Selektiv Høst af Marginating-hepatiske Leukocytter
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
Leveren sinuskurver indeholder talrige leukocytundertyper af forskellige immunaktiviteter kritiske for organismen. For eksempel er marginating hepatisk (MH) naturlige dræber (NK) celler, også kendt som pit celler, kendetegnet morfologisk som store granulære lymfocytter (LGLs) og funktionelt som leukocytter med spontan cytotoksisk kapacitet, der muliggør hepatisk-resistens imod optagelse af blod- bæres tumor metastaser. Målet med fremgangsmåden heri præsenterede er at muliggøre selektiv høst af MH leukocytter, for at studere dette vigtige og unikke cellepopulation (og immun rum), og at belyse virkningen af forskellige manipulationer (f.eks immunaktivering) på disse specifikke celler.
Trods den manglende immunitet at eliminere en udvikling af primær tumor, beviser hos cancerpatienter og dyremodeller viser, at immunsystemet kan styre cirkulerende tumorceller, mikrometastaser og tilbageværende sygdom through cellemedieret immunitet (CMI). Men der er en tilsyneladende uoverensstemmelse mellem disse in vivo kapaciteter og in vitro undersøgelser i mennesker og dyr, som viser, at de fleste autologe tumorceller er resistente over for cytotoksicitet ved at cirkulere eller milt leukocytter 1,2. Denne uoverensstemmelse kan tilskrives, i det mindste delvis, til in vivo foreligger en særskilt leukocyt subpopulation, nemlig marginating-hepatisk (sinusoids) leukocytter og deres subpopulation af aktiverede NK-celler, nemlig pit celler 3. Faktisk upublicerede data fra vort laboratorium viste, at syngene tumorceller (MADB106), som blev fundet resistent over for cirkulerende og milt leukocytter, i F344-rotter blev lyseret ved MH-NK-celler 4. Således kan tumorceller, der er angiveligt "NK-resistente" til cirkulerende leukocytter være kontrolleret af MH-NK-celler. Bemærkelsesværdigt, i mus den forøgede aktivitet af MH-NK er kun tydelig følgende in vivo immunestimulering (fx gennem anvendelse af Poly I: C eller CpG-C) 5.
Lever-specifik NK-celler (MH-NK) er placeret inde i sinusformede lumen, der tilslutter sig de endotelceller og Kupffer celler. MH-NK-celler udelukkende kendetegnet ved sfæriske tætte granuler og stang-kernehus vesikler 6, som indeholder sur phosphatase som lysosomale enzymer og perforin og granzymer som bioaktive stoffer 7,8. Sammenlignet med cirkulerende NK-celler, MH-NK-celler udviser et højere antal og størrelse af granuler og vesikler 9-11. Under inflammatoriske tilstande, blev MH-NK-celler vist at udvise højere ekspression af LFA-1 12, sammenlignet med cirkulerende NK-celler. Denne forøgede ekspression kan udgøre en mekanisme, hvorved MH-NK-celler er mere cytotoksisk over for visse tumorceller end cirkulerende NK-celler 13,14. nterestingly, efter in vitro inkubation med interleukin (IL) -2, MH-NK-celler bliver forstørret, ogderes antal og størrelse af granulater stige, som alle er i overensstemmelse med en fi l af lymfokin-aktiverede dræber (LAK) -celler 15.
Aktive MH leukocytter kan ikke udelukkende opnås gennem standard lever leukocyt høstmetoder, der er baseret på slibning og biologisk nedbrydning af vævet. Vores perfusion tilgang beskrevet heri har to store fordele sammenlignet med de standard fremgangsmåder. Først perfusionen tilgang selektivt høster MH leukocytter, forebygge kontaminering af andre leukocytter fra andre lever rum. For det andet perfusion teknik bedre bevarer integriteten, aktiviteten, og den fysiologiske miljø af MH leukocytter, i modsætning til vævsbehandling nærmer sig skader celler eller ændre deres morfologi, og på grund af vævsskade, inducere frigivelse af forskellige faktorer, der markant modulere immun aktivitet.
Leveren er et vigtigt målorgan for cancermetastase and for forskellige infektioner 16. Som MH-celler udviser unikke egenskaber er det vigtigt at studere denne specifik population under forskellige betingelser med hensyn til disse patologier. For eksempel er det værd at bemærke, at systemiske immunaktivering af forskellige BRMs (f.eks poly I: C eller CpG-C) er blevet vist at aktivere MH-leukocytter mere end cirkulerende leukocytter 5.
Protocol
Representative Results
Discussion
Leveren perfusionsmetode præsenteret heri muliggør en selektiv høst og læsning af den unikke population af marginating hepatiske leukocytter. Nedsat NK-celler, også kaldet pit celler 3, udgør en karakteristisk NK-celle population, bor i de hepatiske sinuskurver. De findes i rotter, mus 17 og i mennesker 18,19. Sammenlignet med isolerede perifere NK-celler, pit celler demonstrerede højere cytotoksicitet mod YAC-1 og CC531s målcellelinjer 20, viste sig at have et højere…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors and this work were supported by NIH/NCI grant # R01CA172138 (to SBE).
Materials
| 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 | |||
| 22W Fluorescent Daylight Magnifier Lamp |
References
- Melamed, R., et al. The marginating-pulmonary immune compartment in rats: characteristics of continuous inflammation and activated NK cells. J Immunother. 33, 16-29 (2010).
- Benish, M., Melamed, R., Rosenne, E., Neeman, E., Sorski, L., Levi, B., Shaashua, L., Matzner, M., Ben-Eliyahu, S. The marginating-pulmonary immune compartment in mice exhibits increased NK cytotoxicity and unique cellular characteristics. Immunologic Research. 58, 28-39 (2014).
- Wisse, E., van’t Noordende, J. M., van der Meulen, J., Daems, W. T. The pit cell: description of a new type of cell occurring in rat liver sinusoids and peripheral blood. Cell Tissue Res. 173, 423-435 (1976).
- Vermijlen, D., et al. Hepatic natural killer cells exclusively kill splenic/blood natural killer-resistant tumor cells by the perforin/granzyme pathway. J Leukoc Biol. 72, 668-676 (2002).
- Gao, B., Radaeva, S., Park, O. Liver natural killer and natural killer T cells: immunobiology and emerging roles in liver diseases. J Leukoc Biol. 86, 513-528 (2009).
- Kaneda, K. Liver-associated large granular lymphocytes: morphological and functional aspects. Arch Histol Cytol. 52, 447-459 (1989).
- Podack, E. R., Hengartner, H., Lichtenheld, M. G. A central role of perforin in cytolysis?. Annu Rev Immunol. 9, 129-157 (1991).
- Kamada, M. M., et al. Identification of carboxypeptidase and tryptic esterase activities that are complexed to proteoglycans in the secretory granules of human cloned natural killer cells. J Immunol. 142, 609-615 (1989).
- Wisse, E., et al. On the function of pit cells, the liver-specific natural killer cells. Semin Liver Dis. 17, 265-286 (1997).
- Bouwens, L., Remels, L., Baekeland, M., Van Bossuyt, H., Wisse, E. Large granular lymphocytes or "pit cells" from rat liver: isolation, ultrastructural characterization and natural killer activity. Eur J Immunol. 17, 37-42 (1987).
- Vanderkerken, K., et al. Origin and differentiation of hepatic natural killer cells (pit cells). Hepatology. 18, 919-925 (1993).
- Luo, D., et al. The role of adhesion molecules in the recruitment of hepatic natural killer cells (pit cells) in rat liver. Hepatology. 24, 1475-1480 (1996).
- Shresta, S., MacIvor, D. M., Heusel, J. W., Russell, J. H., Ley, T. J. Natural killer and lymphokine-activated killer cells require granzyme B for the rapid induction of apoptosis in susceptible target cells. Proc Natl Acad Sci U S A. 92, 5679-5683 (1995).
- Luo, D., et al. Involvement of LFA-1 in hepatic NK cell (pit cell)-mediated cytolysis and apoptosis of colon carcinoma cells. J Hepatol. 31, 110-116 (1999).
- Wiltrout, R. H., et al. 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. J Immunol. 143, 372-378 (1989).
- Schluter, K., et al. Organ-specific metastatic tumor cell adhesion and extravasation of colon carcinoma cells with different metastatic potential. Am J Pathol. 169, 1064-1073 (2006).
- Wiltrout, R. H., et al. Augmentation of organ-associated natural killer activity by biological response modifiers. Isolation and characterization of large granular lymphocytes from the liver. J Exp Med. 160, 1431-1449 (1984).
- Winnock, M., et al. Functional characterization of liver-associated lymphocytes in patients with liver metastasis. Gastroenterology. 105, 1152-1158 (1993).
- Hata, K., et al. Isolation, phenotyping, and functional analysis of lymphocytes from human liver. Clin Immunol Immunopathol. 56, 401-419 (1990).
- Vanderkerken, K., Bouwens, L., Wisse, E. Characterization of a phenotypically and functionally distinct subset of large granular lymphocytes (pit cells) in rat liver sinusoids. Hepatology. 12, 70-75 (1990).
- Sorski, L., Melamed, R., Lavon, H., Matzner, P., Rosenne, E., Ben-Eliyahu, S. . , (2015).
