Name: selective harvesting of marginating-hepatic leukocytes
Uploaded: 2016-07-21T13:00:00
Description: Watch this Scientific Journal Video about Selective Harvesting of Marginating-hepatic Leukocytes at JoVE.com

The authors and this work were supported by NIH/NCI grant # R01CA172138 (to SBE).

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
<ol>
	<li>Preparations
	<ol>
		<li>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.</li>
		<li>Pass heparinized PBS through the peristaltic pump lines and th...

<ol>
	<li>Melamed, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marginating-pulmonary+immune+compartment+in+rats:+characteristics+of+continuous+inflammation+and+activated+NK+cells.">The marginating-pulmonary immune compartment in rats: characteristics of continuous inflammation and activated NK cells.</a> J Immunother. 33, 16-29 (2010).</li><li>Benish, M., Melamed, R., Rosenne, E., Neeman, E., Sorski, L., Levi, B., Shaashua, L., Matzner, M., Ben-Eliyahu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marginating-pulmonary+immune+compartment+in+mice+exhibits+increased+NK+cytotoxicity+and+unique+cellular+characteristics.">The marginating-pulmonary immune compartment in mice exhibits increased NK cytotoxicity and unique cellular characteristics.</a> Immunologic Research. 58, 28-39 (2014).</li><li>Wisse, E., van't Noordende, J. M., van der Meulen, J., Daems, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pit+cell:+description+of+a+new+type+of+cell+occurring+in+rat+liver+sinusoids+and+peripheral+blood.">The pit cell: description of a new type of cell occurring in rat liver sinusoids and peripheral blood.</a> Cell Tissue Res. 173, 423-435 (1976).</li><li>Vermijlen, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+natural+killer+cells+exclusively+kill+splenic/blood+natural+killer-resistant+tumor+cells+by+the+perforin/granzyme+pathway.">Hepatic natural killer cells exclusively kill splenic/blood natural killer-resistant tumor cells by the perforin/granzyme pathway.</a> J Leukoc Biol. 72, 668-676 (2002).</li><li>Gao, B., Radaeva, S., Park, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+natural+killer+and+natural+killer+T+cells:+immunobiology+and+emerging+roles+in+liver+diseases.">Liver natural killer and natural killer T cells: immunobiology and emerging roles in liver diseases.</a> J Leukoc Biol. 86, 513-528 (2009).</li><li>Kaneda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver-associated+large+granular+lymphocytes:+morphological+and+functional+aspects.">Liver-associated large granular lymphocytes: morphological and functional aspects.</a> Arch Histol Cytol. 52, 447-459 (1989).</li><li>Podack, E. R., Hengartner, H., Lichtenheld, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+central+role+of+perforin+in+cytolysis?.">A central role of perforin in cytolysis?.</a> Annu Rev Immunol. 9, 129-157 (1991).</li><li>Kamada, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+carboxypeptidase+and+tryptic+esterase+activities+that+are+complexed+to+proteoglycans+in+the+secretory+granules+of+human+cloned+natural+killer+cells.">Identification of carboxypeptidase and tryptic esterase activities that are complexed to proteoglycans in the secretory granules of human cloned natural killer cells.</a> J Immunol. 142, 609-615 (1989).</li><li>Wisse, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+function+of+pit+cells,+the+liver-specific+natural+killer+cells.">On the function of pit cells, the liver-specific natural killer cells.</a> Semin Liver Dis. 17, 265-286 (1997).</li><li>Bouwens, L., Remels, L., Baekeland, M., Van Bossuyt, H., Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+granular+lymphocytes+or+&amp;#34;pit+cells&amp;#34;+from+rat+liver:+isolation,+ultrastructural+characterization+and+natural+killer+activity.">Large granular lymphocytes or &amp;#34;pit cells&amp;#34; from rat liver: isolation, ultrastructural characterization and natural killer activity.</a> Eur J Immunol. 17, 37-42 (1987).</li><li>Vanderkerken, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+differentiation+of+hepatic+natural+killer+cells+(pit+cells).">Origin and differentiation of hepatic natural killer cells (pit cells).</a> Hepatology. 18, 919-925 (1993).</li><li>Luo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+adhesion+molecules+in+the+recruitment+of+hepatic+natural+killer+cells+(pit+cells)+in+rat+liver.">The role of adhesion molecules in the recruitment of hepatic natural killer cells (pit cells) in rat liver.</a> Hepatology. 24, 1475-1480 (1996).</li><li>Shresta, S., MacIvor, D. M., Heusel, J. W., Russell, J. H., Ley, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+killer+and+lymphokine-activated+killer+cells+require+granzyme+B+for+the+rapid+induction+of+apoptosis+in+susceptible+target+cells.">Natural killer and lymphokine-activated killer cells require granzyme B for the rapid induction of apoptosis in susceptible target cells.</a> Proc Natl Acad Sci U S A. 92, 5679-5683 (1995).</li><li>Luo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+LFA-1+in+hepatic+NK+cell+(pit+cell)-mediated+cytolysis+and+apoptosis+of+colon+carcinoma+cells.">Involvement of LFA-1 in hepatic NK cell (pit cell)-mediated cytolysis and apoptosis of colon carcinoma cells.</a> J Hepatol. 31, 110-116 (1999).</li><li>Wiltrout, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Augmentation+of+mouse+liver-associated+natural+killer+activity+by+biologic+response+modifiers+occurs+largely+via+rapid+recruitment+of+large+granular+lymphocytes+from+the+bone+marrow.">Augmentation of mouse liver-associated natural killer activity by biologic response modifiers occurs largely via rapid recruitment of large granular lymphocytes from the bone marrow.</a> J Immunol. 143, 372-378 (1989).</li><li>Schluter, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ-specific+metastatic+tumor+cell+adhesion+and+extravasation+of+colon+carcinoma+cells+with+different+metastatic+potential.">Organ-specific metastatic tumor cell adhesion and extravasation of colon carcinoma cells with different metastatic potential.</a> Am J Pathol. 169, 1064-1073 (2006).</li><li>Wiltrout, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Augmentation+of+organ-associated+natural+killer+activity+by+biological+response+modifiers.+Isolation+and+characterization+of+large+granular+lymphocytes+from+the+liver.">Augmentation of organ-associated natural killer activity by biological response modifiers. Isolation and characterization of large granular lymphocytes from the liver.</a> J Exp Med. 160, 1431-1449 (1984).</li><li>Winnock, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+liver-associated+lymphocytes+in+patients+with+liver+metastasis.">Functional characterization of liver-associated lymphocytes in patients with liver metastasis.</a> Gastroenterology. 105, 1152-1158 (1993).</li><li>Hata, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+phenotyping,+and+functional+analysis+of+lymphocytes+from+human+liver.">Isolation, phenotyping, and functional analysis of lymphocytes from human liver.</a> Clin Immunol Immunopathol. 56, 401-419 (1990).</li><li>Vanderkerken, K., Bouwens, L., Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+phenotypically+and+functionally+distinct+subset+of+large+granular+lymphocytes+(pit+cells)+in+rat+liver+sinusoids.">Characterization of a phenotypically and functionally distinct subset of large granular lymphocytes (pit cells) in rat liver sinusoids.</a> Hepatology. 12, 70-75 (1990).</li><li>Sorski, L., Melamed, R., Lavon, H., Matzner, P., Rosenne, E., Ben-Eliyahu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (2015).</li></ol>

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 humans18,19. 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 ...

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. immune activation) on these specific cells.
Despite the failure of immunity to eliminate a developing primary tumor, evidence in cancer patients and animal models indicate that the immune system can control circulating tumor cells, micrometastases, and residual disease through cell-mediated immunity (CMI). However, there is an apparent inconsistency between these in vivo capabilities and in vitro studies in humans and in animals, which demonstrate that most autologous tumor cells are resistant to cytotoxicity by circulating or splenic leukocytes1,2. This inconsistency may be ascribed, at least in part, to the in vivo existence of a distinct leukocyte subpopulation, namely the marginating-hepatic (sinusoids) leukocytes and their subpopulation of activated NK cells, namely pit cells3. Indeed, unpublished data from our laboratory indicated that in F344 rats syngeneic tumor cells (MADB106), which were found resistant to circulating and splenic leukocytes, were lysed by MH-NK cells4. Thus, tumor cells that are allegedly &#34;NK-resistant&#34; to circulating leukocytes may be controlled by MH-NK cells. Noteworthy, in mice the enhanced activity of MH-NK is evident only following in vivo immune stimulation (e.g. through the use of Poly I:C or CpG-C)5.
Liver-specific NK cells (MH-NK) are situated inside the sinusoidal lumen, adhering to the endothelial cells and Kupffer cells. MH-NK cells are exclusively characterized by spherical dense granules and rod-cored vesicles6, which contain acid phosphatase as lysosomal enzymes, and perforin and granzymes as bioactive substances7,8. Compared with circulating NK cells, MH-NK cells exhibit a higher number and size of granules and vesicles9-11. Under inflammatory conditions, MH-NK cells were shown to exhibit higher expression of LFA-112, as compared to circulating NK cells. This enhanced expression might constitute a mechanism by which MH-NK cells are more cytotoxic against certain tumor cells than circulating NK cells 13,14. nterestingly, following in vitro incubation with interleukin (IL)-2, MH-NK cells become enlarged, and their number and size of granules increase, all of which are consistent with a pro&#64257;le of lymphokine-activated killer (LAK) cells15.
Active MH leukocytes cannot be exclusively obtained through the standard liver leukocyte harvesting methods, which are based on grinding and biological degradation of the tissue. Our perfusion approach described herein has two major advantages compared to the standard approaches. First, the perfusion approach selectively harvests MH leukocytes, preventing contamination by other leukocytes from other liver compartments. Second, the perfusion technique better preserves the integrity, the activity, and the physiological milieu of MH leukocytes, unlike the tissue processing approaches that damage cells or alter their morphology, and due to tissue damage, induce the release of various factors that markedly modulate immune activity.
The liver is a major target organ for cancer metastasis and for various infections16. As MH cells exhibit unique characteristics it is important to study this specific population under various conditions with respect to these pathologies. For example, it is worthy to note that systemic immune activation by various BRMs (e.g. poly I:C or CpG-C) have been shown to activate MH-leukocytes more than circulating leukocytes5.

<table border="0" cellpadding="0" cellspacing="0" width="605">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Autoclud Peristaltic pump</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Butterfly needle</td>
			<td style="width:103px;">OMG</td>
			<td style="width:119px;"></td>
			<td>26G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Butterfly needle</td>
			<td style="width:103px;">OMG</td>
			<td style="width:119px;"></td>
			<td>21G*3/4&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe</td>
			<td style="width:103px;">Pic solution</td>
			<td style="width:119px;"></td>
			<td>1 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe</td>
			<td style="width:103px;">Pic solution</td>
			<td style="width:119px;"></td>
			<td>2.5 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe</td>
			<td style="width:103px;">Pic solution</td>
			<td style="width:119px;"></td>
			<td>5 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe</td>
			<td style="width:103px;">Pic solution</td>
			<td style="width:119px;"></td>
			<td>10 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe</td>
			<td style="width:103px;">Pic solution</td>
			<td style="width:119px;"></td>
			<td>25 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Blunted-edged forceps</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scissors</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">hemostat</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue forceps</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">22W Fluorescent Daylight Magnifier Lamp</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

selective harvesting of marginating-hepatic leukocytes

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.

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<...

Watch this Scientific Journal Video about Selective Harvesting of Marginating-hepatic Leukocytes at JoVE.com

Selective Harvesting of Marginating-hepatic Leukocytes

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.

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

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