Intravital 2-photon Microscopy of Lymphocytes Migrating on Fibroblastic Reticular Cell Conduit in Lymph Node Cortex, March 31,2015

Intravital Microscopy of T-cell Migration on The FRC Conduit

In addition to rapidly conducting soluble materials across lymphatic tissues, the FRCC supports purposeful migration of lymphocytes during their "search" for compartments in which to reside, interact with other cells, divide or pass out into the efferent lymph stream. Position recognition is aided by molecular markers. The FRCC displays on its surfaces ECM & adhesion proteins that support T- and B-cell migration past dendritic cells to lymphatic tissue compartments.

Rat lymph node, HRP reaction product 1 minute after intralymphatic inoculation. CLICK to see chemokine in FRCC by confocal microscopy

Gif animation modified from video in Bajenoff et al, Immunity, 2007.

This animation is a sample of the kinds of visual analyses that can be made today using multispectral, intravital 2-photon microscopy. This technology confirmed, clarified and added new dynamic dimensions to contributions of earlier workers who laboriously analyzed, in vivo and ex vivo, phenomena in lymphatic tissues during the past 30-40 years. Previously, rather than "real time" analyses, specimens were collected in time-course experiments designed to address the same questions that now can be answered in hours rather than decades. The GFP-transgenic mouse's proteins express green fluorescent protein via a gene that is regulated by the ubiquitin promoter. Interestingly, in this tissue only stromal cells (FRCC), endothelial cells and rare mononuclear cell types are green. Only the transfused syngeneic red fluorescent transgenic lymphocytes can be seen crawling around. The mouse's own lymphocytes are invisible and appear as empty space. Look carefully and you can see indentations made by the invisible autologous cells. That there are not so many of the red fluorescent cells makes it easy to see them crawling on the stroma. This animated gif is from one of Marc Bajenoff's video clips Ron Germain showed when he gave an introduction to my work at my 'retirement' ceremony 23 March 2007.

The reference from which I converted this movie segment to an animated gif follows:

Marc Bajénoff, Jackson G. Egen, Lily Y. Koo, Jean Pierre Laugier, Frédéric Brau, Nicolas Glaichenhaus, and Ronald N. Germain. 2006. Stromal Cell Networks Regulate Lymphocyte Entry, Migration, and Territoriality in Lymph Nodes.Immunity, Vol 25, 989-1001.

References:

Clark, S 1962. The Reticulum of Lymph Nodes in Mice Studied with the Electron Microscope. Am. J. Anat. 110: 217-257.

Moe, RE. 1964. Electron Microscopic Appearance of the Parenchyma of Lymph Nodes. Am J Anat 114:341-369.

Anderson, A. O., and N. D. Anderson. 1975. Studies on the structure and permeability of the microvasculature in normal rat lymph nodes. (see page 401 in:) Amer. J. Path. 80:387-418.

Chang, TW, E Celis, HN Eisen and F Solomon. 1979. Crawling movements of lymphocytes on and beneath fibroblasts in culture. Proc Natl Acad Sci U S A. 76(6): 2917–2921. [PDF]

Anderson, AO and S Shaw. 1993. T cell adhesion to endothelium: the FRC conduit system and other anatomic and molecular features which facilitate the adhesion cascade in lymph node. Sem. Immunol. 5: 271-282.

Anderson, AO, and S Shaw. 1995. "Lymphocyte Trafficking." Chapter 3, In Clinical Immunology, Principles and Practice. RR Rich, TA Fleisher, BD Schwartz, WT Shearer, and W Strober (eds.), pp 39-49, Mosby-Year Book, Inc., St. Louis, MO.

Gretz, JE, EP Kaldjian, AO Anderson, and S Shaw. 1996. Sophisticated strategies for information encounter in the lymph node: The reticular network as a conduit of soluble information and a highway for cell traffic. ("Cutting Edge") J. Immunol. 157:495-499.

Gretz, JE, AO Anderson, and S Shaw. 1997. Cords, channels, corridors and conduits: critical architectural elements facilitating cell interactions in the lymph node cortex. Immunol. Rev. 156:11-24.

Kuprash, DV, MB Alimzhanov, AV Tumanov, AO Anderson, K Pfeffer, and SA Nedospasov. 1999. TNF and Lymphotoxin beta Cooperate in the Maintenance of Secondary Lymphoid Tissue Microarchitecture But Not in the Development of Lymph Nodes. J. Immunol 163: 6575-6580.

Gretz, JE, CC Norbury, AO Anderson, AEI Proudfoot, and S Shaw. 2000. Lymph-borne Chemokines and Other Low Molecular Weight Molecules Reach High Endothelial Venules via Specialized Conduits While a Functional Barrier Isolates the Lymphocyte Microenvironments in Lymph Node Cortex. J. Exp. Med. 192:1425-1440.

Kaldjian, EP, JE Gretz, AO Anderson, Y Shi, S Shaw. 2001. Spatial and molecular organization of lymph node T cell cortex: A labyrinthine cavity bounded by an epithelial sheet of fibroblastic reticular cells anchored to basement membrane-like extracellular matrix. Int Immunol 13:1243-1253.

Kuprash, DV, MB Alimzhanov, AV Tumanov, SI Grivennikov, AN Shakkov, LN Drutskaya, MW Marino, RL Turetskaya, AO. Anderson, K Rajewsky, K Pfeffer and S A Nedospasov. 2002. Redundancy in TNF and LT signaling in vivo: mice with inactivation of the entire TNF/LT locus versus single knockout mice. Mol. Cell. Biol. 22: 8626-8634.

Bajénoff, M, S Granjeaud and S Guerder. 2003. The Strategy of T Cell Antigen-presenting Cell Encounter in Antigen-draining Lymph Nodes Revealed by Imaging of Initial T Cell Activation. J. Exp. Med. 198: 715-724

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Carragher, D, Johal, R, Button, A, White, A, Eliopoulos, A, Jenkinson, E, Anderson, G, and J Caamano. 2004. A Stroma-Derived Defect in NF-{kappa}B2-/- Mice Causes Impaired Lymph Node Development and Lymphocyte Recruitment. J. Immunol. 173(4): 2271-2279.

Pabst, O., Peters, T., Czeloth, N., Bernhardt, G., Scharffetter-Kochanek K., and Förster, R. Cutting Edge: Egress of Newly Generated Plasma Cells from Peripheral Lymph Nodes Depends on beta 2 Integrin. J. Immunol. 174: 7492-7495.

Qi, H., Egen, J.G., Huang, A.Y.C., and Germain, R.N. 2006. Extrafollicular Activation of Lymph Node B Cells by Antigen-Bearing Dendritic Cells. Science 312(5780): 1672-1676

Bajénoff, M., J.G., Egen, L.Y. Koo, Laugier, J.P., Brau, F., Glaichenhaus, N., and Germain, R.N.. 2006. Stromal Cell Networks Regulate Lymphocyte Entry, Migration, and Territoriality in Lymph Nodes. Immunity 25(6): 989-1001.

Roozendaal, R., Mebius, R.E., Kraal, G. 2008. The Conduit System of the Lymph Node. Int Immunol 20: 1483-1487

Mueller, S.N. and Germain, R.N. 2009. Stromal cell contributions to the homeostasis and functionality if the immune system. Nat. Rev. Immunol. 9(9): 618-629

Ruddle, N.H. and Akirav, E.M. 2009. Secondary Lymphoid Organs: Responding to Genetic and Environmental Cues in Ontogeny and the Immune Response. J. Immunol. 183: 2205-2212.

Sinha, R.K., C Park, Il-Y Hwang, M.D. Davis and J.H. Kehrl. 2009. B Lymphocytes Exit Lymph Nodes through Cortical Lymphatic Sinusoids by a Mechanism Independent of Sphingosine-1-Phosphate-Mediated Chemotaxis. Immunity, 30(3): 434-446

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