. J Exp Med. 2004.

Abstract

Primary sclerosing cholangitis (PSC), a chronic inflammatory liver disease characterized by progressive bile duct destruction, develops as an extra-intestinal complication of inflammatory bowel disease (IBD) (Chapman, R.W. 1991. Gut. 32:1433-1435). However, the liver and bowel inflammation are rarely concomitant, and PSC can develop in patients whose colons have been removed previously. We hypothesized that PSC is mediated by long-lived memory T cells originally activated in the gut, but able to mediate extra-intestinal inflammation in the absence of active IBD (Grant, A.J., P.F. Lalor, M. Salmi, S. Jalkanen, and D.H. Adams. 2002. Lancet. 359:150-157). In support of this, we show that liver-infiltrating lymphocytes in PSC include mucosal T cells recruited to the liver by aberrant expression of the gut-specific chemokine CCL25 that activates alpha4beta7 binding to mucosal addressin cell adhesion molecule 1 on the hepatic endothelium. This is the first demonstration in humans that T cells activated in the gut can be recruited to an extra-intestinal site of disease and provides a paradigm to explain the pathogenesis of extra-intestinal complications of IBD.

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Figures

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 < 0.01). 5% of PBLs and virtually all lamina propria CD3+ cells in Crohn's disease expressed CCR9, whereas <2% of LILs in PBC were CCR9+. The graph shows percentage of CCR9+/CD3+ cells as mean ± SEM for each group. (B) CCR9 is coexpressed with α4β7 on PSC LIL. (C) CCR9 was expressed on both CD8 and CD4+ LILs in PSC (CD8 63% vs. CD4 35%; not depicted). PSC CD8+ LILs are predominantly memory cells as demonstrated by their expression of high levels of CD11a (left). Gating of PSC LILs on CCR9 and CD8 reveals that all the CCR9+ cells are CD11ahigh and the majority also express CD45RA, confirming that they are long-lived memory cells. (D) PSC LILs were enriched for CD45RA+ and CCR9+ and their response to PHA stimulation was compared with that of CD45RA+ naive T cells from blood. After 4 h of PHA stimulation, intracellular cytokine staining of CD45RA+ PBLs showed a marked increase in IL-2, but minimal IFNγ production, consistent with a naive phenotype, whereas most CCR9+ CD45RA+ (CD11ahigh)-enriched PSC LILs stained for IFNγ consistent with memory cells. Isotype-matched controls are depicted as white histograms with positive cytokine staining as overlapping black histograms.

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), and in portal tracts where macrophages/dendritic cells (confirmed by CD68 coexpression; not depicted) stained strongly (A3). There was no detectable CCL25 staining of hepatocytes, bile ducts, or vascular endothelium in normal liver (A4) or other chronic inflammatory diseases including primary biliary cirrhosis (A5). Staining with isotype-matched control antibodies was negative (A6). (B) Dual color immunofluorescence (B1/B4, yellow merged image) colocalized staining with CCL25 antibody (B2/B5, green) and CD31 antibody (B3, red) or LYVE-1 antibody (B6, red) to sinusoidal endothelial cells. Nulcei were counterstained with DAPI blue. (C) Western blotting confirmed the immunohistochemistry findings with detection of CCL25 in all PSC livers, but minimal detection in normal or other chronically inflamed livers. P1-7, PSC; NL, normal liver; PB1,2, PBC; ALD, alcoholic liver disease. Protein loading was normalized with β-actin staining. (D) Real-time PCR confirmed a 10-fold increase of CCL25 mRNA in PSC compared with control tissue (normal skin). *, P < 0.001. Modest amounts of CCL25 mRNA were detected in NL and other liver samples (BAT, biliary atresia; ALD and PBC) but the levels were not statistically significant compared with skin.

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 by an antibody to α4β7 or preincubation with pertussis toxin. BSA served as a mock substrate, and MnCl2 was used as a nonsignaling trigger of integrin adhesion. Binding of normal lymphocytes to ICAM-1 in response to Mn confirms that these cells could be triggered to bind via integrins. There was no significant binding to MAdCAM-1 in the absence of CCL25 or Mn. (B) CCL25 (16 ± 7%), CXCL12 (29 ± 10%), and CCL5 (19 ± 6%) all stimulated chemotaxis of PSC liver-derived lymphocytes in invasion chambers in vitro (*, P < 0.02, n = 7). Phenotyping of transmigrated lymphocytes by flow cytometry confirmed that the CCR9+ LILs responded to CCL25, but not to CXCL12 or CCL5, suggesting that CCL25 is the dominant chemokine recruiting CCR9+ LIL. (C) Expression of CXCL12 is restricted to biliary epithelium (C1) and is not detected on portal (C1, arrow) or sinusoidal endothelium (C2) in PSC. No staining was seen in isotype matched control sections (C3). Thus, CXCL12 is unlikely to be important in recruiting CCR9+ cells to the liver, but might be involved in positioning/retaining recruited cells around bile ducts.

References

1. Chapman, R.W. 1991. Aetiology and natural history of primary sclerosing cholangitis–a decade of progress? Gut. 32:1433–1435. - PMC - PubMed
1. Grant, A.J., P.F. Lalor, M. Salmi, S. Jalkanen, and D.H. Adams. 2002. Homing of mucosal lymphocytes to the liver in the pathogenesis of hepatic complications of inflammatory bowel disease. Lancet. 359:150–157. - PubMed
1. Befeler, A.S., T.W. Lissoos, T.D. Schiano, H. Conjeevaram, K.A. Dasgupta, J.M. Millis, K.A. Newell, J.R. Thistlethwaite, and A.L. Baker. 1998. Clinical course and management of inflammatory bowel disease after liver transplantation. Transplantation. 65:393–396. - PubMed
1. Nagata, S., C. McKenzie, S.L. Pender, M. Bajaj-Elliott, P.D. Fairclough, J.A. Walker-Smith, G. Monteleone, and T.T. MacDonald. 2000. Human Peyer's patch T cells are sensitized to dietary antigen and display a Th cell type 1 cytokine profile. J. Immunol. 165:5315–5321. - PubMed
1. von Andrian, U.H., and C.R. Mackay. 2000. T-cell function and migration. Two sides of the same coin. N. Engl. J. Med. 343:1020–1034. - PubMed

Hepatic endothelial CCL25 mediates the recruitment of CCR9+ gut-homing lymphocytes to the liver in primary sclerosing cholangitis - PubMed (original) (raw)