Selective Harvesting of Marginating-hepatic Leukocytes

Summary

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Abstract

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Introduction

The liver sinusoids contain numerous leukocyte subtypes of various immune activities critical to the organism. For example, marginating hepatic (MH) natural killer (NK) cells, also known as pit cells, are characterized morphologically as large granular lymphocytes (LGLs) and functionally as leukocytes with spontaneous cytotoxic capacity, enabling hepatic-resistance against the establishment of blood-borne tumor metastases. The goal of the method presented herein is to enable selective harvesting of MH leukocytes, in order to study this important and unique cell population (and immune compartment), and to elucidate the impact of various manipulations (e.g. imm....

Protocol

Ethics Statement: Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at Tel-Aviv University.

1. Rats Protocol

1. Preparations
   1. Prepare heparinized PBS (30 units/ml) solution to be used at room temperature (RT) by adding 30 units of preservative free heparin per ml of Phosphate buffered saline (PBS) 1x solution. Calculate 35 ml per animal.
   2. Pass heparinized PBS through the peristaltic pump lines and the butterfly needles to eliminate air bubbles.
   3. Sterilize surgical tools – 2 pairs of scissors, blunted-edged forceps, 2 hemostats, and....

Results

In F344 rats we compared the cytotoxicity of MH-NK cells (collected from the liver sinusoids by forced liver perfusion) to cytotoxicity of the entire liver cell population following mechanical grinding of the liver tissue, and to the cytotoxicity of circulating leukocytes. All cell preparations were washed at least 3 times, as routine in immunological assays, and as target cell lines we used the allogenic YAC-1 or the syngeneic MADB106 target cell lines. As indicated in Figures 1<.......

Discussion

The liver perfusion method presented herein enables a selective harvesting and studying of the unique population of marginating hepatic leukocytes. Hepatic NK cells, also called pit cells³, constitute a distinctive NK cell population that reside in the hepatic sinusoids. They are found in rats, mice¹⁷ and in humans^18,19. Compared to isolated peripheral NK cells, pit cells demonstrated higher cytotoxicity against YAC-1 and CC531s target cell lines²⁰, were shown to have a higher .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Autoclud Peristaltic pump
Butterfly needle	OMG		26 G
Butterfly needle	OMG		21 G*3/4"
Syringe	Pic solution		1 ml
Syringe	Pic solution		2.5 ml
Syringe	Pic solution		5 ml
Syringe	Pic solution		10 ml
Syringe	Pic solution		25 ml
Blunted-edged forceps
Scissors
hemostat
Tissue forceps
22 W Fluorescent Daylight Magnifier Lamp