Isolation of murine hepatic lymphocytes using mechanical dissection for phenotypic and functional analysis of NK1.1^+ cells

AIM: To choose an appropriate methods for the isolation of hepatic lymphocytes between the mechanical dissection and the enzymatic digestion and investigate the effects of two methods on phenotype and function of hepaticlymphocytes.METHODS: Hepatic lymphocytes were isolated from untreated, poly (I:C)-stimulated or ConA-stimulated mice using the two methods, respectively. The cell yield per liver was evaluated by direct counting under microscope.Effects of digestive enzymes on the surface markers involved in hepatic lymphocytes were represented by relative change rate [(percentage of post-digestionpercentage of pre-digestion)/percentage of pre-digestion].Phenotypic analyses of the subpopulations of hepatic lymphocytes and intracellular cytokines were detected by flow cytometry. The cytotoxicity of NK cells from wild C57BL/6 or poly (I:C)-stimulated C57BL/6 mice was analyzed with a 4-h ^51Cr release assay.RESULTS: NK1.1^+ cell markers, NK1.1 and DX5, were significantly down-expressed after enzymatic digestion and their relative change rates were about 28% and 32%,respectively. Compared with the enzymatic digestion, the cell yield isolated from unstimulated, poly (I:C)-treated or ConA-treated mice by mechanical dissection was not significantly decreased. Hepatic lymphocytes isolated by the mechanical dissection comprised more innate immune cells like NK, NKT and γδ cells in normal C57BL/6 mice.After poly (I:C) stimulation, hepatic NK cells rose to about 35%, while NKT cells simultaneously decreased. Following ConA injection, the number of hepatic NKT cells was remarkably reduced to 3.67%. Higher ratio of intracellular IFN-γ^+(68%) or TNF-α^+(15%) NK1.1^+ cells from poly (I:C)treated mice was obtained using mechanical dissection method than control mice. There was no difference in viability between the mechanical dissection and the enzymatic digestion, and hepatic lymphocytes obtained with the two methods had similar cytotoxicity against YAC-1 cells.CONCLUSION: There is no difference in the cell yield and viability of the hepatic lymphocyte isolated with the two methods. The mechanical dissection, but not the enzymatic digestion, may be suitable for the phenotypic analysis of hepatic NK1.1^+ cell.

Effect of ligustrum fruit extract on reproduction in experimental diabetic rats

Aim: To study the effect of ligustrum fruit on spermatogenesis and blood gonadal hormones in diabetic rats.Methods: Experimental diabetes was induced in male Wistar rats with streptozotocin. Ligustrum fruit extract wasgiven by gastric gavage at a dose of crude drug 30 g·kg^(-1)·d^(-1) for 110 days. The serum gonadadotropic hormones andtestosterone were determined on d 60 and testicular histology examined on d 110. Results: In the control diabeticrats, the seminiferous tubules were dilated and the spermatogenic cells irregularly arranged. Spermatogenesis was arrest-ed with the number of spermatids highly reduced and spermatozoa not observed. In the treated rats, all types of sper-matogenic cells were practically normal. The serum luteinizing hormone (LH), follicle-stimulating hormone (FSH)and testosterone levels were higher in the treated than in the control rats, but the difference was insignificant. Conclu-sion: In experimental diabetic rats, ligustrum fruit extract protects the damaging effect of experimental diabetes onspermatogenesis. (Asian J Androl 2001 Mar; 3 : 71-73)

Hydrodynamic Gene Delivery of Interleukin-22 Protects the Mouse Liver from Concanavalin A-, Carbon Tetrachloride-,and Fas Ligand-Induced Injury via Activation of STAT3

Interleukin-22 (IL-22) is a recently identified T cell-derived cytokine whose biological significance remains obscure.Previously,we have shown that IL-22 plays a protective role in T cell-mediated hepatitis induced by Concanavalin A (Con A),acting as a survival factor for hepatocytes.In the present paper,we demonstrate that hydrodynamic gene delivery of IL-22 cDNA driven either by a liver-specific albumin promoter or a human cytomegalovirus (CMV) promoter results in IL-22 protein expression,STAT3 activation,and expression of several anti-apoptotic proteins,including Bcl-xL,Bcl-2,and Mcl-1 in the liver.Immunohistochemical analysis reveals that IL-22 protein expression is mainly detected in the cytoplasm of hepatocytes.Overexpression of IL-22 by hydrodynamic gene delivery significantly protects against liver injury,necrosis,and apoptosis induced by administration of Con A,carbon tetrachloride (CCl_4),or the Fas agonist Jo-2 mAb.Western blot analyses show that overexpression of IL-22 significantly enhances activation of STAT3 and expression of Bcl-xL,Bcl-2, and Mcl-1 proteins in liver injury induced by Con A.In conclusion,hydrodynamic gene delivery of IL-22 protects against liver injury induced by a variety of toxins,suggesting the therapeutic potential of IL-22 in treating human liver disease.Cellular & Molecular Immunology.2004;1(1):43-49.

Cytokines,STATs and Liver Disease

The Janus kinase-signal transducers and activators of transcription(JAK-STAT)signaling pathway,activated by more than 50 cytokines or growth factors,plays critical roles in a wide variety of cellular functions in the hematopoietic,immune,neuronal and hepatic systems.In the liver,this signaling pathway,activated by more than 20 cytokines,growth factors,hormones,and hepatitis viral proteins,plays critical roles in antiviral defense,acute phase response,hepatic injury,repair,inflammation,transformation,and hepatitis.This article reviews the biological significance of STAT1,2,3,4,5,6 in hepatic functions and diseases.Cellular & Molecular Immunology. 2005;2(2):92-100.

Hepatitis B Virus X Protein Sensitizes Primary Mouse Hepatocytes to Ethanol-and TNF-α-Induced Apoptosis by a Caspase-3-Dependent Mechanism

It is well-documented that alcohol drinking together with hepatitis viral infection accelerates liver injury;however the underlying mechanisms remain unknown.In this paper,we demonstrated that primary hepatocytes from transgenic mice overexpressing hepatitis B virus X protein(HBX)were more susceptible to ethanol- and TNF-α- induced apoptotic killing.Compared to normal control mouse hepatocytes,ethanol and/or TNF-α treatment led to a significant increase in reactive oxygen species,mitochondrial permeability transition,cytochrome C release, caspase-3 activity,and poly(ADP-ribose)polymerase degradation in hepatocytes from HBX transgenic mice. Blocking caspase-3 activity antagonized ethanol-and TNF-α-induced apoptosis in primary hepatocytes from HBX transgenic mice.Taken together,our findings suggest that HBX sensitizes primary mouse hepatocytes to ethanol- and TNF-α-induced apoptosis by a caspase-3-dependent mechanism,which may partly explain the synergistic effects of alcohol consumption and hepatitis B virus infection on liver injury.Cellular & Molecular Immunology. 2005;2(1):40-48.

MHC Class Ⅰ Antigen Presentation-Recently Trimmed and Well Presented

Presentation of antigenic peptide to T cells by major histocompatibility complex (MHC) class I molecules is the key to the cellular immune response.Non-self intracellular proteins are processed into short peptides and transported into endoplasmic reticulum (ER) where they are assembled with class I molecules assisted by several chaperone proteins to form trimeric complex.MHC class I complex loaded with optimised peptides travels to the cell surface of antigen presentation cells to be recognised by T cells.The cells presenting non-self peptides are cleared by CD8 positive T cells.In order to ensure that T cells detect an infection or mutation within the target cells the process of peptide loading and class I expression must be carefully regulated.Many of the cellular components involved in antigen processing and class I presentation are known and their various functions are now becoming clearer.Cellular & Molecular Immunology.2004;1(1):22-30.