HUMAN ENVIRONMENTS Environ 3
Popular in Course
Popular in Environmental Health, Science, And Policy
This 5 page Class Notes was uploaded by Mrs. Kelly Wilkinson on Saturday September 12, 2015. The Class Notes belongs to Environ 3 at University of California - Irvine taught by Staff in Fall. Since its upload, it has received 47 views. For similar materials see /class/201885/environ-3-university-of-california-irvine in Environmental Health, Science, And Policy at University of California - Irvine.
Reviews for HUMAN ENVIRONMENTS
Report this Material
What is Karma?
Karma is the currency of StudySoup.
You can buy or earn more Karma at anytime and redeem it for class notes, study guides, flashcards, and more!
Date Created: 09/12/15
Lsravr 372 Realtime imaging of lymphocytes in vivo Michael D CahalanIL Ian Parkerf Sindy H Wei and Mark J Miller New preparations fluorescent probes and imaging techniques are providing the means to observe the behavior of cells in the tissue environment of lymphoid organs In particular when combined with two photon laser microscopy intravital imaging of surgically exposed lymph nodes provides a unique view of lymphocyte migration and antigen presentation as it occurs within the living animal The view is emerging that lymphocytes migrate randomly within lymphoid organs and that lymphocyte contact with antigen presenting cells may be a stochastic process rather than one guided by chemokine gradients Addresses Department of Physiology and Biophysics University of California Irvine California 92697 USA Te mail mcahalanuciedu fDepartment of Neurobiology and Behavior University of California Irvine California 92697 USA Current Opinion in Immunology 2003 15372 377 This review comes from a themed issue on Immunological techniques Edited by Ronald N Germain 0952 7915 see front matter 2003 Elsevier Science Ltd All rights reserved DOI 10101680952 79150300079 7 Abbreviations APC antigen presenting cell DC dendritic cell Introduction The immune system consists of a distributed network of trillions of cells that must operate independently to provide antigen speci city and yet function in a coordi nated manner to defend us from a wide variety of patho gens Communication between cells can be initiated by direct cell contact or can take place at some distance within the tissue environment via chemokines Over the past 20 years remarkable progress in molecular immunology has de ned the mechanism of antigen recog nition and identi ed a growing cast of molecules and signaling pathways that link the T cell receptor to the nucleus However we still understand very little about the basics of motility compartmentalization and antigen recognition in viva because these events occur within densely packed lymphoid organs 1 3 How do T cells B cells and dendritic cells DCs move within the native tissue environment How are T and B cell compartmental boundaries established and maintained How does a T cell locate antigen within the lymph node by following chemokine gradients or by random collisions with antigen presenting cells APCs There is increasing recognition that events de ned 272 who may not correspond to the physiological situation 23972 Moo 4 For example contact between a T cell and an APC leads to a redistribution of surface molecules and formation of the immunological synapse 5 7 This type of mole cular redistribution has also been studied in vitro in planar lipid bilayers with de ned molecular constituents Yet it is still unclear whether stable synapses occur in the environ ment of the lymph node or whether antigen recognition naturally involves short lived serial encounters Lymphoid organs have remained a black box into which de ned cell populations can be induced to home but from which we have been able to obtain only snapshot views by extracting cells or analyzing xed tissue Clearly there exists a strong need for imaging approaches to visualize living cells within intact lymphoid tissue Seeing T cells in their native environment new preparations for imaging To illuminate the black box of the tissue environment new 272 Miro preparations have been developed that more faithfully represent the native tissue environment of intact lymphoid organs These include 3D collagen gel matrices 8 monolayers of endothelial cells ECs bathed with owing solution to mimic the forces experienced by cells 9 cultures of clusters or reaggregated tissue frag ments 103911 engineered tissue surrogates 12 and whole lymph node explants Figure 1 1314quot15quot All of these preparations lack intact vascular and lymphatic vessels and therefore cannot be applied to investigate processes such as lymphocyte traf cking Furthermore the lack of blood and lymphatic vessels may disrupt the distribution of important soluble factors or alter the phy siological levels of tissue oxygenation To overcome this limitation several promising methodol ogies have been developed to visualize cells within the in viva tissue environment Non invasive methods include bioluminescence imaging of cells engineered to express luciferase 16 magnetic resonance imaging microscopy to track cells labeled with superparamagnetic particles 17 and positron emission tomography PET 18 Although these methods can be applied to intact animals all three require cell engineering to derive populations that can be detected and presently lack single cell resolu tion Instead optical techniques offer cellular and even sub cellular levels of resolution Intravital preparations of Current Opinion in Immunology 2003 15372 377 wwwcurrent opinioncom Figure 1 In vivo imaging of lymphocytes Cahalan et a o Model system Planar lipid bilayer 3D collagen gels Reaggregates and clusters Lymphoid tissue explants Intravital preparation 3 an 2 Xlt EC monolayer under flow Strengths High spatial and temporal resolution control over molecular interactions Realistic morphology d 3D behavior mimics natural peripheral tissue substra e Permits adhesive and transmigration behaviors to be analyzed on a physiologic substrate under conditions of ow Preserves the immediate contacts and short range factors 3D tissue environment physiological substrates natural cell to cell interactions long and short range environmental effects True physiological environment Weaknesses 2D important native environmental factors are missing nonnative morphology Solution exchange is difficult may not mimic secondary lymphoid tissues Missing physiological blood ow and tissue signals Lacking physiological extracellular substrates and longran e environmental factors Lacking lymphatic inputs and normal blood ow Technically difficult Current Opll llol l ll l immunology The strengths and weaknesses of model systems in current use for the real time imaging of lymphocytes EC endothelial cell exposed lymphoid organs permit light microscopic imag ing in the native tissue environment With intact circula tory elements but require anesthesia and surgery to bring objective lenses close enough to the tissue To image cells at depths of more than about 50 um two photon microscopy is the technique of choice When combined With uorescent probes confocal microscopy or twophoton microscopy can reveal single cells at the plane of focus either by imaging through a pinhole in the case of confocal microscopy or by selectively exciting the fluorophore only at the plane of focus in the case of two photon microscopy Recently we provided a detailed comparison of confocal and twophoton microscopy as wwwcurrent opinioncom Current Opinion in Immunology 2003 15372 377 374 Immunological techniques applied to the imaging ofT cells i Sill 19 Advantages of twophoton microscopy include less photodamage greater sensitivity and deeper imaging within tissue which is possible because the nearinfrared illumination penetrates tissue more effectively Imaging T cells in explanted lymph nodes Advances in imaging techniques and uorescent markers to label or genetically tag cells or speci c proteins are now making it possible to see events in real time that could previously only be inferred Confocal and twophoton imaging approaches have recently provided the rst glimpse of lymphocyte dynamics within the tissue envir onment We used twophoton microscopy to visualize the behavior of individual T and B lymphocytes in explanted lymph node 14quot and spleen 20 maintained in culture Using an i viva adoptive transfer approach Figure 2 T and B cells labeled with green or red CellTrackerTM dyes Molecular Probes Inc Eugene OR USA homed to appropriate locations and exhibited vigorous motility within the intact lymph node with velocities that aver aged 12 umminute and 6 umminute respectively T cells were observed tomigrate in a stopandgo fashion similar to the behavior ofT cells in a collagen gel matrix culture system with alternating episodes of rapid motion when cells were elongated followed by momentary pauses when cells rounded up The period of these cycles averaged 1 2 minutes In the absence of antigen very few T cells were truly stationary although pressure on the lymph node or accumulated photodamage caused the cells to stop moving In healthy preparations a 3D random walk in all three axes emerges over time because of Tcell turning In the absence of antigen naive T cells from ovalbuminspeci c Figure 2 Currenl Opinion in Immunology lntravital two photon microscopy an anesthetized mouse with surgically exposed lymph node on the microscope stage Details of the preparation have been described 23 1301110 transgenic TCR mice moved randomly without evidence of chemokine gradients Very different results were obtained in a parallel study that used a similar adoptive transfer approach but dif ferent imaging and tissue culture conditions Na39ive T cells were immobile when lymph nodes were maintained in culture with atmospheric oxygen tension and imaged using confocal microscopy Possible reasons for the dif ferences observed have been discussed previously 19 2122 and include differences in imaging depth tissue handling photodamage and oxygen tension in the tissue Our study 14quot used lymph nodes immersed in 95 Oz 5 C02 as previous work on brain slice pre parations clearly demonstrated enhanced survival and function when tissue preparations are maintained i vilra under these conditions Stoll at 4 15quot however chose to use 20 OZ reasoning that lymph nodes may possess low oxygen tension under physiological conditions 23 lntravital twophoton imaging In an effort to resolve the discrepancy regarding the motility of naive T cells i viva we adopted twophoton microscopy to perform intravital imaging of the inguinal lymph node in an anesthetized mouse 24quot In this preparation a simple surgical procedure exposed the inguinal node and allowed lymphocytes to be tracked i viva as they move within the microcirculation home into the lymph node and migrate within the Tcell zone of the lymph node Care was taken to maintain intact cir culation of blood and lymph and to avoid microdissection by imaging through naturally occurring windows in the fat pads that lie on top of the node Under these conditions T cells exhibited vigorous motility and migrated randomly without evidence for collective drift or motion along putative chemokine gradients As observed in explanted lymph nodes naive T cells moved in a stopandgo manner elongating while moving rapidly ahead and then pausing every 1 2 min on average Overall the average velocity of movement was 11 ummin very similar to that of T cells in explanted nodes at the same temperature Figure 3 illustrates a eld ofT cells with four cell tracks quot quot 4 pt dd A 4 cell positions In this instance the mouse was breathing room air during the entire experiment in other experiments we used a mask to deliver a stream of 95 OZ 5 C02 to maintain respiratory drive during longterm measure ments The key point is that vigorous motility in a random walk characterizes the behavior of naive T cells i viva We postulate that T cells distribute autonomously through the Tcell zone and that the search for antigen is a stochastic process Visualizing the interaction between T cells and antigenpresenting cells Antigen challenge dramatically alters the behavior of T cells leading to cell enlargement expression of new Current Opinion in Immunology 2003 15372 377 wwwcurrent opinioncom Figure 3 In vivo imaging of lymphocytes Cahalan et a 375 Current Opinion ll l immunology Intravital two photon images of T cells in the inguinal lymph node of an anesthetized mouse a Trajectories of four separate cells at varying times The colors represent cells at different depths ranging from Ni 00 to 150 Mm below the surface of the lymph node with blue representing the bottom and t red the to tetramethylrhodamine dextran Scale bar 50 gm of the imaging volume Scale bar 25 gm b T cells green vessels and fibers both red labeled via tail vein injection WI genes secretion of cytokines and cell proliferation Two different approaches have been used to Visualize changes in Tcell dynamics evoked by antigen When antigen speci c T cells were transferred into animals that had been injected subcutaneously with speci c antigen clus ters and swarms of enlarged T cells were observed one day following adoptive transfer 14quot At later times cells divided and resumed a Vigorous pattern ofmotility Using wwwcurrent opinioncom Current Opinion in Immunology 2003 15372 377 376 Immunological techniques an alternative method of antigen priming in which APCs were differentiated in vim from one marrow cells pulsed with antigen and then injected subcutaneously Stoll 22 d 15quot observed contact between T cells and APCs that lasted gt15 hours in a onetoone pattern of association Recently we have pursued an in viva labeling method to visualize antigenprimed DCs interacting with D44r T cells M Miller SH Wei I Parker MD Cahalan unpub lished data lfthe T cells can be likened to swimming sh s behave effectively as nets they make contact with T cells by throwing out long membrane tethers and rapidly reeling them back in constantly changing their shape and greatly expanding their capture radius It appears from these early studies that the initial encounter between a T cell and a DC relies upon dynamic cell behaviors that are nely tuned to optimize the chance of random collisions Conclusions Twophoton microscopy represents an optimal technique for tracking the behavior of living cells deep within the tissue environment It is already feasible to image T cells and other cells of the immune system within the circula tion or in the tissue environment of lymph node spleen Peyer s patch thymus and peripheral tissues Video pre sentations of the data demonstrate the dynamic behavior of T cells and B cells as they migrate within the lymph node and of DCs as they interact with T cells during antigen presentation Twophoton imaging will be adap table to a wide variety of new probes for second messen gers and gene expression and to a broad range of processes both physiological and pathological Combined with intravital imaging of surgically exposed lymphoid organs twophoton imaging is providing a unique view of lymphocyte dynamics in viva Update A recent study used twophoton microscopy to examine the interaction of dendritic cells labeled in vitro with motile CD cells in an explanted lymph node preparation 25quot T cells made stable longlasting contacts with antigenpulsed DCs rather than a series of short contacts References and recommended reading Papers of particular interest published within the annual period of review have been highlighted as 39 of special interest quot of outstanding interest GretzJE Anderson AO Shaw S Cords channels corridors and conduits cri ical architectural elements facilitating cell interaction n the lymph node cortex Immunol Rev 1997 15611 24 lquot Gretz JE Norbury CC Anderson AO Proudfoot AE Shaw S Lymp orne chemokines and 0t er ow molecular weight molecules reach high endothelial venules via specialized condui s i e a functional barrier limits access 0 lymphocyte microenvironments in lymph node cortex J Exp Med 2000 1921425 1440 3 Oyster JG Chemokines and cell migration in secondary lymphoid organs Science 1999 2862098 2102 4 Jenkins MK Khoruts A Ingulli E Mueller DL McSorley SJ Reinhardt RL Itano A Paper KA vivo activation of antigen specific CD4 T cells Annu Rev Immunol 2001 1923 45 5 Grakoui A Bromley SK Sumen C Davis MM Shaw AS Allen PM Dustin ML The immunological synapse a molecular machine controlling T cell activation Science 1999 285221 227 6 Dustin ML de Fougerolles AR Reprogramming T cells the role of extracellular matrix in coordination of T cell activation and migration Curr Opin Immunol 2001 13286 290 7 Dustin ML Allen PM Shaw AS Environmental control of immunological synapse formation and duration Trends Immunol 2001 22192 194 8 Gunzer M Schafer A Borgmann S Grabbe S Zanker KS Brocker EB Kam gen E Friedl P Antigen presentation in extracellular matrix interactions of T cells with dendritic cells are dynamic short lived and sequential Immunity 2000 323 332 9 Kantele JM Kurk S Juntila MA Effects of continuous exposure to stromal cell derived factor 1 alpha on T cell rolling and tight adhesion to monolayers of activated endothelial cells J lmmunol2000 1645035 5040 10 Bousso P Bhakta NR Lewis RS Robey E Dynamics of o t y yte stromal cell interactions visualized by two photon microscopy Science 2002 2961876 1880 Two photon microscopy was emplo ed to image motility of and inter actions between thymocytes and stromal cells in a reaggregated thymic organ culture system during positive selection 11 Hommel M Kyewski B Dynamic changes during the immune response in T cell antigen presenting cell clusters isolated from lymph nodes J Exp Med 2003 197269 280 12 Poznansky MC Evans RH Foxall RB Olszak IT Piascik AH Hartman KE Brander 0 Meyer TH Pykett MJ Chabner KTet al Efficient generation of uman T cells from a tissue engineered thymic organoid Nat Biotechnol 2000 18729 734 13 Tan JT Dudl E LeRoy E Murray R SprentJ Weinberg Kl Surh CD IL 7 is critical for homeostatic proliferation and survival of naive T cells Proc Natl Acad Sci USA 2001 988732 8737 14 Miller MJ Wei SH Parker Cahalan MD Two photon imaging of p ocyte motility and dynamic antigen responses in intact lymph node Science 2002 2961869 1873 This aper introduces two o on microscopy to examine lymphocyte motility and antigen responses in an explanted lymph node preparation Highly motile T and B cells were observed and shown to have differing velocities in their respective compartments 39 antigen included T cell enlargement formation of stable cu s ms and a resumption of vigorous motility following cell division 15 Stoll S Delon J Brotz TM Germain RN Dynamic imaging of T on cell dendritic cell interactions in lymph nodes Science 2002 Confocal imaging was used to image T cells interacting with DCs in explanted lymph node cultures Na39I39ve T cells were reportedly immotile and stable conjugates between T cells and APCs were observe 16 Contag CH Bachmann MH Advances in in vivo bioluminescence imaging of gene expression Annu Rev Biomed Eng 2002 4235 260 17 Dodd SJ Williams M Suhan JP Williams DS Koretsky AP Ho 0 Detection of sing e mammalian cells by high resolution magnetic resonance imaging Biophys J 1999 76103 109 18 Dubey P Su H Adonai N Du S Rosato A Braun J Gambhir SS Witte C 39 39 39 39 the T quot 39 lespunse by positron emission tomography Proc NatlAcad Sci USA 2003 1001232 1237 19 Cahalan MD Parker Wei SH Miller MJ Two photon tissue 0 imaging seeing the immune response in a fresh light eviews Immunology 2002 2872 880 This review provi es a technical description and a comparison of two photon and confocal imaging methods applied to the immune system 20 Wei SH Miller MJ Cahalan MD Parker Two photon imaging in intact lymphoid tissue Adv Exp Med Biol 2002 512203 208 Current Opinion in Immunology 2003 15372 377 wwwcurrent opinioncom
Are you sure you want to buy this material for
You're already Subscribed!
Looks like you've already subscribed to StudySoup, you won't need to purchase another subscription to get this material. To access this material simply click 'View Full Document'