Selective Harvesting of Marginating-pulmonary Leukocytes

Summary

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Abstract

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

Introduction

Leukocytes adhering to the capillaries of the lungs (i.e., marginating-pulmonary (MP) leukocytes)¹ were shown to exhibit distinct leukocyte composition and unique activity compared to leukocytes from other immune compartments (e.g., circulation, spleen, bone-marrow)^2-4. For example, MP-leukocytes exhibit higher natural killer (NK) cells cytotoxicity against various tumor cells, compared to circulating and splenic NK cells, as well as differentiated messenger RNA (mRNA) levels and increased secretion of pro- and anti-inflammatory cytokines. The composition of cells is also differentiated from circulating leukocytes as MP-leukocyt....

Protocol

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

1. Rat Protocol

1. Preparations
   1. Arrange 2 butterfly 21 G needles. Optionally, prepare 2 blunt-edged butterfly 21 G needles by filing the sharp edge of the needle.
   2. Sterilize surgical tools: 2 pairs of scissors, blunted-edged forceps, hemostat, tooth-tissue forceps sterilized by autoclave at 121 °C for at least 30 min on gravity (dry) setting.
   3. Prepare heparinized PBS (30 units/ml) by adding 30 units of heparin per ml of phosphate buffered saline (1x PBS) solution. Use at RT. 35 m....

Results

The MP-compartment exhibit a different leukocyte subset composition compared to circulating leukocytes. Using flow cytometry analysis, leukocyte subpopulations were identified and quantified to characterize the composition of both circulating and MP leukocytes. Granulocytes and lymphocytes were identified based on forward and side scatters. Within the lymphocytes, NKRP-1^bright cells were identified as NK cells, CD3⁺ as T cells, RM1⁺ as monocytes, and CD4

Discussion

The method presented herein enables the selective harvesting and studying of the unique population of MP-leukocytes. Compared to circulating or splenic leukocytes, the MP population is characterized by a distinct composition of leukocyte subpopulations, higher activation levels, higher release of various cytokines, and higher mRNA levels of pro- and anti-inflammatory cytokines^2,3. Specifically, we have shown that MP-NK cells are more cytotoxic than circulatory NK cells against various target cells^3,4

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Autoclud Peristaltic pump
Butterfly needle	OMG		26G
Butterfly needle	OMG		21G*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
Heparin sodium porcine mucosa (perservative free)	Sigma Aldrich