Isolation of lymphocytes from normal adult human liver suitable for phenotypic and functional characterisation

Curry, Michael P.; Norris, Suzanne; Golden-Mason, Lucy; Doherty, Derek G.; Deignan, Tina; Collins, C D; Traynor, O.; McEntee, Gerry; Hegarty, John E.; O’Farrelly, Cliona

doi:10.1016/s0022-1759(00)00204-0

Cited by 56 publications

(54 citation statements)

References 20 publications

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“…5,[19][20][21] The mechanical protocol resulted in poor viability, which was improved by enzymatic dispersal. However, the modified protocol yielded the highest percentage of viable cells.…”

Section: Discussionmentioning

confidence: 99%

“…Liver retrieval was achieved by a standard operation after in situ perfusion with University of Wisconsin (UW) solution. 25 UW has no significant effect on lymphocyte viability and functional characteristics 21,26 and removes the intrasinusoidal lymphocytes that constitute up to 20% of the hepatic lymphocyte pool, while the resident IHL remain. 17 Fresh liver biopsies (B10 g each) were collected from unused liver segments of 23 donors during liver reduction or splitting at the time of transplantation.…”

Section: Methodsmentioning

confidence: 99%

“…Each donor biopsy was divided into three equal pieces (B3 g each) for isolation of IHL using (a) mechanical, (b) enzymatic digestion, 5,21 and (c) a modified enzymatic dispersal protocol.…”

Section: Isolation Protocolsmentioning

confidence: 99%

“…5,[19][20][21] Mechanical methods have yielded very low cell numbers, with low viability and contamination with dead cells and cell debris. Enzymatic methods initially failed to improve cell yields due to long periods of incubation with collagenase.…”

mentioning

confidence: 99%

“…Recently, modified procedures for isolation and enrichment of nonparenchymal cells from human livers have been described. 5,21 These approaches improved yield and viability but resulted in time-dependent enzyme-induced alteration of certain markers. 21 Hence, further efforts are needed to improve the purity of single-cell suspensions and to ensure that there are sufficient cell yields with acceptable viability for phenotyping and functional analysis.…”

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confidence: 99%

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Isolation, purification and flow cytometric analysis of human intrahepatic lymphocytes using an improved technique

Morsy

Norman

Mitry

et al. 2005

Laboratory Investigation

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Intrahepatic lymphocytes (IHL) with their diverse and distinctive subsets emphasise the importance of the liver as a site of immunological activity, but special care is required for their isolation and characterisation. Protocols for IHL isolation, purification and FACS analysis were devised and compared with published extraction protocols. We have reduced the time that IHL are exposed to potentially damaging enzymes during extraction and purified specific subsets using monoclonal antibody (mAb)-coated magnetic microbeads. This has yielded IHL populations with higher viability than previously described protocols (92-95%, compared with 39-86%). Flow cytometric characterisation of IHL subset immunophenotypes was optimised by combining CD45 staining (fluorescence gating) with traditional light scatter properties. Using a panel of mAb and liver biopsies obtained from 23 cadaveric liver transplant donors, we show that the normal liver contains a heterogeneous IHL population with distinctive phenotypes. CD8 þ IHL was the predominant population with a mean CD4/CD8 ratio of 1:1.7. Up to 40% of IHL expressed cdTCR and a third expressed CD56 NK marker; indicating a site of intense immunological activity. The techniques described will allow these cell types to be isolated, fully characterised and their physiological functions to be determined. The histologically normal liver contains heterogeneous and diverse IHL with large numbers of CD8 þ , NK, NKT and cd þ cells.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Each donor biopsy was divided into three equal pieces (B3 g each) for isolation of IHL using (a) mechanical, (b) enzymatic digestion, 5,21 and (c) a modified enzymatic dispersal protocol.…”

Section: Isolation Protocolsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Isolation, purification and flow cytometric analysis of human intrahepatic lymphocytes using an improved technique

Morsy

Norman

Mitry

et al. 2005

Laboratory Investigation

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Characterization of E‐NTPDase (EC 3.6.1.5) activity in hepatic lymphocytes: A different activity profile from peripheral lymphocytes

Doleski

Adefegha

Cabral

et al. 2017

Cell Biochemistry & Function

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Extracellular purine nucleotides are able to interact with specific receptors and trigger a number of important physiological functions in cells. This interaction is controlled by ectonucleoside triphosphate diphosphohydrolase (E-NTPDase), enzyme that present their catalytic site at the extracellular space and degrades nucleotides. This purinergic signaling has important functions in peripheral lymphocytes and may represent an important new therapeutic target for the treatment of immunological diseases. However, there is dearth of information on the involvement of E-NTPDase in liver lymphocytes. The liver is an important organ, which performs both metabolic and toxicological roles in living organism, and hepatic lymphocytes may play crucial action in the regulation of immune responses in the liver tissue. Furthermore, various chronic diseases such as cirrhosis may be treated with novel pharmacotherapy by targeting the modulation of hepatic lymphocytes. Thus, the significance of this study is to evaluate the activity of E-NTPDase in liver lymphocyte and compare its activity with the peripheral lymphocytes.

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Phenotypic and Functional Characterization of Intrahepatic T Lymphocytes During Chronic Hepatitis C

et al. 2003

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The pathogenesis of liver cell injury during chronic hepatitis C (CHC) is poorly understood. The cellular immune response is thought to play a key role in both inhibition of viral replication and liver pathology. However, little is currently known about which lymphocyte populations and which immune effectors contribute to or control liver damage. We investigated a panel of 15 phenotypic and functional markers of intrahepatic T-lymphocyte subsets irrespective of their antigen specificity in 48 hepatitis C virus (HCV)-infected patients and 8 healthy control subjects. Lymphocyte characteristics were evaluated from liver biopsy specimens both at gene expression level by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) and by immunochemistry, in relation with the degree of liver injury and with intrahepatic HCV-RNA levels. As compared with controls, we found major changes in T-lymphocyte subsets in HCV-infected patients, with a significant decrease of T-cell antigen receptor (TCR) delta and CD56 gene expression, associated with a concomitant increase of TCRalpha and CD8beta that were correlated with cytotoxic factors, proinflammatory chemokines, and chemokine receptors including peforin, interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), RANTES, and CXCR3. The gene expression of CD8beta, a specific marker for conventional TCRalpha+CD8+ lymphocytes, was correlated by multivariate analysis with both alanine aminotransferase (ALT) serum levels and histologic activity index. Furthermore, CD8 staining was observed by immunochemistry in the areas of lobular and piecemeal necrosis. In contrast, no lymphocyte marker was correlated with viral load, measured both in serum and in liver. In conclusion, these results strongly suggest key roles for CD8+ T cells as effectors of liver damage during chronic HCV infection and for their inability to control viral replication.

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Isolation of lymphocytes from normal adult human liver suitable for phenotypic and functional characterisation

Cited by 56 publications

References 20 publications

Isolation, purification and flow cytometric analysis of human intrahepatic lymphocytes using an improved technique

Isolation, purification and flow cytometric analysis of human intrahepatic lymphocytes using an improved technique

Characterization of E‐NTPDase (EC 3.6.1.5) activity in hepatic lymphocytes: A different activity profile from peripheral lymphocytes

Phenotypic and Functional Characterization of Intrahepatic T Lymphocytes During Chronic Hepatitis C

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