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In vivo imaging of leukocyte trafficking in blood vessels
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Thorsten R Mempel, M Lucila Scimone, J Rodrigo Mora a...
In vivo imaging of leukocyte trafficking Mempel et al. 407




information can be generated with three-dimensional        ...
408 Immunological techniques




 Table 1

 List of mouse models in which green fluorescent protein is expressed in leukoc...
In vivo imaging of leukocyte trafficking Mempel et al. 409




 Table 2

 Selected intravital microscopy models in murine ...
410 Immunological techniques




antigen 1 (LFA-1; aLb2), which is critical for mediating       Limitations and strategies...
In vivo imaging of leukocyte trafficking Mempel et al. 411




Figure 1                                                   ...
412 Immunological techniques




Figure 2


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In vivo imaging of leukocyte trafficking Mempel et al. 413




essentially motionless while maintaining physiologic       ...
414 Immunological techniques




Figure 3                                                                                 ...
In vivo imaging of leukocyte trafficking Mempel et al. 415




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10. Ley K, Bullard DC, Arbon...
416 Immunological techniques




49. Kunkel EJ, Campbell DJ, Butcher EC: Chemokines in lymphocyte                  67. Pel...
In vivo imaging of leukocyte trafficking Mempel et al. 417




    intravascular trafficking in lung microvessels. Am J Pat...
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  1. 1. In vivo imaging of leukocyte trafficking in blood vessels and tissues Thorsten R Mempel, M Lucila Scimone, J Rodrigo Mora and Ulrich H von Andrianà Selective recruitment of blood-borne leukocytes to Introduction tissues and their proper positioning within them is Recent progress in biology has frequently gained momen- crucial for the many integrated functions of the immune tum from knowledge integration that reconciles findings system. Intravital microscopy (IVM) techniques have been obtained by reductionist in vitro approaches with obser- employed for more than a century to study these events vations from maximum complexity in vivo models, and at the single-cell level in living animals. Conventional vice versa. In this sense, the use of intravital microscopy video-based IVM allows the visualization of extremely rapid (IVM) to observe single leukocytes in blood vessels and adhesion events at the interface between blood and tissue. tissues of live animals is a maximum complexity Multiphoton IVM is a relatively new tool for imaging approach. A carefully chosen combination of IVM tech- the slower dynamics of cell migration and cell–cell interactions niques with pharmacological and genetic interventions in the extravascular space in three dimensions. Fueled can provide a sensitive ‘reality check’ allowing the critical by the burgeoning development of sophisticated fluorescent auditing and refinement of concepts derived from reduc- markers and increasingly powerful imaging tools, we are tionist in vitro studies. currently witnessing the emergence of a new field in immuno-imaging, in which leukocyte function and The use of microscopes to study the behavior of leuko- cell–cell communication is explored in a truly physiological cytes in sufficiently translucent tissues of experimental context. animals goes back to the 19th century [1,2]. This early work showed that leukocytes, unlike erythrocytes, undergo adhesive interactions with endothelial cells in Addresses The CBR Institute for Biomedical Research and the Department of postcapillary venules and that inflammation dramatically Pathology, Harvard Medical School, Boston, Massachusetts increases the frequency and alters the character of these 02115, USA interactions [2]. IVM studies during the past three dec- à e-mail: uva@cbr.med.harvard.edu ades have used brightfield transillumination (whereby light-absorbing structures appear dark before a bright background) and increasingly epifluorescence to deepen Current Opinion in Immunology 2004, 16:406–417 our understanding of the unique biophysical conditions to This review comes from a themed issue on which leukocytes are exposed in microvessels [3] and to Immunological techniques define the molecular events leading to inflammation- Edited by Peter Friedl induced leukocyte recruitment to peripheral tissues [4–10]. More recently, IVM was instrumental in explain- ing the physiological recruitment of lymphocytes to sec- 0952-7915/$ – see front matter ondary lymphatic organs (SLOs; [11–15]). This work has ß 2004 Elsevier Ltd. All rights reserved. led to the discovery and subsequent refinement of the concept that multi-step adhesion cascades determine DOI 10.1016/j.coi.2004.05.018 where and when leukocytes are permitted access to a given tissue in the body [16–18]. Abbreviations 3D three-dimensional The past few years have seen the advent of several novel DC dendritic cell imaging modalities. In comparison to conventional single- GFP green fluorescent protein photon epifluorescence IVM, these new techniques fea- HEV high endothelial venule ture markedly increased tissue penetration, thus allowing IFN interferon detailed and dynamic views of leukocytes ‘at work’ deep IL interleukin IR infrared within solid tissues. They include bioluminescence ima- i.v. intravenous ging; magnetic resonance imaging; positron emission IVM intravital microscopy tomography; and single photon emission computed tomo- LN lymph node graphy. These approaches allow for the non-invasive MP multiphoton PLN peripheral LN tracking of leukocyte populations over long periods of PP Peyer’s patch time. However, they still lack the spatial and/or temporal SLO secondary lymphatic organs resolution to visualize single-cell dynamics in situ. Such Current Opinion in Immunology 2004, 16:406–417 www.sciencedirect.com
  2. 2. In vivo imaging of leukocyte trafficking Mempel et al. 407 information can be generated with three-dimensional colorless diffractive spheres of fairly uniform size. This fluorescence imaging techniques using multiphoton circumstance has been exploited by microscopists for over (MP) microscopy [19], which can be combined with 165 years [1,2]. Ideal tissues for the study of leukocytes in second harmonic generation imaging [20] to study brightfield IVM must be translucent and devoid of inter- immune cell migration and cell–cell communication in nal structures that strongly scatter or absorb visible light. the interstitium of solid organs [21,22]. This article will Thus, most early work has been conducted in membranous highlight some recent technical and conceptual advances tissues using monochrome cameras to generate recordings in the IVM field and discuss how these techniques may be for off-line analysis. A few studies have used color record- harnessed to see and understand how immunity happens. ings under conventional transillumination or grayscale imaging with oblique transillumination to observe migrat- Visualization techniques ing leukocytes in the extravascular space [23] and in ‘The more precisely the position is determined, the less precisely superficial sinusoids of the liver [24]. However, at phy- the momentum is known in this instant, and vice versa’ Werner siological blood flow rates in transillumination, free-flow- Heisenberg, 1927. ing and fast rolling leukocytes are indistinguishable from the rapidly moving red blood cells, which outnumber Although Heisenberg’s famous uncertainty principle leukocytes $1000:1. Therefore, only cells that are sta- describes a phenomenon of quantum physics, it rings tionary or sufficiently slowed by adhesive interactions all-too-familiar in the ears of intravital microscopists bent with the vessel wall can be detected with this technique. on studying leukocyte migration. Measurements of dynamic biological phenomena typically dictate that Fluorescence microscopy techniques investigators find a compromise between their needs for Intravital fluorophores allow a more complete analysis of optimal temporal resolution versus spatial and depth reso- the entire leukocyte flux in microvessels. Reagents such lution. Methods that yield the highest resolution of struc- as rhodamine 6G, acridine orange or acridine red, which ture, such as electron microscopy, necessitate the most stain nuclei and/or intracellular organelles that are not invasive sample manipulation, such as fixation procedures, found in red blood cells, can be injected intravenously prohibiting dynamic observations of live cells or tissues. (i.v.) and accumulate selectively in leukocytes (and to some degree in platelets and endothelial cells). However, Surgical preparation the intense excitation light that is needed to obtain In IVM experiments, the very process of making a phy- detectable fluorescent signals can cause phototoxic effects sical measurement can interfere with the object under that may manifest as enhanced leukocyte adhesion, study. A conditio sine qua non of any IVM preparation is the microvascular thrombosis, or changes in microvascular requirement for an interpretable optical signal from the diameter and perfusion. The degree of photodamage focal plane of the microscope objective. Suitable optical depends on the type of fluorophore, the intensity and conditions must be created in almost all experimental wavelength of the excitation light, and the duration of models by dissection of the tissue of interest and/or light exposure [25,26]. Strategies to avoid phototoxicity surrounding anatomical structures. The associated trauma include the use of sensitive video cameras that can reliably inevitably acts as a local, and sometimes even systemic, detect weak fluorescent signals with minimal excitation pro-inflammatory stimulus, a well-known phenomenon in intensities. Video-triggered xenon-arc stroboscopes that post-operative surgical care of human patients. Also, even emit a single microsecond pulse of excitation light on a brief drying of surgically exposed tissues causes massive each video frame allow further substantial ($33 000-fold) microvascular dysfunction, which must be prevented by reduction in light exposure compared to continuous superfusion with physiological buffers. The buffer’s com- illumination (video cameras operating according to the position, pH, dissolved gas content (ambient pO2 reg- National Television System Committee [NTSC] standard ulates arteriolar diameter) and potential contaminants acquire 30 frames per second, thus requiring 30 ms cum- (especially bacterial endotoxins) must be carefully con- ulative flash duration per second recording). trolled. Surgery also mandates the use of anesthetics, which can alter respiratory as well as macro- and micro- A further refinement of standard fluorescence IVM meth- hemodynamic conditions, and might modulate the ods is achieved with strategies that allow observers to release or action of inflammatory cells and mediators. identify distinct leukocyte subsets. For example, rather Thus, minimally traumatizing microsurgical techniques than injecting a fluorescent dye i.v., which labels all and a prudent choice and dosing of anesthetics are key leukocytes indiscriminately, leukocyte populations can elements in IVM experimentation. first be purified and fluorescently tagged in vitro. After re- injection into a recipient animal, the transferred cells are Brightfield microscopy easily detected because they are the only fluorescent When a tissue is properly exposed, leukocytes within it particles in the circulation [8]. More recently, leukocyte can be detected by various optical means. In brightfield subset-specific IVM studies have been performed with transillumination, intravascular leukocytes appear as transgenic animals in which a fluorescent protein, such as www.sciencedirect.com Current Opinion in Immunology 2004, 16:406–417
  3. 3. 408 Immunological techniques Table 1 List of mouse models in which green fluorescent protein is expressed in leukocytes in a regulated or subset-specific fashion. Name of mouse strain Type of mutation Target cell type Reference 4 get Knock-in GFP linked to IL4-gene via IRES element. GFP expression [61] correlates with IL-4 transcription. 7.2fms-EGFP Transgene GFP expression in mononuclear phagocytes. [62] CCL17-GFP Knock-in In heterozygous mice, GFP expression correlates with CCL17 activity. [63] LN DC subsets, but not spleen DCs, express variable levels of GFP. CD2-EGFP Transgene CD8þ T cells are GFPbright, subsets of CD4þ T cells and NK cells are GFPdim [27] CX3CR1-GFP Knock-in In heterozygous mice, GFP expression correlates with CX3CR1 [64] (fraktalkine receptor) transcription. Monocytes are GFPbright; subsets of NK cells, microglia and DCs are GFPint. IL-2-GFPki Knock-in In heterozygous mice, GFP expression correlates with IL-2 transcription. [65] lys-EGFP Knock-in In heterozygous mice, GFP expression correlates with lysozyme activity. [66] In peripheral blood fluorescence is restricted to neutrophil granulocytes. mEGFP/mb-1(inv) Knock-in Heterozygous animals express GFP selectively in B cells. [67] MHC II-EGFP Knock-in MHC class II–GFP fusion protein. DCs are GFPbright, B cells GFPint, [68] and macrophages are GFPdim. NG-BAC BAC-transgene Regulatory elements are conserved in BAC. GFP expression correlates [69] with Rag2 transcription in developing lymphocytes. T-GFP Transgene GFP expressed under control of CD4 promoter. Intronic silencer is lacking. [70] All T cells are GFPbright, but lose expression upon activation. Yeti (YFP-enhanced Knock-in YFP expressed spontaneously in NK and NKT cells and, after [71] transcript for IFN-g) stimulation, in Th1 cells. BAC, bacterial artificial chromosome; EGFP, enhanced green fluorescent protein; IRES, internal ribosomal entry site; NK, natural killer; YFP, yellow fluorescent protein. green fluorescent protein (GFP), is expressed under mouse IVM models has been described, some of which control of a subset-specific promoter (Table 1; [13,27,28]). are summarized in Table 2. Animal models Data analysis and interpretation The necessity for sufficient tissue translucency to detect Modes of intravascular leukocyte–endothelial cell inter- leukocytes in brightfield IVM has caused early intravital actions that are uniquely quantifiable by IVM include: the microscopists to perform their studies primarily in mem- formation of adhesive tethers that slow fast moving cells; branous tissues including the interdigital web [1], mesen- subsequent rolling, which occurs at much slower velocity tery and tongue of frogs [2]; bat wing [29]; hamster cheek than the hydrodynamic velocity [35]; sticking (typically pouch [30]; mesentery in various species [3,5,6,31]; defined as firm arrest for at least 30 seconds); and extra- implanted ear chambers [32] and tenuissimus muscle vasation [2]. Each of these parameters reflects the action in rabbits [4]; and the cremaster muscle in rats [33,34]. of one or several distinct molecular pathways with unique biophysical characteristics [4–6]. Thus, it makes sense to With the advent of fluorescence microscopy and low-light compare results from studies that have measured these detection technology, IVM of leukocyte traffic in solid parameters in different models. organs has become feasible, thus allowing comparative analyses of tissue-specific recruitment events. Moreover, However, there are caveats. For example, many studies the recent explosion of available biological tools, such as have measured the rate at which leukocytes roll or stick in antibodies, recombinant proteins, small molecule inhibi- venules before and after an experimental manipulation, tors and genetically engineered cells and animals has such as application of an antibody or inflammatory stim- enabled investigators to go beyond the mere description ulus. Such measurements can be misleading unless they of microvascular phenomena to hypothesis-driven dissec- are correlated to concomitant local blood flow rates and the tion of distinct molecular events. Specifically, selective subset frequency and total number of leukocytes passing functional modulation (primarily of adhesion and signal- through the same vessel segment or, as a surrogate para- ing molecules) by these means, either in adoptively meter, the systemic and differential white blood cell count, transferred leukocytes or host tissues, has greatly con- which together determine the flux of cells that are deliv- tributed to the definition of their precise physiological ered to the site of observation. Also, the adhesion pathways role in vivo. Given the relative paucity of such tools in that mediate any given step of the leukocyte recruitment most mammalian species other than the mouse, it is no cascade can vary substantially between different tissues, surprise that recent innovations in the IVM field have and even within the same tissue, depending on the type focused on murine models. Indeed, a growing selection of and kinetics of the pro-inflammatory stimulus and the Current Opinion in Immunology 2004, 16:406–417 www.sciencedirect.com
  4. 4. In vivo imaging of leukocyte trafficking Mempel et al. 409 Table 2 Selected intravital microscopy models in murine tissues. Organ Special endothelial Imaging Field(s) of study Reference(s) features/adhesiveness modality Bone marrow/bone NVA, sinusoids EF, MP Hematopoiesis, oncology, BM transplantation. [72] CNS: cortex VA, BBB EF, MP Cerebral blood flow, EAE, stroke, [73] neurodegeneration, neurobiology, encephalitis. spinal cord [74] Cremaster muscle VA TI, EF Inflammation, thrombosis ischemia-reperfusion [10,75] injury, cancer metastasis. Eye: iris VA, NVA EF, reflected TI Uveitis. [76,77] retina BBB EF, CF Diabetes. [78] Intestine VA EF IBD, ischemia-reperfusion injury, GvHD. [79] Knee (synovium) VA EF Arthritis. [80] Kidney (hydronephrotic) Glomeruli TI Electrophysiology. [81] Liver NVA, portal and hepatic TI Blood flow regulation, metastasis, inflammation, [82] microvessels, sinusoids Kupffer cell function, leukocyte homeostasis, platelet survival. Lung (transplant to VA, NVA EF Allotransplantation, [83] dorsal chamber) pulmonary blood flow. Lymph node: inguinal (subiliac) VA, HEV EF, CF, MP Lymphocyte recirculation, immune response. [12,13,15,37] mesenteric [56] popliteal [55] Mesentery VA TI, EF Leukocyte traffic, endothelial permeability, [3,9] ischemia -reperfusion injury. Pancreas/islets VA TI, EF Diabetes, pancreatitis. [84–86] Peyer’s patch HEV EF Lymphocyte recirculation. [11,14,15,36] Skin: ear VA EF DTH, psoriasis. [87–89] back (dorsal window) VA EF Inflammation, transplant rejection, ischemia- [86,90] reperfusion injury, tumor biology, atherosclerosis. Spleen NVA, marginal zone, TI, CF Lymphocyte recirculation. [56,91] red pulp sinusoids BBB, blood brain barrier; CF, confocal microscopy; CNS, central nervous system, DTH, delayed-type hypersensitivity; EAE, experimental allergic encephalomyelitis; EF, epifluorescence; GvHD, graft-versus-host disease; HEV, high endothelial venules; IBD, inflammatory bowel disease; MP, multiphoton microscopy; NVA, adhesiveness not restricted to venules; TI, transillumination; VA, venular adhesiveness only. nature of the responsive leukocyte subset(s). Measure- level, such as flow cytometry- or histology-based homing ments of interstitial cell migration and cell–cell interac- assays, is that the precise step of the recruitment cascade, tions in a scanned volume of tissue are also subject to at which a particular molecule is acting, can be identified limitations that are only beginning to be explored. For in vivo. Two mouse IVM models have been particularly instance, the most motile migrating cells are more likely to instrumental in deciphering the molecules that mediate leave (and re-enter) an imaged tissue volume than slow ¨ the multi-step cascade that guides naıve T- and B-cell moving or stationary cells. When measuring average velo- homing to peripheral lymph nodes (PLNs) and Peyer’s cities or frequency distributions of migratory velocities, Patches (PPs; [36,37]). These studies have shown that, highly motile cells may be measured more than once and during homing to PLNs, both T and B cells use L-selectin are consequently over-represented in the sample. These (CD62L) and its counter-receptor, peripheral node examples highlight the need for careful consideration of addressin (PNAd), to tether and roll in high endothelial the experimental conditions when interpreting or compar- venule (HEVs), but not other segments of the microvas- ing data derived from different IVM studies. ¨ cular bed [12]. When they are tethered, naıve T cells must encounter CCR7 ligands (CCL19 or CCL21) expressed In vivo dissection of lymphocyte ¨ on HEVs to home to PLNs or PPs, whereas naıve B cells homing pathways and central memory T cells can also be stimulated by Naıve lymphocyte recruitment to secondary ¨ CXCL12 in these organs [13,15,38]. In addition, B cells lymphoid organs can be recruited by the CXCR5 ligand, CXCL13 [14,15, The major strength of IVM compared to techniques that ¨ 39]. Upon activation by chemokines, naıve T and B allow insights into homing mechanisms at the population cells activate the integrin leukocyte function-associated www.sciencedirect.com Current Opinion in Immunology 2004, 16:406–417
  5. 5. 410 Immunological techniques antigen 1 (LFA-1; aLb2), which is critical for mediating Limitations and strategies to study immediate firm arrest in PLN HEVs (reviewed in [22]). antigen-experienced lymphocytes L-selectin also plays a role during T- and B-cell rolling in Despite these phenotypic distinctions, the behavior of PP HEVs, but it is less essential than in PLNs because effector/memory cells in situ is still largely unexplored. the integrin a4b7 and its ligand, MAdCAM (for mucosal As it is not (yet) possible to determine the history and addressin cell adhesion molecule 1), also contribute to phenotype of endogenous lymphocytes in immunized rolling in PPs. The a4b7/MAdCAM pathway is particu- animals by direct in situ observation, investigators must larly essential in this tissue because it allows tethered resort to purifying a cell population of interest from donor cells to slow down and, upon chemokine activation, animals and then transferring tagged cells to a recipient collaborates with LFA-1 to mediate firm arrest [11,40]. to assess their distribution (by homing experiments) and Our current understanding of these intricate tissue-spe- intravascular behavior (by IVM). The problem with this cific multi-step recruitment cascades is nearly entirely approach is the scarcity and inhomogeneity of antigen- based on IVM analysis. experienced lymphocytes that can be isolated from immunized animals. Adoptive transfer experiments of Effector and memory cell trafficking labeled lymphocyte subsets for homing or IVM experi- ¨ Although naıve lymphocytes are selectively equipped to ments requires at least several million highly purified recirculate through SLOs, antigen encounter induces cells. This problem is exacerbated upon i.v. injection their differentiation into effector cells, which express (the route of choice for homing experiments) because new traffic molecules and migrate to peripheral tissues leukocytes must first pass through the pulmonary circu- [18]. Most effector T cells die after clearing their anti- lation where many cells become transiently trapped and gen, leaving behind a small population of memory will only enter the systemic circulation after some delay. T cells that respond more rapidly when antigen is The need for large cell numbers can be somewhat re-encountered. The molecular mechanisms governing alleviated in IVM studies by injecting fluorescent cells the migration of effector cells are less well characterized into a feeding artery close to the site of observation ¨ than those guiding naıve lymphocytes. Effector cells [8,37]. The number of cells that can be observed under acquire different profiles of adhesion molecules and high power magnification, however, is naturally limited chemokine receptors depending on the SLO in which by the blood flow rate in postcapillary venules; for ¨ their naıve precursors are primed by dendritic cells example, the average blood flow in cortical HEVs in (DCs). Thus, antigen presentation by DCs from gut- mouse inguinal LNs is $1.8 nl/s [37]. To avoid undesired associated SLOs induces effector cells that home to the hemodynamic effects during cell injections, fluorescent intestine [41–44], whereas DCs from skin-draining cells must be injected slowly (over several minutes) and lymph nodes (LNs) induce a skin-homing effector phe- at a high concentration to detect a sufficient number notype (JR Mora and UH von Andrian, unpublished). passing through each microvessel. The required number Thus, whereas skin homing T cells use the cutaneous of cells doubles if a control population is to be compared lymphocyte-associated antigen (CLA) and respond to to a test sample that has been exposed to experimental CCR4- and CCR10-activating chemokines, gut-homing manipulations such as treatment with an enzyme or T cells express a4b7 and respond to CCL25, the CCR9 antibody. ligand [45,46]. To circumvent these problems, we have sought to Because lymphocyte recruitment molecules are selec- develop tissue culture methods to generate specialized tively and constitutively expressed in the skin and gut, memory and effector T-cell subsets from T-cell receptor tissue-tropic effector/memory cells can migrate to these ¨ (TCR) transgenic naıve T cells under controlled in vitro sites (at least to some degree) in the absence of inflam- conditions (Figure 1). To this end, we have described a mation [43]. However, antigen-experienced T cells also method of producing virtually unlimited numbers of express receptors for inflammation-induced traffic sig- antigen-specific cytotoxic effector or central memory nals and migrate efficiently to sites of inflammation CD8þ T cells by culturing peptide-stimulated lympho- [47,48]. The type, pattern and quantity of inflamma- blasts in a high concentration of IL-2 or IL-15, respec- tion-seeking traffic molecules depends on the type of tively [50,51]. More recently, we discovered that effector effector cells; for example, polarized T helper 1 (Th1) cells with preferential gut- or skin-homing properties cells express different chemokine receptors and bind ¨ can be generated from naıve T cells by coculture with more efficiently to endothelial selectins than Th2 cells, DCs from PPs and cutaneous LNs, respectively (JR and central memory cells are distinguished from effector Mora and UH von Andrian, unpublished; [43]). We memory subsets by their expression of the lymph node are now routinely using these ‘custom-differentiated’ homing receptors, CCR7 and L-selectin (reviewed in T-cell subsets to study the molecular mechanisms that [18]). Similarly, IgA-producing plasmablasts express control their selective recruitment to lymphoid and non- mucosal homing receptors, whereas their IgG-producing lymphoid tissues [48,52] and to sites of inflammation cousins do not [49]. [48,53]. Current Opinion in Immunology 2004, 16:406–417 www.sciencedirect.com
  6. 6. In vivo imaging of leukocyte trafficking Mempel et al. 411 Figure 1 efficiently than the surrounding tissue. Thus, the dark (or red) vessel outlines provide convenient landmarks to which blood-borne leukocyte movement is confined. TCR transgenic Therefore, reasonably exact measurements of intravas- naïve CD8+ T cell cular cell velocity can be obtained from two-dimensional IVM recordings of microvessels that are positioned within Coculture with the focal plane of a microscope objective. Moreover, most Stimulate splenocytes peptide-pulsed intravascular adhesion events occur within seconds or with peptide Ag (48 hours) DCs from PPs quicker, and are best analyzed from recordings obtained at conventional video frame rates (i.e. 30 frames per second for NTSC or 25 frames per second in Phase Coculture with Alternation by Line [PAL] standard systems). At this peptide-pulsed acquisition speed, proper tissue positioning is usually DCs from sufficient to minimize movements of the preparation cutaneous LNs due to respiration or muscle action, thus avoiding image Lymphoblast Gut-homing blurring or distortions. Slow tissue movements in the X–Y effector cell direction, ranging from tens to hundreds of micrometers, Wash are often acceptable without precluding data extraction. Culture in These facilitating factors are of little use for IVM studies Culture in IL-15 or IL-7 of extravascular leukocytes. In principle, cells in tissues IL-2 (20 ng/ml) (5 ng/ml) are free to migrate in any direction, thus requiring accu- 5+ days rate imaging in three dimensions to analyze their trajec- Skin-homing tories. Therefore, stacks of optical sections must be effector cell acquired and reassembled to render a three-dimensional (3D) image of a volume of tissue surrounding the migrat- ing cell. Repeated scanning of the same volume of interest allows detection of the displacement of each migrating cell over time. The larger the number of Z- Cytotoxic Central sections and the smaller the vertical step size, the better effector cell memory cell the 3D resolution and the longer the interval during Current Opinion in Immunology which a migrating cell can be tracked. However, the acquisition of large image stacks takes much longer than Schematic diagram of in vitro strategies to generate tissue-tropic conventional video recordings. Moreover, as interstitial T-cell subsets from TCR-transgenic naıve CD8þ T cells. Effector cells ¨ produced by culture of antigen-activated lymphoblasts in a high cells migrate at velocities that are two to three orders of concentration of IL-2 exhibit potent antigen (Ag)-specific cytotoxicity magnitude slower than those in microvessels, much and IFN-g production and, upon i.v. injection, accumulate in liver, lungs, longer observation periods are necessary for accurate spleen and at sites of inflammation, but not in LNs or PPs [48,50]. By measurements. Thus, even slow and subtle tissue move- contrast lymphoblast culture in IL-15 (or IL-7) generates central- ments (as small as a few micrometers) can render 3D memory-like cells that give rise to effector cells upon re-exposure to Ag and confer long-lived memory upon adoptive transfer to naıve ¨ images virtually uninterpretable. mice [50]. In vitro generated central memory cells home to all SLOs and can also migrate to sites of inflammation, albeit less efficiently We encountered these problems when we attempted to than effector cells [48,53]. T cell activation in the presence of purified adapt our established mouse inguinal LN model [37] to DCs from PPs or skin-draining LNs generates effector T cells that 3D imaging using two-photon microscopy (see below). In migrate preferentially to the skin or gut, respectively (JR Mora and UH von Andrian, unpublished; [43]). this model, a microsurgically dissected skin flap with the embedded LN is gently stretched by tension sutures over a glass slide next to the anesthetized mouse’s flank In vivo imaging of interstitial leukocyte migration and (Figure 2a). Thoracic and abdominal movements from cell–cell communication the animal’s normal respiration can be transmitted to Although IVM techniques to examine intravascular leu- the skin flap to cause macroscopically imperceptible kocyte–endothelial cell interactions have evolved over intermittent tissue displacement, which was of little one and a half centuries, in situ extravascular leukocyte consequence for conventional epifluorescence videomi- behavior is largely unexplored territory. The traditional croscopy, but turned out to be detrimental when we emphasis on intravascular leukocytes is partly explained attempted 3D recordings. To alleviate this, we tested a by the fact that blood-filled microvessels are easily variety of restraining devices akin to what has been detected because the abundant hemoglobin in the vessel described by others [54]. However, in our hands, lumen absorbs transmitted and fluorescent light more mechanical restriction of soft tissue motion often caused www.sciencedirect.com Current Opinion in Immunology 2004, 16:406–417
  7. 7. 412 Immunological techniques Figure 2 (a) Jugular vein (drugs) Femoral artery (cell injection) Carotid artery (ABP, HR, blood) (b) (c) (iii) Scan head BCU Ti:S laser Direct detectors (ii) PC (i) (vii) 20X/NA 0.95 (ii) (iii) (iv) (v) Thermistor Heated circulator (vi) Intravital microscopy techniques to study leukocyte traffic in peripheral lymph nodes. (a) The inguinal LN model [37] makes use of a skin flap preparation. This preparation is ideally suited for IVM studies of lymphocyte adhesion in HEV because it has a well-defined venular tree that is readily visualized in this position. Interstitial lymph fluid from the flank and upper hind leg is normally drained towards this LN (white arrow), but may also drain via lymphatic anastomoses into the ipsilateral axillary LN or to contralateral PLNs (gray arrows). (b) The popliteal LN receives afferent lymph from the lower hind leg (white arrow). This preparation is ideally suited for MP-IVM because its small dimensions allow imaging in the deep cortex [55]. (c) A small skin incision in the hollow of the knee provides access to the LN, which is immersed in saline surrounded by vacuum grease (i) and sealed with a cover slip (ii). The cover slip is glued to an adjustable metal holder (iii). A metallic heating coil (iv) is positioned on top of the cover slip together with a water droplet (v) for immersion of a dipping objective. Tissue temperature is monitored through a microthermocouple (vi) in close vicinity to the LN and regulated by adjusting water flow through the heating coil using a circulating hot water bath (not shown). Tissue movement is prevented by percutaneous clamps attached to superficial osseous structures of the spine, pelvis and hind leg (not shown). Blood and efferent lymph flow leave at the LN hilus (vii), and afferent lymph vessels (viii) enter the node at its distal pole. Pulsed infrared laser light from a titanium:sapphire (Ti:S) laser is raster-scanned over the field of view of a high numerical aperture lens. The beam conditioning unit (BCU) serves to adjust the laser power through a Pockel’s cell and to blank the beam when no signal is recorded. ABP, arterial blood pressure; HR, heart rate. uncontrollable loss of physiological lymph flow to the Our efforts led us to establish a new IVM model in mouse prepared node, presumably because differential intersti- popliteal LN (Figure 2b; [55]). This organ sits in the tial pressure gradients allowed lymph fluid that accumu- dorsum of the knee, and the relevant neighboring bones lated in the vicinity of the inguinal LN to be drained to can be firmly immobilized without excessive manipula- other PLNs. As lymph flow is known to influence leu- tion of soft tissues. Moreover, this LN receives afferent kocyte traffic in PLNs (reviewed in [22]), we sought an lymph from the lower leg, which has no alternative alternative to the inguinal LN preparation. direction of drainage. Thus, our new preparation remains Current Opinion in Immunology 2004, 16:406–417 www.sciencedirect.com
  8. 8. In vivo imaging of leukocyte trafficking Mempel et al. 413 essentially motionless while maintaining physiologic entire sample above and below the focal plane is exposed blood and lymph flow [22]. to excitation light, fluorescence is only induced in the focal plane. During repetitive acquisition of vertical Another important factor in 3D imaging of cell migration image stacks for 3D rendering, photobleaching and is the choice of visualization technique. Imaging ap- phototoxicity are greatly reduced, allowing many hours proaches that can be used to obtain spatial information of imaging without compromising specimen viability. from thick fluorescent samples include widefield detec- Second, due to the inherent optical sectioning effect, tion combined with deconvolution processing, confocal there is no need to reject out of focus light by placement and MP-IVM. Both widefield fluorescence and confocal of a pinhole as in confocal microscopy. Therefore, non- microscopy use single-photon excitation; that is, the descanned photodetectors (i.e. those without a pinhole in sample is exposed to visible light (typically in the green the light path) can be used to collect even scattered or blue spectrum) in which photons have sufficient energy emitted light, which would be rejected during confocal to excite a fluorochrome, which then emits light of a imaging. This increases the signal:noise ratio and sensi- slightly longer wavelength (typically in the green or red tivity of the imaging system. Third, infrared excitation spectrum, respectively). light becomes much less scattered and absorbed than light at shorter wavelengths, thus allowing deeper optical Because single-photon excitation requires intense short penetration of tissues. Indeed, MP imaging allows at least wavelength (i.e. high energy) excitation light, its use for fivefold deeper tissue penetration in mouse lymph nodes IVM studies is limited by several disadvantages, includ- than confocal imaging [54,55,57–59]. Fourth, MP exci- ing a high risk of phototoxicity, rapid photobleaching, and tation can excite several different fluorophores at a single restriction of imaging depth to less than $100 mm in soft excitation wavelength, thus allowing the simultaneous tissues such as PLNs (for a detailed discussion, see [21]). detection of several differentially tagged fluorescent To our knowledge, deconvolution strategies have not objects [22,55]. For example, our MP-IVM system per- been explored to image leukocyte migration in living mits simultaneous signal acquisition from three channels, tissues, such as PLNs. Confocal microscopy has been but by labeling some target structures with a single employed in murine spleen [56] and to record T cells and fluorophore and others with a combination of dyes (e.g. DCs in the superficial cortex of excised LNs [57]. green plus red will appear as yellow) it becomes possible, in principle, to distinguish as many as six differentially The application of MP excitation to optical microscopy tagged objects in a single recording. Finally, fifth, the has overcome many of the limitations of single-photon spontaneous interaction of infrared light with large ani- fluorescence techniques [19]. This technology makes use sotropic macromolecules, particularly collagen, leads to of a few well-known principles of quantum physics. When frequency doubling, which elicits so-called second- two (or more) photons with an energy that is insufficient to harmonic generation of UV-photons that can be regis- excite a fluorescent molecule arrive at the same point in tered with appropriate photodetectors. This enables us to space virtually at the same time (within 10À16 s), they can visualize extracellular matrix fibers in a separate channel, co-operate. By virtue of near-colocalization, two or more which can serve as a convenient tool to identify landmark low-energy (i.e. long wavelength) photons can combine structures in complex tissues and to analyze interactions their energy to excite a fluorescent molecule in essentially of migrating cells with extracellular matrix components the same way as a single high-energy photon. Simply [20,22,55,60]. speaking, when two photons of 1000 nm wavelength interact simultaneously with a fluorescent molecule, the In our popliteal LN model, MP-IVM routinely yields 3D fluorophore behaves as if it encountered one photon with time-lapse videos of fluorescent signals from up to a depth twice the energy (i.e. $500 nm wavelength). To generate of 400 mm below the surface of the organ, permitting cell this MP effect, infrared (IR) or near-IR light from a migration and cell–cell interactions to be tracked over titanium:sapphire (Ti:S) laser is condensed into ultrashort several hours (for examples of video footage that can ($100 fs) pulses, each pulse having a photon density that be generated with this approach, the reader is referred is $125 000-fold higher than a continuous beam from a to http://cbr.med.harvard.edu/investigators/vonandrian/ laser with the same average output power. These IR laboratory/Pages/Videos%20Page.html). These videos photon packages are focused on a specimen through an can be analyzed with automated image tracking software objective with high numerical aperture. Optical focusing to yield quantitative measurements of 3D T-cell and DC further increases the photon density to a degree that MP motility from thousands of individual measurements fluorescence excitation is achieved in a small volume [55]. It has also been possible to characterize the quality ($1 fl) of space around the focal point of the objective. and duration of T cell–DC interactions in the presence and absence of a cognate antigen during the first 48 hours In our view, there are several distinct features that make ¨ after a naıve T cell has homed to a PLN (Figure 3). We MP-IVM the technique of choice for 3D imaging of living found that, during the first eight hours after entry from the tissues. First, unlike single-photon microscopy, where the blood, T cells undergo multiple short encounters with www.sciencedirect.com Current Opinion in Immunology 2004, 16:406–417
  9. 9. 414 Immunological techniques Figure 3 optical component technology continue to push the limits of what can be visualized with this technique. One area that is still in its infancy is the investigation into how the many gigabytes of digital data are best processed, pre- sented and interpreted to distill a maximum of infor- CD44 mation from highly complex multi-agent processes. A CD25 discussion about the choice and relevance of different IL-2/IFN-γ From CD69 measurement parameters of leukocyte behavior and their blood Proliferation immunological and pathophysiological ‘meaning’ will be Brief, serial Stable Brief, serial encounters necessary when a critical amount of data from different encounters contacts investigations has reached the public domain. 0h 8h 20h 44h To Phase I Phase II Phase III blood Another exciting possibility is the use of MP-IVM to Hours (h) study in situ changes in immune-cell function and differ- T-cell motility entiation. For example, transgenic mice have been Current Opinion in Immunology described that express fluorescent proteins as surrogate markers of effector cytokine production (Table 1). How- ¨ A schematic diagram of the three phases of naıve T-cell priming by ever, the inevitable lag time of several hours between dendritic cells in peripheral lymph nodes, as revealed by multiphoton signal-induced reporter gene activation and detectable intravital microscopy [55]. Upon entry into a LN containing antigen- fluorescent protein expression limits the usefulness of presenting mature DCs, the T cells engages in highly motile these tools for studying T-cell responses in real time. For behavior with repeated short-lasting contacts with multiple DCs this application, in situ visualization of intracellular ion during the first $8 hours (Phase 1). Antigen encounter during this phase induces detectable upregulation of the activation markers fluxes or redistribution of chimeric fluorescent signaling CD44 and CD69. During Phase 2 ($8–24 hours) T cells form stable molecules might be as scientifically rewarding as it is conjugates with individual DCs that last at least 60 minutes and technically challenging. probably much longer. During this period, T cells upregulate CD25 and begin to produce IL-2 and IFN-g, but do not yet proliferate. On the second day (Phase 3) T cells again become highly motile, References and recommended reading form only short contacts with DCs and divide rapidly. Eventually, Papers of particular interest, published within the annual period of review, have been highlighted as: they assume full effector function (after three–four days) and return to the blood stream. of special interest of outstanding interest 1. Wagner R: Erla¨uterungstafeln zur Physiologie und Entwicklungsgeschichte. 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The acceler- Lectin-like cell adhesion molecule 1 mediates leukocyte rolling in mesenteric venules in vivo. Blood 1991, 77:2553-2555. ating evolution of powerful new imaging technologies combined with an increasing diversity of biological tools 7. Kubes P, Suzuki M, Granger DN: Nitric oxide: An endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci USA 1991, has transformed IVM experimentation from a mainly 88:4651-4655. descriptive activity to an hypothesis-driven examination 8. von Andrian UH, Hansell P, Chambers JD, Berger EM, Filho IT, of fundamental immunological processes in real time and Butcher EC, Arfors KE: L-selectin function is required for real life. In particular, we expect exciting new develop- b2-integrin-mediated neutrophil adhesion at physiological shear rates in vivo. Am J Physiol 1992, 263:H1034-H1044. ments from the future use of MP-IVM in lymphoid and 9. Mayadas TN, Johnson RC, Rayburn H, Hynes RO, Wagner DD: non-lymphoid tissues. 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