Name: selective harvesting of marginating-pulmonary leukocytes
Uploaded: 2016-03-11T15:00:00
Description: Watch this Scientific Journal Video about Selective Harvesting of Marginating-pulmonary Leukocytes at JoVE.com

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

Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at Tel-Aviv University.
1. Rat Protocol
<ol>
	<li>Preparations
	<ol>
		<li>Arrange 2 butterfly 21 G needles. Optionally, prepare 2 blunt-edged butterfly 21 G needles by filing the sharp edge of the needle.</li>
		<li>Sterilize surgical tools: 2 pairs of scissors, blunted-edged forceps, hemostat, tooth-tissue forceps sterilized by autoclave at 121 &#176;C for at least 30 min on gravity (dry) setting....

<ol>
	<li>Kuebler, W. M., Goetz, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marginated+pool.">The marginated pool.</a> Eur Surg Res. 34, 92-100 (2002).</li><li>Benish, 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=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> Immunol Res. 58, 28-39 (2014).</li><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>Shakhar, G., 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=Amelioration+of+operation-induced+suppression+of+marginating+pulmonary+NK+activity+using+poly+IC:+a+potential+approach+to+reduce+postoperative+metastasis.">Amelioration of operation-induced suppression of marginating pulmonary NK activity using poly IC: a potential approach to reduce postoperative metastasis.</a> Ann Surg Oncol. 14, 841-852 (2007).</li><li>Smyth, M. J., Godfrey, D. I., Trapani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fresh+look+at+tumor+immunosurveillance+and+immunotherapy.">A fresh look at tumor immunosurveillance and immunotherapy.</a> Nat Immunol. 2, 293-299 (2001).</li><li>Talmadge, J. E., Meyers, K. M., Prieur, D. J., Starkey, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+NK+cells+in+tumour+growth+and+metastasis+in+beige+mice.">Role of NK cells in tumour growth and metastasis in beige mice.</a> Nature. 284, 622-624 (1980).</li><li>Grimm, E. A., Mazumder, A., Zhang, H. Z., Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphokine-activated+killer+cell+phenomenon.+Lysis+of+natural+killer-resistant+fresh+solid+tumor+cells+by+interleukin+2-activated+autologous+human+peripheral+blood+lymphocytes.">Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes.</a> J Exp Med. 155, 1823-1841 (1982).</li><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=Marginating+pulmonary-NK+activity+and+resistance+to+experimental+tumor+metastasis:+suppression+by+surgery+and+the+prophylactic+use+of+a+beta-adrenergic+antagonist+and+a+prostaglandin+synthesis+inhibitor.">Marginating pulmonary-NK activity and resistance to experimental tumor metastasis: suppression by surgery and the prophylactic use of a beta-adrenergic antagonist and a prostaglandin synthesis inhibitor.</a> Brain Behav Immun. 19, 114-126 (2005).</li><li>O'Dea, K. P., 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=Lung-marginated+monocytes+modulate+pulmonary+microvascular+injury+during+early+endotoxemia.">Lung-marginated monocytes modulate pulmonary microvascular injury during early endotoxemia.</a> Am J Respir Crit Care Med. 172, 1119-1127 (2005).</li><li>Sorski, L., Melamed, R., Ben-Eliyahu, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+role+for+marginating-pulmonary+and+marginating-hepatic+NK+cells+in+cancer+anti-metastatic+surveillance+and+in+immunotherapy.">A unique role for marginating-pulmonary and marginating-hepatic NK cells in cancer anti-metastatic surveillance and in immunotherapy.</a> , (2014).</li><li>Levi, B., 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=Continuous+stress+disrupts+immunostimulatory+effects+of+IL-12).">Continuous stress disrupts immunostimulatory effects of IL-12).</a> Brain Behav Immun. 25, 727-735 (2011).</li></ol>

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 cytokines2,3. Specifically, we have shown that MP-NK cells are more cytotoxic than circulatory NK cells against various target cells3,4</sup...

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-leukocytes have a higher ratio of innate/adaptive immunity compared to circulating leukocytes (50% vs. 30%, respectively). The goal of the method presented herein is to enable selective harvesting of MP-leukocytes, in order to study this important and unique immune compartment (cell population), and to elucidate the impact of various manipulations (e.g., immune activation) on these specific cells.
To understand the significance of this unique population, it is important to note that the immune system can control circulating tumor cells, micrometastases, and residual disease through in vivo functions of cell-mediated immunity (CMI). This ability is evident despite the precedent failure of the immune system to control the primary tumor, and supported by ample in vivo evidence in cancer patients and animal models5. Importantly, these findings are often inconsistent with in vitro and ex vivo studies, which report that most autologous tumor cells are resistant to cytotoxicity by circulating leukocytes in blood samples from humans and animals (measured by cytotoxicity assays) 6,7. This discrepancy may be attributed to the in vivo existence of distinct leukocyte populations, such as the aforementioned MP leukocyte population, and specifically its subpopulation of activated NK cells3. Indeed, syngeneic tumor cells (MADB106), which were found to be resistant to circulating and splenic leukocytes, were shown to be lysed by MP-NK cells3,8. Thus, the allegedly &#39;NK-resistant&#39; MADB106 cells that metastasize to lungs of fischer344 (F344) rats are controlled by MP-NK cells, but not by circulating or splenic NK cells, which are commonly studied given their ease of access.
Purified and active MP-leukocytes are inaccessible through the standard harvesting methods of leukocytes from the lungs, which are based on lung tissue grinding or biological degradation9. Our approach has two major advantages compared to tissue processing approaches. First, the perfusion approach selectively harvests MP-leukocytes, separating them from other cells that originate from the lung parenchymal, interstitial, and broncho-alveolar compartments. Second, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, unlike the grinding and biological processing approaches that damage cells, alter their morphology, and induce the production and release of various factors that modulate immune activity and specifically suppress NK cytotoxicity10.
The lungs are a major target organ for cancer metastasis and for various infectious diseases. All circulating malignant cells and infected cells pass through the lung capillaries, where they need to deform and interact with capillary endothelial cells and resident leukocytes. Under these conditions, circulating cells can be easily targeted by resident MP-leukocytes. It thus seems biologically advantageous to have activated leukocytes in this immune compartment, and it is important to study this unique MP-population in different biological, experimental, and clinical settings. It is worthy to note that systemic immune activation by various biological-response-modifiers (e.g., polyinosinic-polycytidylic acid (poly I:C) or type-C CpG oligodeoxynucleotides (CpG-C ODN)) have been shown to activate MP-leukocytes more than circulating leukocytes3,8,11.

<table border="0" cellpadding="0" cellspacing="0" width="640">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">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:251px;">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:251px;">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:251px;">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:251px;">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:251px;">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:251px;">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:251px;">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:251px;">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:251px;">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:251px;">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:251px;">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:251px;">Heparin sodium porcine mucosa (perservative free)</td>
			<td style="width:103px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

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-1bright cells were identified as NK cells, CD3+ as T cells, RM1+ as monocytes, and CD4<s...

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

Selective Harvesting of Marginating-pulmonary Leukocytes

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 ...

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