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 ...

The overall goal of this procedure is to selectively harvest marginating-hepatic leukocytes, immune cells that adhere to the sinusoids of the liver, by forced perfusion from mouse or rat livers. This method allows the study of the hepatic leukocytes which exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. The main advantage of this technique is that it yields leukocytes exclusively from the liver sinusoid uncontaminated by cells from other liver compartments such as parenchyma.Demonstrating the procedure will be Liat Sorski and Lee Shaashua, two amazing graduate students from my laboratory. Before beginning the liver perfusion procedure flush heparinized PBS through the peristaltic pump lines and the butterfly needles to remove any air bubbles. Then, immediately upon the cessation of respiration use sterile scissors and tooth tissue forceps to make a midline abdominal incision starting at the lower abdomen of the euthanized rat up to the xiphoid process taking care not to damage the internal organs.Next, use the forceps to lift the sternum and carefully cut the diaphragm and rib cage on both sides. Clamp a hemostat on the sternum and fold the rib cage rostrally to expose the cardiopulmonary complex. Then, prepare a 21 gauge butterfly needle connected to a 10 milliliter syringe and insert it into the right ventricle of the heart.Next, use the 10 milliliter syringe to collect as much blood as possible. When all of the blood has been collected, use a hemostat to clamp the caudal vena cava as close as possible to the heart. Then, move the intestines out of the abdominal cavity to the left side of the animal to expose the portal vein.Next, prepare a 25 gauge IV catheter connected to the outflow pipe of the peristaltic pump and insert it into the portal vein as caudal as possible while remaining rostral to the splenic vein. Then, ready a 21 gauge butterfly needle connected to a five milliliter syringe and insert it into the inferior vena cava caudal to the hemostat. Turn on the peristaltic pump at a speed of approximately three milliliters per minute and collect the first milliliters of perfusate contaminated with blood into the syringe.Place the five milliliter harvesting syringe with the 20 milliliter harvesting syringe without stopping the pump when the perfusate becomes a pale red color. Then, collect at least 20 milliliters of liver perfusate at seven milliliters per minute. When the whole liver is light brown, terminate the perfusion.Then, centrifuge the perfusate and aspirate the supernatant. To collect mouse marginating-hepatic leukocytes, immediately upon the cessation of respiration, use sterile scissors and tooth tissue forceps to make a midline abdominal incision up to the xiphoid process. Next, use the forceps to lift the sternum and carefully cut the diaphragm and rib cage on both sides.Clamp a hemostat on the sternum and fold the rib cage rostrally to expose the cardiopulmonary complex. Move the intestines outside of the abdominal cavity to the left of the animal exposing the portal vein. Then, connect a 30 gauge needle to a peristaltic pump and insert it into the portal vein rostral to the splenic vein.Cut the inferior vena cava above the diaphragm to allow the drainage and collection of the perfusate from the chest cavity. Then, turn on the peristaltic pump at a speed of approximately three milliliters per minute and collect the first milliliters of blood contaminated perfusate from the chest cavity pool. When three milliliters have been collected, turn off the pump.Then, after inserting a 21 gauge butterfly needle connected to a 10 milliliter syringe, reinitiate the perfusion at a speed of up to four milliliters per minute until 10 milliliter of perfusate are collected from the chest cavity. When the liver turns light brown, terminate the perfusion. Next, transfer the perfusate to a 15 milliliter tube.Centrifuge it. And then aspirate the supernatant. Whereas marginating-hepatic NK cells exhibit a profound cytotoxicity against allogeneic target cell lines both the entire liver cell population and the circulating leukocytes display profoundly lower levels of cytotoxicity and almost no killing against a syngeneic target cell line.The same relative lower levels of cytotoxicity are evident when mechanically processed livers undergo washing and density gradient separation. Facts analysis of specific leukocyte populations reveals profound differences in the composition of the immune cell populations from the circulating and liver cell compartments. Once mastered, this technique can be completed in a few minutes if it is performed properly.Following this procedure, the cell can be used for flow cytrometry, PCR, or any other methods for characterizing this important leukocyte population. This technique enables the selective harvesting of marginating-hepatic leukocytes to advance our understanding of the biological and clinical significance. Specifically we expect to controlling circulating aberrant cells and liver related diseases.After watching this video, you should have a good understanding of how to collect margating-hepatic leukocytes.

selective-harvesting-of-marginating-hepatic-leukocytes

Research

JoVE Journal

Immunology and Infection

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Selective Harvesting of Marginating-pulmonary Leukocytes

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.

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.

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets

Even in the absence of antiretroviral drugs, only ~15% of infants breastfed by HIV-infected mothers become infected, suggesting a strong protective effect of breast milk (BM). Unless access to clean water and appropriate infant formula is reliable, the WHO does not recommend cessation of breastfeeding for HIV-infected mothers. Numerous factors likely work in tandem to reduce BM transmission. Breastfed infants ingest ~105&#8722;108 maternal leukocytes daily, though what remains largely unclear is the contribution of these cells to the antiviral qualities of BM. Presently we aimed to isolate cells from human BM in order to measure antibody-dependent cellular phagocytosis (ADCP), one of the most essential and pervasive innate immune responses, by BM phagocytes against HIV targets. Cells were isolated from 5 human BM samples obtained at various stages of lactation. Isolation was carried out via gentle centrifugation followed by careful removal of milk fat and repeated washing of the cell pellet. Fluorescent beads coated with HIV envelope (Env) epitope were used as targets for analysis of ADCP. Cells were stained with the CD45 surface marker to identify leukocytes. It was found that ADCP activity was significant above control experiments and reproducibly measurable using an HIV-specific antibody 830A.

Breast milk transmits human immunodeficiency virus (HIV), though only ~15% of infants breastfed by HIV-infected mothers become infected. Breastfed infants ingest ~105&#8722;108 maternal leukocytes daily, though these cells are understudied. Here we describe the isolation of breast milk leukocytes and an analysis of their phagocytic capacity.

Even in the absence of antiretroviral drugs, only ~15% of infants breastfed by HIV-infected mothers become infected, suggesting a strong protective effect ...