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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

B cells are present within chronically inflamed liver tissue and recent evidence implicates them in the progression of liver disease. In addition, a large proportion of hepatic lymphomas are of B-cell origin. The molecular signals that regulate normal and malignant B-cell recruitment into peripheral tissue from blood are poorly understood, leading us to study human B-cell migration through hepatic sinusoidal endothelial cells in flow-based adhesion assays. In such assays, human blood-derived B cells were captured from shear flow without a previous rolling phase and underwent firm adhesion mediated by vascular cell adhesion molecule-1 (VCAM-1). Unlike T cells, which displayed vigorous crawling behavior on the endothelium, B cells remained static before a proportion underwent transendothelial migration mediated by a combination of intercellular adhesion molecule-1 (ICAM-1), vascular adhesion protein-1, common lymphatic endothelial and vascular endothelial receptor-1/stabilin-1, and the chemokine receptors, CXCR3 and CXCR4. B-cell lymphoma cell lines and primary malignant B cells from patients with chronic lymphocytic leukemia and marginal zone B cell lymphoma also underwent integrin-mediated firm adhesion involving ICAM-1 and/or VCAM-1 and demonstrated ICAM-1-dependent shape-change and crawling behavior. Unlike primary lymphocytes, the malignant cells did not undergo transendothelial migration, which could explain why lymphomas are frequently characterized by the intravascular accumulation of malignant cells in the hepatic sinusoids. Conclusion: Our findings demonstrate that distinct combinations of signals promote B-cell recruitment to the liver, suggesting the possibility of novel targets to modulate liver inflammation in disease. Certain features of lymphocyte homing are maintained in lymphoma recruitment to the liver, suggesting that therapeutic targets for lymphocyte recruitment may also prevent hepatic lymphoma dissemination. (HEPATOLOGY 2012)

The liver is a unique environment for lymphocyte recruitment, which occurs within the low-shear environment of the hepatic sinusoids mediated by interactions with hepatic sinusoidal endothelial cells (HSECs).1, 2 Leukocytes entering from the blood through the sinusoids undergo sequential adhesive interactions with HSECs, but these differ in important respects when compared to the classical endothelial adhesion cascade. In particular, classical rolling adhesion is not observed and the initial tethering step is brief and selectin independent. In addition to integrin- and chemokine-mediated steps common to all vascular beds, nonclassical adhesion molecules have been reported to be involved in the hepatic sinusoids. These include vascular adhesion protein-1 (VAP-1) and the common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1)/stabilin-1.1, 3, 4 To date, the molecular basis of B-cell recruitment to the liver is unknown, and most studies have focussed on T cells.3, 5-7 Here, we report that human B cells can be captured from flow by HSECs and that they use a distinct combination of endothelial adhesion molecules to adhere to and migrate through HSECs under flow.

A large proportion of liver-infiltrating lymphomas are B cell in origin,8, 9 and this led us to compare the behavior of primary B cells with malignant B cells. The patterns of lymphoma infiltration within the liver can be broadly divided into nodular, portal, and sinusoidal, and hepatic involvement is an important part of staging, being associated with poor prognostic markers and “B” symptoms.8, 10 Homing mechanisms must contribute to these hepatic lymphomas, because the majority arise as a consequence of dissemination from a primary site and malignant B cells maintain their ability to traffic.10-12 We thus extended our studies to investigate the behavior of two non-Hodgkin's lymphoma (NHL) B-cell lines, as well as circulating primary malignant B cells, and report on distinct differences in the molecular mechanisms of adhesion, crawling, and transmigration in these cells.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Isolation and Culture of Human HSECs.

Liver endothelial cells (ECs) were isolated from human liver tissue obtained from explanted livers or donor tissue surplus to surgical requirements, as described previously.4 All tissue was collected from patients in the Liver Unit at the Queen Elizabeth Hospital in Birmingham (Birmingham, UK) with informed consent and under local ethics committee approval. In brief, approximately 30 g of tissue underwent collagenase digestion (0.2% collagenase type Ia; Sigma-Aldrich, St. Louis, MO). The digested tissue was placed over a 33%/77% Percoll (Amersham Biosciences, GE Healthcare, Little Chalfont, UK) density gradient. ECs were isolated by immunomagnetic selection using antibodies (Abs) against CD31 conjugated to Dynabeads (Invitrogen, Paisley, UK). ECs were then cultured in medium composed of human endothelial basal growth medium (Invitrogen), 10% AB human serum (HD Supplies, Glasgow, UK), 10 ng/mL of vascular endothelial growth factor (VEGF), and 10 ng/mL of hepatocyte growth factor (HGF) (PeproTech, Peterborough, UK). Cells were grown in collagen-coated culture flasks and were maintained at 37°C in a humidified incubator with 5% CO2 until confluence. Using this protocol, a sufficient number of cells were isolated from either diseased or healthy tissue for use in functional assays.

Isolation of Primary Human B Cells.

Peripheral blood lymphocytes were isolated as previously described by density-gradient centrifugation over Lympholyte (VH Bio, Gateshead, UK) for 25 minutes at 800×g.13 Harvested lymphocytes were washed in phosphate-buffered saline (PBS) and resuspended in RPMI 1640 with 10% fetal calf serum (FCS). CD4, CD8, and B-cell populations were isolated by using negative immunomagnetic selection kits (Invitrogen). Kits were used as per the manufacturer's instructions. Highly pure populations of untouched peripheral blood B cells were obtained. Flow cytometry demonstrated greater than 98% expression of CD19 on isolated populations.

Lymphoma Cell Lines.

Non-Hodgkin's B-cell lymphoma lines, CRL-2261,14 and Karpas 42215 as well as the Jurkat T-cell line, obtained from Cancer Research UK Cell Services (London, UK), were cultured in medium composed of RPMI 1640 and 10% FCS.

Isolation of Primary Malignant B Cells.

Patients with high levels of circulating malignant B cells were identified, one with chronic lymphocytic leukemia (CLL) and one with a marginal zone B-cell lymphoma (MZL). Blood was collected from these patients with informed consent and under local ethics committee approval. Peripheral blood lymphocytes were isolated, as previously described,13 by density-gradient centrifugation over Lympholyte (VH Bio) for 25 minutes at 800×g. Harvested lymphocytes were washed in PBS and resuspended in RPMI 1640 with 10% FCS. T cells were depleted using anti-CD3 Abs (OKT3; Janssen Cilag, High Wycombe, UK) and antimouse immunoglobulin G (IgG)-coated beads (Invitrogen). Flow cytometry demonstrated that >90% of the isolated peripheral lymphocyte population in these patients was positive for the B-cell marker, CD19.

Flow Cytometry.

Cell lines and peripheral blood mononuclear cells were washed, resuspended, and labeled with different fluorochrome-labeled primary Abs against chemokine receptors at optimal dilutions at 4°C, followed by a washing step with PBS and 5% FCS. Samples were analyzed on a Dako Cyan Flow cytometer using Summit 4.3 Software (DakoCytomation, Glostrup, Denmark). The following Abs were used for fluorescence-activated cell-sorting (FACS) analysis of chemokine receptors and B-cell subsets: CCR6 (CTC5/FAB 1802P); CCR7 (150503/FAB197A); CXCR3 (49801/FAB160A); CXCR4 (12G5/FAB170P); and CXCR5 (51505/FAB190P) and were purchased from R&D Systems (Abingdon, UK). CD19 (MOPC-21/555413) was purchased from BD Pharmingen (Swindon, UK), and CD27 (O323/302822) was purchased from BioLegend (Cambridge, UK). The following Abs were used for integrin expression: alpha L/CD11a (clone 345913); beta 2/CD18 (clone 212701); beta 1/CD29 (clone P5D2); and alpha 4/CD49d (clone 265329) and were all purchased from R&D Systems.

Flow Adhesion Assays.

B-cell interaction with human HSECs was studied in flow-based adhesion assays using confluent monolayers of HSECs grown in chamber slides (Ibidi, Munich, Germany) and stimulated with tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ) for 24 hours at 10 ng/mL. We have previously demonstrated that cytokine treatment of human HSECs with TNF-α and IFN-γ led to increased cell-surface expression of intercellular adhesion molecule-1 (ICAM-1) and CLEVER-1, whereas VAP-1 expression was unaffected by these cytokines.3, 4, 13

In some experiments, the endothelial monolayers were incubated with CXCL12 (300 ng/mL; Peprotech EC Ltd., London, UK) 2 hours before assays. Chamber slides were connected to a flow system, as previously described.4 Purified populations of B cells (1 × 106 cells/mL), lymphoma cell lines Karpas 422 and CRL-2261 (0.5 × 106 cells/mL), or primary malignant B cells (1 × 106 cells/mL) were perfused in flow media (endothelial-basal media supplemented with 0.01% human serum; Invitrogen) through the chamber slides over the ECs at a shear stress of 0.05 Pa, which mimics physiological flow in the sinusoids.3 Phase-contrast video recordings were made during lymphocyte/cell-line perfusion and allowed offline analysis to determine the proportion of rolling, adherent, and transmigrated cells. Cells appearing phase bright were above the endothelial monolayer, whereas phase dark cells had undergone transmigration through the monolayer. To determine the molecular basis of the interactions in some assays, lymphocytes were incubated with pertussis toxin (200 ng/mL; Sigma-Aldrich) before perfusion to block chemokine activity by G-protein-coupled (GPC) receptors or blocking Abs to specific chemokine receptors for 30 minutes, and HSEC monolayers were incubated with blocking Abs for 30 minutes. Abs used were against CXCR3 (clone 49801, 10 μg/mL; R&D Systems, Minneapolis, MN), CXCR4 (clone 12G5, 10 μg/mL; R&D Systems), ICAM-1 (BBIG-I1,10 μg/mL; R&D Systems), vascular cell adhesion molecule-1 (VCAM-1) (BBIG-V1, 10 μg/mL; R&D systems), VAP-1 (TK8-14, 10 μg/mL; Biotie Therapies, Turku, Finland), CLEVER-1/stabilin-1 (20 μg/mL),16 and isotype-matched controls (mouse IgG1; Dako, Stockport, UK, and IgG2a; R&D Systems). In some experiments, cell lines were pretreated with mitomycin C (Sigma-Aldrich) in RPMI and 10% FCS at a concentration of 25 μg/mL over 24 hours. Cell viability was confirmed by trypan blue staining.

Analysis of Lymphocyte Crawling.

After adhering to the endothelium, in vivo lymphocytes undergo intravascular crawling on the endothelium before undergoing transendothelial migration in response to signals presented on the endothelial surface. To investigate this phenomenon, we analyzed the crawling behavior of T cells, B cells, and B-cell lymphoma cell lines that had adhered to HSECs under flow. Cell-migratory behavior was quantified using ImageJ software (Reference Rasband, W.S., ImageJ, 1997-2011; National Institutes of Health, Bethesda, MD) to manually track each lymphocyte in a field of view over a set period of time. A chemotaxis tool (ibidi, Munich, Germany) allowed this tracking to be plotted graphically.

Static Transmigration Assays.

To study preferential transendothelial migration of B-cell subsets, we performed static transmigration experiments across monolayers of HSECs. HSECs were grown until confluent on collagen-coated 3-μm-pore cell-culture transwell inserts (BD Biosciences, Oxford, UK) and incubated with TNF-α and IFN-γ for 24 hours at 10 ng/mL. A total of 1.2 million peripheral blood lymphocytes in 400 μL of flow media were transferred on the transwell inserts and allowed to transmigrate to the bottom chamber, containing 700 μL of flow medium over 4 hours. The starting population and the cells that had transmigrated were stained for CD19 and CD27 and analyzed by FACS.

To study the migration of CRL-2261 and Karpas 422 cell lines toward CXCL12 and CXCL10, a total of 1.2 million cells were placed in 3-μm-pore cell-culture transwell inserts (BD Biosciences), without an endothelial monolayer, in 24-well plates with flow media or flow media supplemented with either 300 ng/mL of CXCL12 or 300 ng/mL of CXCL10. Cells that had migrated to the bottom well after 4 hours were collected, pelleted, and counted in a manual counting chamber. The Jurkat T-cell line was used as a positive control.

Immunohistochemistry.

All immunostaining was performed on archival formalin-fixed, paraffin-embedded tissues using a Dako automated immunostainer and epitope unmasking carried out using the Dako PT link. After hydrogen peroxide and protein blocks, mouse monoclonal anti-CD20 (M0755; Dako) was applied for 1 hour at a 1:1,000 dilution and visualized with ImmPact Nova Red (Vector Laboratories, Burlingame, CA) or the EnVision HRP kit (Dako). Double immunolabeling was carried out after low-temperature epitope unmasking (ALTER).17 CD20 was visualized with ImmPACT DAB nickel (Vector Laboratories), and, after an avidin/biotin block (Vector Laboratories) and a second protein block, rabbit polyclonal pan-cytokeratin (Z0622; Dako) was applied at a 1:100 dilution for 1 hour and detected with alkaline phosphatase avidin biotin and Vector blue (Vector Laboratories).

Statistical Analysis.

Paired two-tailed t tests were used to assess significance in single treatments; for multiple treatments, variation was assessed using analysis of variance, followed by Dunnett's test for comparison of control. Statistical calculations were performed using Prism 5 software (GraphPad Software, Inc., La Jolla, CA). A P value <0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

B-Cell Interactions With HSECs Under Flow.

When B cells were perfused over monolayers of TNF-α- and IFN-γ-treated HSECs, they were captured from flow and underwent firm adhesion. Unlike T cells, they did not undergo a tethering step before adhesion, but appeared to arrest and bind directly from flow. In addition, after arresting, they displayed minimal crawling across the endothelial monolayer, which was in marked contrast to adherent T cells that demonstrate clear crawling behavior (Fig. 1A).

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Figure 1. Molecular determinants of B-cell transmigration across human HSECs. B cells and T cells were perfused separately over monolayers of cytokine-treated HSECs at a shear stress of 0.05 Pa. (A) Adherent cells within a single field were manually tracked over 5 minutes using ImageJ software. Tracking data were analyzed by plotting the movement of each T cell and each B cell from a common point. Each dot represents an individual lymphocyte, black labeling demonstrates overall movement against the direction of flow, and red labeling demonstrates overall movement in the direction of flow. (B) The effect of blocking of adhesion molecules and chemokine receptors on the number of adherent cells and (C and D) the proportion of adherent B cells transmigrating across HSEC under flow are shown. (E) Static transmigration assays with HSECs were used to determine preferential migration of CD27+ and CD27 CD19+ B cells by comparing the proportion of each subset in pre- and post-transmigration populations. Results are expressed as the means ± standard error of the mean of three independent experiments using different HSECs. *P < 0.05. IMC, isotype-matched control; IC, intercellular adhesion molecule-1; VC, vascular adhesion molecule-1; CL, CLEVER-1/stabilin-1; PT, pertussis toxin; VAP, vascular adhesion protein-1.

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We analyzed the molecules involved in B-cell recruitment by HSECs. We studied classical adhesion receptors ICAM-1 and VCAM-1 as well as VAP-1 and CLEVER-1, which our group has shown mediate the transendothelial migration of T cells across HSECs.3, 4 VCAM-1 was the predominant capture receptor for primary B cells (Fig. 1B). Furthermore, B-cell capture and adhesion was chemokine independent, because pertussis blockade had no effect on the numbers of B cells undergoing firm adhesion (Fig. 1B), although it did inhibit transendothelial migration (Fig. 1C). We have reported previously that the proportion of adherent CD4+ and CD8+ T cells undergoing transendothelial migration across HSECs ranges from 12% to 23%.4 In this study, the proportion of adherent B cells undergoing transmigration ranged from 6.5% to 8.6%. The transmigration of B cells was reduced significantly by the blockade of ICAM-1, VAP-1, and CLEVER-1, and blocking all three receptors had an additive effect and abolished 75% of the transmigration (Fig. 1C).

Pertussis blockade led to a 50% reduction in B-cell transmigration, confirming the role of chemotactic signals in B-cell transendothelial migration. We studied the specific chemotactic signals that contribute to transendothelial migration by blocking CXCR3 and CXCR4. These receptors were chosen because their ligands are expressed in inflamed hepatic sinusoids.13, 18 Both CXCR3 and CXCR4 contributed to B-cell migration, although only CXCR3 blockade led to a statistically significant reduction in transendothelial migration (Fig. 1D).

Other groups have demonstrated the accumulation of CD27+ memory B cells expressing CXCR3 in chronic hepatitis C, suggesting that CD27+ B cells are preferentially recruited to the inflamed liver.19 Transwell assays with human HSECs demonstrated an enrichment of the CD27+ population after transmigration, but transmigration was not an exclusive property of the CD27+ population (Fig. 1E).

To assess whether B-cell recruitment is associated with specific liver diseases, we analyzed B cells in inflamed liver tissue from several different liver diseases. B cells were detected throughout the hepatic parenchyma and in aggregates in tertiary follicles in primary biliary cirrhosis (PBC), autoimmune liver disease, hepatitis C, and nonalcoholic steatohepatitis, confirming that B-cell infiltration is a characteristic of many chronic liver diseases (Fig. 2 A,B).

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Figure 2. B cells are found in the parenchyma and in lymphoid aggregates in chronically inflamed human liver. (A) Immunohistochemical staining of paraffin-embedded liver tissue demonstrating the presence of CD20-positive B cells in hepatic parenchyma from PBC, autoimmune hepatitis (AIH), hepatitis C (HCV), and NASH (nonalcoholic steatohepatitis). Red arrows indicate extrasinusoidal CD20+ B cells (black) in close contact with cytokeratin-marked hepatocytes (blue). (B) Immunohistochemical staining of paraffin-embedded liver tissue demonstrating lymphoid aggregates containing CD20+ B cells (red) in chronically inflamed liver tissue from PBC, AIH, HCV, and in healthy (normal) liver (NL). (A) Original magnification: x200; (B) original magnification: x400.

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Lymphoma Cell Lines.

B-cell lines (e.g., CRL-2261 and Karpas 422) underwent firm adhesion to TNF-α- and IFN-γ-treated HSECs (Fig. 3A,B). Karpas 422 cells behaved similarly to primary B cells, with VCAM-1 playing the predominant role in firm adhesion (Fig. 3A). In contrast, VCAM-1 did not play a significant role in CRL-2261 cell adherence, in which ICAM-1 was the major adhesion receptor (Fig. 3B). Karpas 422 cells also demonstrated minimal crawling, whereas CRL-2261 demonstrated significant crawling behavior across the endothelial monolayer, which was completely inhibited by ICAM-1 blockade (Fig. 3C). We noted that neither cell line underwent transendothelial migration across the monolayer, in contrast to primary cells. Analysis of integrin expression by flow cytometry demonstrated abundant alphaL/beta2 (CD11a/CD18) on the CRL-2261 cell line and alpha4/beta1 (CD49d/CD29) on the Karpas 422 cell line (Fig. 3D).

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Figure 3. Molecular determinants of B-lymphoma cell-line recruitment by human hepatic sinusoidal endothelium. Flow assays using monolayers of cytokine-treated HSECs were performed with the two B-cell NHL cell lines, CRL-2261 and Karpas 422. (A and B) The number of adherent cells after incubation of HSECs with function-blocking Abs to VCAM-1, ICAM-1, VAP-1, and CLEVER-1/stabilin-1 to lymphoma cells was assessed. VCAM-1 blockade led to a significant reduction in Karpas 422 cell adhesion (A), whereas ICAM-1 was the main adhesion receptor for CRL-2261 (B). Results are expressed as the means ± SEM of three independent experiments using different HSECs. (C) CRL-2261 cells were perfused over monolayers of cytokine-treated HSECs at a shear stress of 0.05 Pa. Adherent cells within a single field were manually tracked over 5 minutes using ImageJ software. Each dot represents an individual cell, black labeling demonstrates overall movement against the direction of flow, and red labeling demonstrates overall movement in the direction of flow. Experiments were repeated after the incubation of HSECs with ICAM-1-blocking Abs. (D) Flow cytometry demonstrated strong expression of CD11a/CD18 (LFA-1, alphaL beta2 integrin) on CRL-2261 and expression of CD49d/CD29 (VLA-4, alpha4 beta1 integrin) on Karpas 422. (E) Mitomycin C treatment (25 μg/mL over 24 hours) led to a reduction in total adhesion, but did not promote the transendothelial migration of B cell lines across monolayers of HSECs under flow. Means ± SEM of two independent experiments are shown. *P < 0.05. IMC, isotype-matched control; M, mitomycin C; SEM, standard error of the mean.

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It has been reported that cells actively undergoing cell division are unable to transmigrate across the endothelium.20 Flow assays were therefore repeated after pretreatment with mitomycin C to block cell division. Although it led to a reduction in the adherence of the cell lines to HSECs, it did not promote transmigration (Fig. 3E).

Chemokines play a vital role in lymphocyte adhesion and subsequent transmigration, and it has been reported that they continue to play an important role in the homing of lymphocytes that have undergone malignant transformation.12 We therefore analyzed the chemokine receptor expression of the cell lines to investigate whether the malignant cells were lacking a chemokine signal necessary for transendothelial migration. FACS analyses demonstrated that both the cell lines expressed CXCR3, which we have previously reported to be important for T-cell transendothelial migration across HSECs, and CXCR4 (Fig. 4A). Our assays involved cytokine stimulation with IFN-γ and TNF-α, which, we have shown previously, leads to the presentation of CXCR3 ligands promoting the transendothelial migration of CXCR3+ lymphocytes.13 We assessed the functional activity of CXCR3 and CXCR4 on the B-cell lines by measuring chemotaxis to CXCL12 and CXCL10 in transwell assays. Only Karpas 422 showed dose-dependent migration toward CXCL12 (Fig. 4B), and neither cell line migrated to CXCL10. The addition of CXCL12 to the flow-based adhesion assays resulted in a reduction in the round adherent cells and an increase in shape-changed cells, reflecting increased motility and intravascular crawling in both cell lines. However, there was still no detectable transendothelial migration (Fig. 4C). We also carried out flow assays with primary malignant B cells. Samples from patients with CLL and MZL demonstrated adhesion to cytokine-treated HSECs under conditions of flow (Fig. 4D). ICAM-1 and VCAM-1 contributed to the CLL adhesion to HSECs, whereas VCAM-1 predominated in the adhesion of the MZL (Figure 4E). Less than 1% of cells demonstrated transendothelial migration, in keeping with our findings with the lymphoma cell lines (data not shown). Immunostaining of liver sections from a patient with hepatic B-cell lymphoma demonstrated a sinusoidal pattern of infiltration consistent with a failure of the infiltrating cell to transmigrate across the sinusoidal endothelium in vivo (Fig. 4F).

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Figure 4. Recruitment of lymphoma cell lines and primary malignant B cells by human hepatic sinusoidal endothelium. (A) Chemokine receptor expression of primary B cells and the CRL-2261 and Karpas 422 cell lines was assessed using flow cytometry. Both cell lines were highly positive for CXCR3 and CXCR4. (B) Transwell assays were performed to assess the migration of lymphoma cell lines across a chemotactic gradient with CXCL12 or CXCL10 (300 ng/mL) through 3-μm pores. Karpas 422 cell lines demonstrated significant chemotaxis toward CXCL12, but this was not observed with CRL-2261. Neither cell line migrated toward CXCL10. Jurkat T cells were used as positive controls. Results are expressed as the mean ± SEM of two sets of triplicates. (C) Flow-based adhesion assays in which Karpas 422 and CRL-2261 cells were flowed over HSECs treated with 300 ng/mL of CXCL12. The addition of CXCL12 significantly increased the proportion of shape-changed cells, but did not lead to transendothelial migration. Results are expressed as a percentage of adherent cells. Results are the mean ± SEM of three independent experiments using different HSECs. (D) Flow assays using monolayers of cytokine-treated HSECs were performed with primary malignant B cells from patients with B-cell CLL and MZL. Total adhesion of primary malignant B cells was compared to the adhesion of primary B cells and the NHL cell lines, CRL 2261 and Karpas 422. (E) The contribution of VCAM-1 and ICAM-1 to the adhesion of malignant B cells to HSECs under flow was assessed using function-blocking Ab. Graphs represent mean ± SEM of two experiments using different HSECs. (F) Immunohistochemical staining of paraffin-embedded liver tissue from a patient with hairy cell leukemia showing diffuse sinusoidal infiltration by CD20-positive B cells. Original magnification: x200. *P < 0.05; ***P < 0.0005. SEM, standard error of the mean.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Previous studies of lymphocyte recruitment to the liver have concentrated on T cells, but there is currently a gathering interest in the role of B cells in the development and progression of chronic inflammatory liver disease. The frequency of B cells in the healthy liver has been reported to be less than 10% of the intrahepatic lymphocyte population,21 although one study found that B cells represent approximately half of the intrahepatic lymphocyte population in the adult mouse.22 In chronic inflammatory liver diseases, these numbers increase markedly because of clonal expansion of resident cells and increased recruitment of B cells from the blood. B cells are found throughout the liver, but at particularly high frequencies in portal lymphoid aggregates in chronic hepatitis C and chronic inflammatory diseases, such as PBC.23, 24 Despite this, there is a paucity of information describing the molecular mechanisms guiding B-cell recruitment to hepatic tissue. Here, we demonstrate that primary B cells use predominantly VCAM-1 to bind HSECs from flow. This differs from T cells, which use ICAM-1 and beta1 integrins in the same system. The absence of an effect of pertussis toxin on B-cell adhesion to the sinusoidal endothelium is another difference, when compared to T cells. This indicates that chemokine-mediated signals are not required for arrest/adhesion under flow.25 This could be because the cells do not require integrin triggering in a low-flow environment or because integrins are activated by a G-protein-independent mechanism. For instance, beta1 integrin-dependent pathways could be responsible for tethering directly,26 as well as mediating stable adhesion as they become more activated.26, 27

An additional difference between B- and T-cell behavior is the markedly reduced motility of B cells on and through the endothelial monolayer. A smaller proportion of adherent B cells subsequently undergo transmigration, when compared to T cells. This may be a consequence of the greatly reduced crawling behavior exhibited by B cells on HSECs in our tracking studies (Fig. 1A). Intravital studies have shown that leukocyte crawling is an essential step before efficient transmigration, and thus reduced B-cell motility on the endothelium will lead to a reduction in transendothelial migration.28 The numbers of B cells that underwent transmigration was significantly reduced by pertussis toxin, implicating GPC receptors, and by blocking ICAM-1, VAP-1, or CLEVER-1/stabilin-1, but not VCAM-1. Combined inhibition of all three adhesion molecules reduced transmigration by 75%. We have previously shown that VAP-1 is implicated in the adhesion of several leukocyte types to HSECs, where it contributes to sialic-acid–dependent tethering and transendothelial migration.3, 5, 29, 30 CLEVER-1 supports lymphocyte adhesion and transmigration to the endothelium in lymphoid tissues,16 and it is expressed by the sinusoidal endothelium in the healthy and inflamed liver. We have recently reported its ability to support transendothelial migration of CD4 regulatory T cells, but not CD4 effectors or CD8 T cells through HSECs,4 and its close homolog, stabilin-2, was also shown to support lymphocyte adhesion to the hepatic endothelium.31 Thus, in our system, B cells and CD4 regulatory T cells use the same combination of ICAM-1/VAP-1 and CLEVER-1 for transendothelial migration through HSECs. This is interesting in light of the evidence that B cells may have immunoregulatory functions within the liver, as demonstrated by the exacerbation of disease activity observed in murine models of PBC when B cells are depleted.24 Pertussis blockade reduced B-cell transmigration by 50%, and antibody blockade implicates both CXCR3 and CXCR4 in transmigration.

We went on to study the behavior of lymphoma cell lines. After secondary lymphoid tissue, the liver is the most-common site for lymphoma infiltration and the majority of hepatic lymphomas are of B-cell origin.8 However, little is known about the molecular mechanisms that underlie this process. NHLs show conserved homing capabilities, most strikingly illustrated by studies reporting that lymphomas arising from gut-associated lymphoid tissue disseminate to the gut, whereas those arising in the skin preferentially traffic to the skin.32 This may be the result of the maintained expression of tissue-specific homing receptors on the malignant cells.33, 34 Our results demonstrate that several features of B-lymphocyte interactions with HSECs are maintained in lymphomas, including the requirement for endothelial activation by proinflammatory cytokines and a preserved role for integrin-mediated firm adhesion under flow. Interestingly, ICAM-1, but not VCAM-1, was involved in capturing the CRL-2261 cell line, whereas VCAM-1 predominated with the Karpas 422 line. Furthermore, the CRL-2261 cell line demonstrated higher motility on ECs, which was also ICAM-1 mediated. Detailed analysis demonstrated that the migratory capabilities of the lymphoma cell lines on the surface of the HSECs overlapped with properties observed in primary lymphocytes. We noted shape change and motility of CRL-2261 cells on the endothelium under flow, and this migration was completely inhibited by ICAM-1 blockade. However, Karpas 422 cells did not display crawling on the endothelium under flow. We excluded the possibility that these cells are unable to migrate because they showed a marked chemotactic response to CXCL12, which has been demonstrated to be a chemoattractant factor for follicular center lymphoma, CLL, and lymphoblastic leukemia.34-37

After stable arrest, leukocytes undergo intravascular crawling and transendothelial migration across endothelial barriers into tissue. To our surprise, we found that the lymphoma cell lines were unable to undergo transendothelial transmigration under flow on HSECs. Even supplementation of the chemokine signal with exogenous CXCL12 failed to induce transendothelial migration, despite inducing shape change. Furthermore, blocking cell division with mitomycin C did not promote transmigration. Thus, it appears that these malignantly transformed cells have lost the ability to transmigrate through the sinusoidal endothelium. If so, this could explain why hepatic lymphomas are often associated with a sinusoidal infiltration pattern in which the malignant cells are observed to remain within the sinusoidal channels (Fig. 4F).8

To confirm our findings in lymphoma cell lines, we studied circulating populations of primary malignant lymphocytes from patients with CLL and MZL. In keeping with the cell-line data, primary malignant cells were able to adhere to human HSECs using ICAM-1 or VCAM-1, but were unable to transmigrate across HSECs.

In conclusion, we have demonstrated the molecular mechanisms involved in primary B-cell recruitment by the hepatic sinusoidal endothelium, and that these molecules could be potential therapeutic targets for chronic inflammatory liver disease. Certain aspects of lymphocyte homing are maintained in lymphoma recruitment to the liver, suggesting that therapeutic targets for lymphocyte recruitment may also prevent lymphoma dissemination to the liver.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  • 1
    Wong J, Johnston B, Lee SS, Bullard DC, Smith CW, Beaudet AL, et al. A minimal role for selectins in the recruitment of leukocytes into the inflamed liver microvasculature. J Clin Invest 1997; 99: 2782-2790.
  • 2
    Xu XD, Ueta H, Zhou S, Shi C, Koga D, Ushiki T, et al. Trafficking of recirculating lymphocytes in the rat liver: rapid transmigration into the portal area and then to the hepatic lymph. Liver Int 2008; 28: 319-330.
  • 3
    Lalor PF, Edwards S, McNab G, Salmi M, Jalkanen S, Adams DH. Vascular adhesion protein-1 mediates adhesion and transmigration of lymphocytes on human hepatic endothelial cells. J Immunol 2002; 169: 983-992.
  • 4
    Shetty S, Weston CJ, Oo YH, Westerlund N, Stamataki Z, Youster J, et al. Common lymphatic endothelial and vascular endothelial receptor-1 mediates the transmigration of regulatory T cells across human hepatic sinusoidal endothelium. J Immunol 2011; 186: 4147-4155.
  • 5
    Bonder CS, Norman MU, Swain MG, Zbytnuik LD, Yamanouchi J, Santamaria P, et al. Rules of recruitment for Th1 and th2 lymphocytes in inflamed liver: a role for alpha-4 integrin and vascular adhesion protein-1. Immunity 2005; 23: 153-163.
  • 6
    Eksteen B, Grant AJ, Miles A, Curbishley SM, Lalor PF, Hubscher SG, et al. Hepatic endothelial CCL25 mediates the recruitment of CCR9+ gut-homing lymphocytes to the liver in primary sclerosing cholangitis. J Exp Med 2004; 200: 1511-1517.
  • 7
    Shetty S, Lalor PF, Adams DH. Lymphocyte recruitment to the liver: molecular insights into the pathogenesis of liver injury and hepatitis. Toxicology 2008; 254: 136-146.
  • 8
    Loddenkemper C, Longerich T, Hummel M, Ernestus K, Anagnostopoulos I, Dienes HP, et al. Frequency and diagnostic patterns of lymphomas in liver biopsies with respect to the WHO classification. Virchows Arch 2007; 450: 493-502.
  • 9
    Baumhoer D, Tzankov A, Dirnhofer S, Tornillo L, Terracciano LM. Patterns of liver infiltration in lymphoproliferative disease. Histopathology 2008; 53: 81-90.
  • 10
    Jaffe ES. Malignant lymphomas: pathology of hepatic involvement. Semin Liver Dis 1987; 7: 257-268.
  • 11
    Kim H, Dorfman RF. Morphological studies of 84 untreated patients subjected to laparotomy for the staging of non-Hodgkin's lymphomas. Cancer 1974; 33: 657-674.
  • 12
    Pals ST, de Gorter DJ, Spaargaren M. Lymphoma dissemination: the other face of lymphocyte homing. Blood 2007; 110: 3102-3111.
  • 13
    Curbishley SM, Eksteen B, Gladue RP, Lalor P, Adams DH. CXCR3 activation promotes lymphocyte transendothelial migration across human hepatic endothelium under fluid flow. Am J Pathol 2005; 167: 887-899.
  • 14
    Fitzgibbon J, Iqbal S, Davies A, O'Shea D, Carlotti E, Chaplin T, et al. Genome-wide detection of recurring sites of uniparental disomy in follicular and transformed follicular lymphoma. Leukemia 2007; 21: 1514-1520.
  • 15
    Dyer MJ, Fischer P, Nacheva E, Labastide W, Karpas A. A new human B-cell non-Hodgkin's lymphoma cell line (Karpas 422) exhibiting both t(14;18) and t(4;11) chromosomal translocations. Blood 1990; 75: 709-714.
  • 16
    Salmi M, Koskinen K, Henttinen T, Elima K, Jalkanen S. CLEVER-1 mediates lymphocyte transmigration through vascular and lymphatic endothelium. Blood 2004; 104: 3849-3857.
  • 17
    Reynolds GM, Billingham LJ, Gray LJ, Flavell JR, Najafipour S, Crocker J, et al. Interleukin 6 expression by Hodgkin/Reed-Sternberg cells is associated with the presence of 'B' symptoms and failure to achieve complete remission in patients with advanced Hodgkin's disease. Br J Haematol 2002; 118: 195-201.
  • 18
    Mendt M, Cardier JE. Stromal-derived factor-1 and its receptor, CXCR4, are constitutively expressed by mouse liver sinusoidal endothelial cells: implications for the regulation of hematopoietic cell migration to the liver during extramedullary hematopoiesis. Stem Cells Dev 2012 Jan 26. doi: 10.1089/scd.2011.0565.
  • 19
    Mizuochi T, Ito M, Saito K, Kasai M, Kunimura T, Morohoshi T, et al. Possible recruitment of peripheral blood CXCR3+ CD27+ CD19+ B cells to the liver of chronic hepatitis C patients. J Interferon Cytokine Res 2010; 30: 243-252.
  • 20
    Berg LP, James MJ, Alvarez-Iglesias M, Glennie S, Lechler RI, Marelli-Berg FM. Functional consequences of noncognate interactions between CD4+ memory T lymphocytes and the endothelium. J Immunol 2002; 168: 3227-3234.
  • 21
    Racanelli V, Rehermann B. The liver as an immunological organ. HEPATOLOGY 2006; 43( 2 Suppl 1): S54-S62.
  • 22
    Novobrantseva TI, Majeau GR, Amatucci A, Kogan S, Brenner I, Casola S, et al. Attenuated liver fibrosis in the absence of B cells. J Clin Invest 2005; 115: 3072-3082.
  • 23
    Racanelli V, Sansonno D, Piccoli C, D'Amore FP, Tucci FA, Dammacco F. Molecular characterization of B cell clonal expansions in the liver of chronically hepatitis C virus-infected patients. J Immunol 2001; 167: 21-29.
  • 24
    Dhirapong A, Lleo A, Yang GX, Tsuneyama K, Dunn R, Kehry M, et al. B cell depletion therapy exacerbates murine primary biliary cirrhosis. HEPATOLOGY 2011; 53: 527-535.
  • 25
    Lalor PF, Curbishley SM, Adams DH. Identifying homing interactions in T-cell traffic in human disease. Methods Mol Biol 2010; 616: 231-252.
  • 26
    Lalor PF, Clements JM, Pigott R, Humphries MJ, Spragg JH, Nash GB. Association between receptor density, cellular activation, and transformation of adhesive behavior of flowing lymphocytes binding to VCAM-1. Eur J Immunol 1997; 27: 1422-1426.
  • 27
    Vajkoczy P, Laschinger M, Engelhardt B. Alpha4-integrin-VCAM-1 binding mediates G protein-independent capture of encephalitogenic T cell blasts to CNS white matter microvessels. J Clin Invest 2001; 108: 557-565.
  • 28
    Phillipson M, Heit B, Colarusso P, Liu L, Ballantyne CM, Kubes P. Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade. J Exp Med 2006; 203: 2569-2575.
  • 29
    McNab G, Reeves JL, Salmi M, Hubscher S, Jalkanen S, Adams DH. Vascular adhesion protein 1 mediates binding of T cells to human hepatic endothelium. Gastroenterology 1996; 110: 522-528.
  • 30
    Yoong KF, McNab G, Hubscher SG, Adams DH. Vascular adhesion protein-1 and ICAM-1 support the adhesion of tumor-infiltrating lymphocytes to tumor endothelium in human hepatocellular carcinoma. J Immunol 1998; 160: 3978-3988.
  • 31
    Jung MY, Park SY, Kim IS. Stabilin-2 is involved in lymphocyte adhesion to the hepatic sinusoidal endothelium via the interaction with alphaMbeta2 integrin. J Leukoc Biol 2007; 82: 1156-1165.
  • 32
    Drillenburg P, Pals ST. Cell adhesion receptors in lymphoma dissemination. Blood 2000; 95: 1900-1910.
  • 33
    Drillenburg P, van der Voort R, Koopman G, Dragosics B, van Krieken JH, Kluin P, et al. Preferential expression of the mucosal homing receptor integrin alpha 4 beta 7 in gastrointestinal non-Hodgkin's lymphomas. Am J Pathol 1997; 150: 919-927.
  • 34
    Noorduyn LA, Beljaards RC, Pals ST, van Heerde P, Radaszkiewicz T, Willemze R, et al. Differential expression of the HECA-452 antigen (cutaneous lymphocyte associated antigen, CLA) in cutaneous and non-cutaneous T-cell lymphomas. Histopathology 1992; 21: 59-64.
  • 35
    Bradstock KF, Makrynikola V, Bianchi A, Shen W, Hewson J, Gottlieb DJ. Effects of the chemokine stromal cell-derived factor-1 on the migration and localization of precursor-B acute lymphoblastic leukemia cells within bone marrow stromal layers. Leukemia 2000; 14: 882-888.
  • 36
    Burger JA, Burger M, Kipps TJ. Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells. Blood 1999; 94: 3658-3667.
  • 37
    Corcione A, Ottonello L, Tortolina G, Facchetti P, Airoldi I, Guglielmino R, et al. Stromal cell-derived factor-1 as a chemoattractant for follicular center lymphoma B cells. J Natl Cancer Inst 2000; 92: 628-635.