Activation of hepatic stellate cells after phagocytosis of lymphocytes: A novel pathway of fibrogenesis - PubMed (original) (raw)

Activation of hepatic stellate cells after phagocytosis of lymphocytes: A novel pathway of fibrogenesis

Nidal Muhanna et al. Hepatology. 2008 Sep.

Abstract

Increased CD8-T lymphocytes and reduced natural killer (NK) cells contribute to hepatic fibrosis. We have characterized pathways regulating the interactions of human hepatic stellate cells (HSCs) with specific lymphocyte subsets in vivo and in vitro. Fluorescence-activated cell sorting (FACS) was used to characterize human peripheral blood lymphocytes (PBLs) and intrahepatic lymphocytes (IHLs) obtained from healthy controls and from patients with either hepatitis B virus (HBV) or hepatitis C virus (HCV) with advanced fibrosis. Liver sections were analyzed by immunohistochemistry and confocal microscopy. To investigate in vitro interactions, PBLs from healthy controls or patients with HCV cirrhosis were co-cultured with an immortalized human HSC line (LX2 cells) or with primary HSCs. Significant alterations in lymphocyte distribution were identified in IHLs but not PBLs. The hepatic CD4/CD8 ratio and NK cells were significantly reduced in HBV/HCV patients. Expression of alpha-smooth muscle actin and infiltration of CD4, CD8, and NK cells were readily apparent in liver sections from patients with cirrhosis but not in healthy controls. Lymphocytes from each subset were in proximity to HSCs primarily within the periportal regions, and some were directly attached or engulfed. In culture, HSC activation was stimulated by HCV-derived CD8-subsets but attenuated by NK cells. Confocal microscopy identified lymphocyte phagocytosis within HSCs that was completely prevented by blocking intracellular adhesion molecule 1 (ICAM-1) and integrin molecules, or by irradiation of HSCs. LX2 knockdown of either Cdc42 or Rac1 [members of the Rho-guanosine triphosphatase (GTPase) family] prevented both phagocytosis and the activation of HSC by HCV-derived lymphocytes.

Conclusion: The CD4/CD8 ratio and NK cells are significantly decreased in livers with advanced human fibrosis. Moreover, disease-associated but not healthy lymphocytes are engulfed by cultured HSCs, which is mediated by the Rac1 and Cdc42 pathways. Ingestion of lymphocytes by HSCs in hepatic fibrosis is a novel and potentially important pathway regulating the impact of lymphocytes on the course of hepatic fibrosis.

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Conflict of interest statement

Potential conflict of interest: Nothing to report.

Figures

Fig. 1

Lymphocyte alterations in fibrotic patients. FACS analysis of isolated intrahepatic and peripheral blood lymphocytes from healthy donors (black bars), HCV (open bars), and HBV (gray bars) fibrotic patients are illustrated (see Patients and Methods). Significant differences were confined to intrahepatic lymphocytes, in which CD4 decreased, leading to an increase of CD4/CD8 ratio (Fig. 1); NK cells decreased in both fibrotic groups.

Fig. 2

Direct lymphocyte-hepatic stellate cell adhesion in situ: Liver biopsies from fibrotic HCV patients (B–D) and healthy controls (A) were stained with primary antibodies. Cy-5 conjugated α-SMA in red was used to stain HSCs, and FITC-CD45 served as a common leukocyte marker. Confocal laser scanning microscopy was used to visualize stained sections as described in Patients and Methods. HSCs (red arrows) were stained as small red cells adjacent to hepatocytes. Stained lymphocytes (white arrows) in blue/purple were only located in a direct proximity to the α-SMA-positive cells along fibrotic septa but not elsewhere. FITC-conjugated CD45 with the classic blue color became pink when merged with the red Cy-5 α-SMA.

Fig. 3

The profibrogenic and antifibrogenic effect of lymphocyte subsets in culture: LX2 cells were co-cultured 24 hours with lymphocytes from either HCV patients or healthy controls, either as mixed or isolated subsets. (A) Results from two individuals of each group. Protein extracts were evaluated for α-SMA (upper lanes) as a marker for HSC activation and compared with β-actin (second lanes). The upper panel demonstrates the activation of LX2 cells after co-culture with mixed HCV-derived PBLs. The middle panel indicates that HCV CD8 cells, and to a lesser extent CD4 cells as well as mixed HCV lymphocytes, activate HSCs, as manifested by a more intense expression of α-SMA protein as assessed by western blot. NK cells barely activate HSCs. Healthy lymphocytes, in the lower part of the figure, fail to activate HSCs. Results from mRNA analysis for expression of α-SMA (B) correspond to results from western blot (LX2 in the upper panel and primary isolated HSCs in the lower one). Experiments were repeated three times with nearly identical results. Moreover, although this figure derived from two HCV and two healthy donors, results are reproducible when the experiment is repeated with lymphocytes from other cases (data not shown). Intracellular cytokine analysis of cultured lymphocytes was studied by FACS using healthy or HCV-derived PBLs (C). Results show decreased TGF-β and increased IFN-γ secretion by NK cells, decreased TGF-β from CD4 and CD8 cells with increased IFN-γ secretion only in the CD8 subsets.

Fig. 4

Direct contact ends as phagocytosis of lymphocytes by HSC: LX2 cells were co-cultured with HCV or healthy lymphocytes for 6 hours. LX2 cells were stained red by the Cy-5 conjugated to α-SMA (red arrows) in direct proximity to HCV lymphocytes (white arrows) for FITC-conjugated CD4 (A; blue), PE-CD8 (B; green), as well as PE-CD16 (NK) HCV-derived cells (C; green). This interaction was not demonstrated using healthy PBL controls (D).

Fig. 5

Similar results of Fig. 4 were seen using primary HSCs instead of LX2 cells.

Fig. 6

After 12, 24, and 72 hours of co-culture, all CD45+ cells underwent phagocytosis inside the HSC (A–B, with LX2 cells; C-D, with primary isolated HSCs), which were also demonstrated in each lymphocyte subset separately (data not shown). Moreover, phagocytosis was associated with α–SMA-positive encapsulation around ingested lymphocytes (green arrows, B and D). This experiment was repeated more than four times.

Fig. 7

Confocal microscopy documents phagocytosis: Serial 1-µm section images showed HSC and CD45+ cells appear and disappear at the same confocal channel, confirming phagocytosis (selective sections are presented in A–D with LX2 cells and in G-J with primary isolated HSCs). HCV-derived lymphocytes were preincubated with DiOC, washed, and then cultured with HSC cells. (E–F) LX2 cells and (K and L) primary isolated HSCs demonstrate lymphocytes inside the HSCs, with release of their DiOC into the HSC cytoplasm. The same was also demonstrated for each lymphocyte subset individually (data not shown). The experiment was repeated four times.

Fig. 8

The apoptosis end death of lymphocytes increased after phagocytosis by HSCs: The apoptosis of lymphocytes was evaluated by FACS after HCV-derived PBL culture for 48 hours either alone or with HSCs (LX2 in panels A-B as well as primary isolated HSCs in panels C-D). Apoptotic PBLs (A), defined as CD45+ Annexin+ and PI(−), were significantly increased in the case of engulfed lymphocytes. Dyed lymphocytes (B) defined as CD45+ Annexin+ and PI(+) were also increased, suggesting a rapid killing of lymphocytes inside the HSCs.

Fig. 9

Phagocytosis is mediated by a ligand/receptor adhesion. Compared with the nonmanipulated co-culture and unstimulated phagocytosis (A), prevention of phagocytosis was achieved when the HSC-related ICAM1 and integrin alphaV were blocked before co-culture as described in Patients and Methods (D and E, respectively). Blocking of HSC-related class I as well as class II before co-culture with lymphocytes did not affect phagocytosis (B and C, respectively). Blocking of lymphocyte-related T9cell receptor before co-culture with lymphocytes did not affect phagocytosis (F). Only when HSC (H) but not lymphocytes (G) were irradiated was phagocytosis blocked. (I) Absence of phagocytosis when HSC were co-cultured with healthy lymphocytes.

Fig. 10

Primary HSCs displayed the same responses as LX2 cells in Fig. 9. The experiment was repeated four times.

Fig. 11

The blocking of ICAM-1 or integrin before co-culture with HCV-derived PBL functionally accompanied with a decrease of the HSC activation: The bands of α-SMA expression by western blotting were decreased after ICAM-1 or integrin blocking in the presence of equal β-actin (lower panel). Expression of α-SMA mRNA (Fig. 10, upper panel) corresponded to western blot results.

Fig. 12

Rac1 & Cdc42 are recruited to the phagocytosis contact area. Cdc42 and Rac1 recruitment in the phagocytosis and cell-to-cell adhesion area are demonstrated in HSC/HCV-derived PBL co-cultures (the left two panels showing co-culture with LX2 cells and the right two panels with primary HSCs). (B and E) FITC-stained Cdc42 and Rac1 (respectively) in blue; (C and F) PE-stained lymphocytes in green. (A and D) Overlay of both stains together with that of Cy-5 red α-SMA staining of LX2 cells (respectively). The experiment was repeated more than four times.

Fig. 13

Rac1 and Cdc42 are required for phagocytosis of lymphocytes by HSC. Silencing of either Cdc42-related or Rac1-related LX2 cells was achieved using specific siRNA transfection. Cdc42 down-regulation was confirmed by western blotting after the specific siRNA silencing compared with negative siRNA control (lower panel). As a result, there was decreased activation of HSC as assessed by α-SMA expression compared with negative siRNA control (upper panel). Results of the Rac1 siRNA silencing were in line with the Cdc42 effects (data not shown). This experiment was repeated four times.

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Activation of hepatic stellate cells after phagocytosis of lymphocytes: A novel pathway of fibrogenesis - PubMed (original) (raw)