Endogenous Regulatory T Cells Adhere in Inflamed Dermal Vessels via ICAM-1: Association with Regulation of Effector Leukocyte Adhesion.

J Immunol. 2012 Jan 25;

Authors: Deane JA, Abeynaike LD, Norman MU, Wee JL, Kitching AR, Kubes P, Hickey MJ

Abstract
Regulatory T cells (Tregs) must express appropriate skin-homing adhesion molecules to exert suppressive effects on dermal inflammation. However, the mechanisms whereby they control local inflammation remain unclear. In this study we used confocal intravital microscopy in wild-type and Foxp3-GFP mice to examine adhesion of effector T cells and Tregs in dermal venules. These experiments examined a two-challenge model of contact sensitivity (CS) in which Treg abundance in the skin progressively increases during the course of the response. Adhesion of CD4(+) T cells increased during CS, peaking 8-24 h after an initial hapten challenge, and within 4 h of a second challenge. At these time points, 40% of adherent CD4(+) T cells were Foxp3(+) Tregs. CD4(+) T cell adhesion was highly dependent on ICAM-1, and consistent with this finding, anti-ICAM-1 prevented Treg adhesion. Skin TGF-β levels were elevated in skin during both challenges, in parallel with Treg adhesion. In the two-challenge CS model, inhibition of ICAM-1 eliminated Treg adhesion, an effect associated with a significant increase in neutrophil adhesion. Similarly, total CD4(+) T cell depletion caused an increase in adhesion of CD8(+) T cells. Because Treg adhesion was restricted by both of these treatments, these experiments suggest that adherent Tregs can control adhesion of proinflammatory leukocytes in vivo. Moreover, the critical role of ICAM-1 in Treg adhesion provides a potential explanation for the exacerbation of inflammation reported in some studies of ICAM-1-deficient mice.

PMID: 22279104 [PubMed - as supplied by publisher]

Assessment of mucus thickness and production in situ.

Methods Mol Biol. 2012;842:217-27

Authors: Holm L, Phillipson M

Abstract
The nature of the mucus gel layer covering the gastrointestinal tract makes it difficult to study outside its natural site attached to the mucosa. Here, we describe a technique for intravital microscopy studies of the mucus gel layer from the stomach down to the colon in anesthetized rats and mice. Mucus thickness and accumulation rate in each segment of the gastrointestinal tract is measured with a micropipette technique under observation through a stereomicroscope. In this way, the nature of the mucus gel in vivo is readily studied, and effects of interventions or disease on the mucus can be determined in longitudinal studies or by comparing animals. Using this technique, we have been able to demonstrate that there are two forms of mucus gel adherent to the stomach and colon mucosa: one layer which is removable by suction and an underlying firm adherent gel layer, while in the small intestine, all mucus adhering to the mucosa can easily be removed.

PMID: 22259138 [PubMed - in process]

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy.

J Vis Exp. 2012;(59)

Authors: Caetano SS, Teixeira T, Tadokoro CE

Abstract
Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus. Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation. Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KI(gfp/gfp) mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

PMID: 22258059 [PubMed - in process]

Intravital multiphoton imaging of immune responses in the mouse ear skin.

Nat Protoc. 2012;7(2):221-34

Authors: Li JL, Goh CC, Keeble JL, Qin JS, Roediger B, Jain R, Wang Y, Chew WK, Weninger W, Ng LG

Abstract
Multiphoton (MP) microscopy enables the direct in vivo visualization, with high spatial and temporal resolution, of fluorescently tagged immune cells, extracellular matrix and vasculature in tissues. This approach, therefore, represents a powerful alternative to traditional methods of assessing immune cell function in the skin, which are mainly based on flow cytometry and histology. Here we provide a step-by-step protocol describing experimental procedures for intravital MP imaging of the mouse ear skin, which can be easily adapted to address many specific skin-related biological questions. We demonstrate the use of this procedure by characterizing the response of neutrophils during cutaneous inflammation, which can be used to perform in-depth analysis of neutrophil behavior in the context of the skin microanatomy, including the epidermis, dermis and blood vessels. Such experiments are typically completed within 1 d, but as the procedures are minimally invasive, it is possible to perform longitudinal studies through repeated imaging.

PMID: 22240584 [PubMed - in process]

Difference in Th1 and Th17 Lymphocyte Adhesion to Endothelium.

J Immunol. 2012 Jan 4;

Authors: Alcaide P, Maganto-Garcia E, Newton G, Travers R, Croce KJ, Bu DX, Luscinskas FW, Lichtman AH

Abstract
T cell subset-specific migration to inflammatory sites is tightly regulated and involves interaction of the T cells with the endothelium. Th17 cells often appear at different inflammatory sites than Th1 cells, or both subsets appear at the same sites but at different times. Differences in T cell subset adhesion to endothelium may contribute to subset-specific migratory behavior, but this possibility has not been well studied. We examined the adhesion of mouse Th17 cells to endothelial adhesion molecules and endothelium under flow in vitro and to microvessels in vivo and we characterized their migratory phenotype by flow cytometry and quantitative RT-PCR. More Th17 than Th1 cells interacted with E-selectin. Fewer Th17 than Th1 cells bound to TNF-α-activated E-selectin-deficient endothelial cells, and intravital microscopy studies demonstrated that Th17 cells engage in more rolling interactions with TNF-α-treated microvessels than Th1 cells in wild-type mice but not in E-selectin-deficient mice. Th17 adhesion to ICAM-1 was dependent on integrin activation by CCL20, the ligand for CCR6, which is highly expressed by Th17 cells. In an air pouch model of inflammation, CCL20 triggered recruitment of Th17 but not Th1 cells. These data provide evidence that E-selectin- and ICAM-1-dependent adhesion of Th17 and Th1 cells with endothelium are quantitatively different.

PMID: 22219321 [PubMed - as supplied by publisher]

Regulation of Eosinophil Trafficking by SWAP-70 and Its Role in Allergic Airway Inflammation.

J Immunol. 2011 Dec 30;

Authors: Bahaie NS, Hosseinkhani MR, Ge XN, Kang BN, Ha SG, Blumenthal MS, Jessberger R, Rao SP, Sriramarao P

Abstract
Eosinophils are the predominant inflammatory cells recruited to allergic airways. In this article, we show that human and murine eosinophils express SWAP-70, an intracellular RAC-binding signaling protein, and examine its role in mediating eosinophil trafficking and pulmonary recruitment in a murine model of allergic airway inflammation. Compared with wild-type eosinophils, SWAP-70-deficient (Swap-70(-/-)) eosinophils revealed altered adhesive interactions within inflamed postcapillary venules under conditions of blood flow by intravital microscopy, exhibiting enhanced slow rolling but decreased firm adhesion. In static adhesion assays, Swap-70(-/-) eosinophils adhered poorly to VCAM-1 and ICAM-1 and exhibited inefficient leading edge and uropod formation. Adherent Swap-70(-/-) eosinophils failed to translocate RAC1 to leading edges and displayed aberrant cell surface localization/distribution of α4 and Mac-1. Chemokine-induced migration of Swap-70(-/-) eosinophils was significantly decreased, correlating with reduced intracellular calcium levels, defective actin polymerization/depolymerization, and altered cytoskeletal rearrangement. In vivo, recruitment of eosinophils to the lungs of allergen-challenged Swap-70(-/-) mice, compared with wild-type mice, was significantly reduced, along with considerable attenuation of airway inflammation, indicated by diminished IL-5, IL-13, and TNF-α levels; reduced mucus secretion; and improved airway function. These findings suggest that regulation of eosinophil trafficking and migration by SWAP-70 is important for the development of eosinophilic inflammation after allergen exposure.

PMID: 22210919 [PubMed - as supplied by publisher]

In vivo characterization of tumor and tumor vascular network using multi-modal imaging approach.

J Biophotonics. 2011 Sep;4(9):645-9

Authors: Kalchenko V, Madar-Balakirski N, Meglinski I, Harmelin A

Abstract
We present a multi-modal optical diagnostic approach utilizing a combined use of Fluorescence Intravital Microscopy (FIM), Dynamic Light Scattering (DLS) and Spectrally Enhanced Microscopy (SEM) modalities for in vivo imaging of tumor vascular network and blood microcirculation. FIM is used for imaging of tumor surroundings and microenvironment, SEM provides information regarding blood vessels topography, whereas DLS is applied for functional imaging of vascular network and blood microcirculation. This complementary combination of the imaging approaches is extremely useful for functional in vivo imaging of blood vasculature and tumor microenvironment. The technique has also a great potential in vascular biology and can significantly expand the capabilities of tumor angiogenesis studies and notably contribute to the development of cancer treatment.

PMID: 21714099 [PubMed - indexed for MEDLINE]

PTP1B Deficiency Exacerbates Inflammation and Accelerates Leukocyte Trafficking In Vivo.

J Immunol. 2011 Dec 9;

Authors: Berdnikovs S, Pavlov VI, Abdala-Valencia H, McCary CA, Klumpp DJ, Tremblay ML, Cook-Mills JM

Abstract
It is reported that PTP1B limits cytokine signaling in vitro. However, PTP1B's function during inflammation in vivo is not known. In this report, we determined whether PTP1B deficiency affects allergic inflammation in vivo. Briefly, lungs of OVA-challenged PTP1B(-/-) mice had elevated numbers of eosinophils and eosinophil progenitors at 6 h after one OVA challenge and at 24 h after a third OVA challenge as compared with OVA-challenged wild-type mice. There was also an increase in numbers of CD11b(+)SiglecF(+)CD34(+)IL-5Rα(+) eosinophil progenitors in the bone marrow, peripheral blood, and spleens of OVA-challenged PTP1B(-/-) mice. Intravital microscopy revealed that, in OVA-challenged PTP1B(-/-) mice, blood leukocytes rapidly bound to endothelium (5-30 min), whereas, in wild-type mice, blood leukocytes bound to endothelium at the expected 6-18 h. Consistent with early recruitment of leukocytes, lung eotaxin and Th2 cytokine levels were elevated early in the PTP1B(-/-) mice. Interestingly, spleen leukocytes from PTP1B(-/-) mice exhibited an increased chemotaxis, chemokinesis, and transendothelial migration in vitro. In summary, PTP1B functions as a critical negative regulator to limit allergic responses.

PMID: 22156494 [PubMed - as supplied by publisher]

Cell-surface sensors for real-time probing of cellular environments.

Nat Nanotechnol. 2011 Aug;6(8):524-31

Authors: Zhao W, Schafer S, Choi J, Yamanaka YJ, Lombardi ML, Bose S, Carlson AL, Phillips JA, Teo W, Droujinine IA, Cui CH, Jain RK, Lammerding J, Love JC, Lin CP, Sarkar D, Karnik R, Karp JM

Abstract
The ability to explore cell signalling and cell-to-cell communication is essential for understanding cell biology and developing effective therapeutics. However, it is not yet possible to monitor the interaction of cells with their environments in real time. Here, we show that a fluorescent sensor attached to a cell membrane can detect signalling molecules in the cellular environment. The sensor is an aptamer (a short length of single-stranded DNA) that binds to platelet-derived growth factor (PDGF) and contains a pair of fluorescent dyes. When bound to PDGF, the aptamer changes conformation and the dyes come closer to each other, producing a signal. The sensor, which is covalently attached to the membranes of mesenchymal stem cells, can quantitatively detect with high spatial and temporal resolution PDGF that is added in cell culture medium or secreted by neighbouring cells. The engineered stem cells retain their ability to find their way to the bone marrow and can be monitored in vivo at the single-cell level using intravital microscopy.

PMID: 21765401 [PubMed - indexed for MEDLINE]

In vivo time-course imaging of tumor angiogenesis in colorectal liver metastases in the same living mice using two-photon laser scanning microscopy.

J Oncol. 2012;2012:265487

Authors: Tanaka K, Morimoto Y, Toiyama Y, Matsushita K, Kawamura M, Koike Y, Okugawa Y, Inoue Y, Uchida K, Araki T, Mizoguchi A, Kusunoki M

Abstract
In vivo real-time visualization of the process of angiogenesis in secondary tumors in the same living animals presents a major challenge in metastasis research. We developed a technique for intravital imaging of colorectal liver metastasis development in live mice using two-photon laser scanning microscopy (TPLSM). We also developed time-series TPLSM in which intravital TPLSM procedures were performed several times over periods of days to months. Red fluorescent protein-expressing colorectal cancer cells were inoculated into the spleens of green fluorescent protein-expressing mice. First- and second-round intravital TPLSM allowed visualization of viable cancer cells (red) in hepatic sinusoids or the space of Disse. Third-round intravital TPLSM demonstrated liver metastatic colonies consisting of viable cancer cells and surrounding stroma with tumor vessels (green). In vivo time-course imaging of tumor angiogenesis in the same living mice using time-series TPLSM could be an ideal tool for antiangiogenic drug evaluation, reducing the effects of interindividual variation.

PMID: 22131993 [PubMed - in process]

Intravital imaging of thrombus formation in small and large mouse arteries: experimentally induced vascular damage and plaque rupture in vivo.

Methods Mol Biol. 2012;788:3-19

Authors: Kuijpers MJ, Heemskerk JW

Abstract
Intravital fluorescence microscopy is increasingly used to measure experimental arterial thrombosis in large and small arteries of mice in vivo. This chapter describes protocols for applying this technology to detect and measure thrombi formed by: (1) ultrasound-induced rupture of an atherosclerotic plaque in the carotid artery of adult Apoe ( -/- ) mice; (2) FeCl(3) or ligation in the carotid artery of nonatherosclerotic mice; and (3) FeCl(3) in the mesenteric venules and arterioles of young mice. In addition, we describe a protocol using two-photon laser scanning microscopy for intraluminal scanning of thrombi formed in the carotid artery. These approaches provide important information that cannot be obtained with ex vivo methods and thus are likely to lead to new insights into the complex process of thrombosis.

PMID: 22130696 [PubMed - in process]

Engagement of αIIbβ3 (GPIIb/IIIa) with ανβ3 mediates interaction of melanoma cells with platelets - a connection to hematogeneous metastasis.

J Biol Chem. 2011 Nov 18;

Authors: Lonsdorf AS, Kraemer BF, Fahrleitner M, Schoenberger T, Gnerlich S, Ring S, Gehring S, Schneider SW, Kruhlak MJ, Meuth SG, Nieswandt B, Gawaz M, Enk AH, Langer HF

Abstract
A mutual relationship exists between metastasizing tumor cells and components of the coagulation cascade. The exact mechanisms as to how platelets influence blood-bourne metastasis, however, remain poorly understood. Here, we used murine B16 melanoma cells to observe functional aspects of how platelets contribute to the process of hematogenous metastasis. We found that platelets interfere with a distinct step of the metastasis cascade, as they promote adhesion of melanoma cells to the endothelium in vitro under shear conditions. Constitutively active platelet receptor GPIIb/IIIa (integrin αIIbβ3) expressed on Chinese Hamster Ovary (CHO) cells promoted melanoma cell adhesion in the presence of fibrinogen, whereas blocking antibodies to ανβ3 integrin on melanoma cells or to GPIIb/IIIa significantly reduced melanoma cell adhesion to platelets. Furthermore, using intravital microscopy, we observed functional platelet-melanoma cell interactions, as platelet depletion resulted in significantly reduced melanoma cell adhesion to the injured vascular wall in vivo. Using a mouse model of hematogenous metastasis to the lung we observed decreased metastasis of B16 melanoma cells to the lung by treatment with a mAb blocking the aν subunit of ανβ3 integrin. This effect was significantly reduced, when platelets were depleted in vivo. Thus, the engagement of GPIIb/IIIa with ανβ3 integrin interaction mediates tumor cell-platelet interactions, and highlights how this interaction is involved in hematogenous tumor metastasis.

PMID: 22102277 [PubMed - as supplied by publisher]

Rapid tumoritropic accumulation of systemically injected plateloid particles and their biodistribution.

J Control Release. 2011 Oct 26;

Authors: van de Ven AL, Kim P, Haley O, Fakhoury JR, Adriani G, Schmulen J, Moloney P, Hussain F, Ferrari M, Liu X, Yun SH, Decuzzi P

Abstract
Nanoparticles for cancer therapy and imaging are designed to accumulate in the diseased tissue by exploiting the Enhanced Permeability and Retention (EPR) effect. This limits their size to about 100nm. Here, using intravital microscopy and elemental analysis, we compare the in vivo localization of particles with different geometries and demonstrate that plateloid particles preferentially accumulate within the tumor vasculature at unprecedented levels, independent of the EPR effect. In melanoma-bearing mice, 1000×400nm plateloid particles adhered to the tumor vasculature at about 5% and 10% of the injected dose per gram organ (ID/g) for untargeted and RGD-targeted particles respectively, and exhibited the highest tumor-to-liver accumulation ratios (0.22 and 0.35). Smaller and larger plateloid particles, as well as cylindroid particles, were more extensively sequestered by the liver, spleen and lungs. Plateloid particles appeared well-suited for taking advantage of hydrodynamic forces and interfacial interactions required for efficient tumoritropic accumulation, even without using specific targeting ligands.

PMID: 22062689 [PubMed - as supplied by publisher]

Leukocyte-endothelial cell interaction is necessary for photodynamic therapy induced vascular permeabilization.

Lasers Surg Med. 2011 Sep;43(7):696-704

Authors: Debefve E, Mithieux F, Perentes JY, Wang Y, Cheng C, Schaefer SC, Ruffieux C, Ballini JP, Gonzalez M, van den Bergh H, Ris HB, Lehr HA, Krueger T

Abstract
BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) affects vascular barrier function and thus increases vessel permeability. This phenomenon may be exploited to facilitate targeted drug delivery and may lead to a new clinical application of photodynamic therapy. Here, we investigate the role of leukocyte recruitment for PDT-induced vascular permeabilization. STUDY DESIGN/MATERIAL AND METHODS: Fluorescein isothiocyanate dextran (FITC-D, 2,000 kDa) was injected intravenously 120 minutes after focal PDT on striated muscle in nude mice bearing dorsal skinfold chambers (Visudyne® 800 µg/kg, fluence rate 300 mW/cm(2) , light dose of 200 J/cm(2) ). Leukocyte interaction with endothelial cells was inhibited by antibodies functionally blocking adhesion molecules ("MABS-PDT" group, n = 5); control animals had PDT but no antibody injection (group "PDT", n = 7). By intravital microscopy, we monitored leukocyte rolling and sticking in real-time before, 90 and 180 minutes after PDT. The extravasation of FITC-D from striated muscle vessels into the interstitial space was determined in vivo during 45 minutes to assess treatment-induced alterations of vascular permeability.
RESULTS: PDT significantly increased the recruitment of leukocytes and enhanced the leakage of FITC-D. Neutralization of adhesion molecules before PDT suppressed the rolling of leukocytes along the venular endothelium and significantly reduced the extravasation of FITC-D as compared to control animals (156 ± 27 vs. 11 ± 2 (mean ± SEM, number of WBC/30 seconds mm vessel circumference; P < 0.05) at 90 minutes after PDT and 194 ± 21 vs. 14 ± 4 at 180 minutes after PDT). In contrast, leukocyte sticking was not downregulated by the antibody treatment.
CONCLUSION: Leukocyte recruitment plays an essential role in the permeability-enhancing effect of PDT. Lasers Surg. Med. 43:696-704, 2011. © 2011 Wiley-Liss, Inc.

PMID: 22057497 [PubMed - in process]

Morphology and Hemodynamics during Vascular Regeneration in Critically Ischemic Murine Skin Studied by Intravital Microscopy Techniques.

Eur Surg Res. 2011 Nov 2;47(4):222-230

Authors: Schweizer R, Merz K, Schlosser S, Spanholtz T, Contaldo C, Stein JV, Enzmann V, Giovanoli P, Erni D, Plock JA

Abstract
Background: With the understanding of angiogenesis and arteriogenesis, new theories about the orchestration of these processes have emerged. The aim of this study was to develop an in vivo model that enables visualization of vascular regenerating mechanisms by intravital microscopy techniques in collateral arteriolar flap vascularity. Methods: A dorsal skin flap (15 × 30 mm) was created in mice and fixed into a skinfold chamber to allow for assessment of morphology and microhemodynamics by intravital fluorescence microscopy (IVFM). Laser scanning confocal microscopy (LSCM) was utilized for three-dimensional reconstruction of the microvascular architecture. Results: Flap tpO(2) was 5.3 ± 0.9 versus 30.5 ± 1.2 mm Hg in controls (p < 0.01). The collateral arterioles in the flap tissue were dilated (29.4 ± 5.3 μm; p < 0.01 vs. controls) and lengthened in a tortuous manner (tortuosity index 1.00 on day 1 vs. 1.35± 0.05 on day 12; p < 0.01). Functional capillary density was increased from 121.00 ± 25 to 170 ± 30 cm/cm(2) (day 12; p < 0.01) as a result of angiogenesis. Morphological evidence of angiogenesis on capillary level and vascular remodeling on arteriolar level could be demonstrated by IVFM and LSCM. Conclusions: Present intravital microscopy techniques offer unique opportunities to study structural changes and hemodynamic effects of vascular regeneration in this extended axial pattern flap model.

PMID: 22056471 [PubMed - as supplied by publisher]

In Vivo SPECT Reporter Gene Imaging of Regulatory T Cells.

PLoS One. 2011;6(10):e25857

Authors: Sharif-Paghaleh E, Sunassee K, Tavaré R, Ratnasothy K, Koers A, Ali N, Alhabbab R, Blower PJ, Lechler RI, Smyth LA, Mullen GE, Lombardi G

Abstract
Regulatory T cells (Tregs) were identified several years ago and are key in controlling autoimmune diseases and limiting immune responses to foreign antigens, including alloantigens. In vivo imaging techniques including intravital microscopy as well as whole body imaging using bioluminescence probes have contributed to the understanding of in vivo Treg function, their mechanisms of action and target cells. Imaging of the human sodium/iodide symporter via Single Photon Emission Computed Tomography (SPECT) has been used to image various cell types in vivo. It has several advantages over the aforementioned imaging techniques including high sensitivity, it allows non-invasive whole body studies of viable cell migration and localisation of cells over time and lastly it may offer the possibility to be translated to the clinic. This study addresses whether SPECT/CT imaging can be used to visualise the migratory pattern of Tregs in vivo. Treg lines derived from CD4(+)CD25(+)FoxP3(+) cells were retrovirally transduced with a construct encoding for the human Sodium Iodide Symporter (NIS) and the fluorescent protein mCherry and stimulated with autologous DCs. NIS expressing self-specific Tregs were specifically radiolabelled in vitro with Technetium-99m pertechnetate ((99m)TcO(4) (-)) and exposure of these cells to radioactivity did not affect cell viability, phenotype or function. In addition adoptively transferred Treg-NIS cells were imaged in vivo in C57BL/6 (BL/6) mice by SPECT/CT using (99m)TcO(4) (-). After 24 hours NIS expressing Tregs were observed in the spleen and their localisation was further confirmed by organ biodistribution studies and flow cytometry analysis. The data presented here suggests that SPECT/CT imaging can be utilised in preclinical imaging studies of adoptively transferred Tregs without affecting Treg function and viability thereby allowing longitudinal studies within disease models.

PMID: 22043296 [PubMed - in process]

Chemokines control naive CD8+ T cell selection of optimal lymph node antigen presenting cells.

J Exp Med. 2011 Oct 31;

Authors: Hickman HD, Li L, Reynoso GV, Rubin EJ, Skon CN, Mays JW, Gibbs J, Schwartz O, Bennink JR, Yewdell JW

Abstract
Naive antiviral CD8(+) T cells are activated in the draining LN (DLN) by dendritic cells (DCs) presenting viral antigens. However, many viruses infect LN macrophages, which participate in initiation of innate immunity and B cell activation. To better understand how and why T cells select infected DCs rather than macrophages, we performed intravital microscopy and ex vivo analyses after infecting mice with vaccinia virus (VV), a large DNA virus that infects both LN macrophages and DCs. Although CD8(+) T cells interact with both infected macrophages and DCs in the LN peripheral interfollicular region (PIR), DCs generate more frequent and stable interactions with T cells. VV infection induces rapid release of CCR5-binding chemokines in the LN, and administration of chemokine-neutralizing antibodies diminishes T cell activation by increasing T cell localization to macrophages in the macrophage-rich region (MRR) at the expense of PIR DCs. Similarly, DC ablation increases both T cell localization to the MRR and the duration of T cell-macrophage contacts, resulting in suboptimal T cell activation. Thus, virus-induced chemokines in DLNs enable antiviral CD8(+) T cells to distinguish DCs from macrophages to optimize T cell priming.

PMID: 22042976 [PubMed - as supplied by publisher]

Intravital two-photon microscopy of immune cell dynamics in corneal lymphatic vessels.

PLoS One. 2011;6(10):e26253

Authors: Steven P, Bock F, Hüttmann G, Cursiefen C

Abstract
BACKGROUND: The role of lymphatic vessels in tissue and organ transplantation as well as in tumor growth and metastasis has drawn great attention in recent years.
METHODOLOGY/PRINCIPAL FINDINGS: We now developed a novel method using non-invasive two-photon microscopy to simultaneously visualize and track specifically stained lymphatic vessels and autofluorescent adjacent tissues such as collagen fibrils, blood vessels and immune cells in the mouse model of corneal neovascularization in vivo. The mouse cornea serves as an ideal tissue for this technique due to its easy accessibility and its inducible and modifiable state of pathological hem- and lymphvascularization. Neovascularization was induced by suture placement in corneas of Balb/C mice. Two weeks after treatment, lymphatic vessels were stained intravital by intrastromal injection of a fluorescently labeled LYVE-1 antibody and the corneas were evaluated in vivo by two-photon microscopy (TPM). Intravital TPM was performed at 710 nm and 826 nm excitation wavelengths to detect immunofluorescence and tissue autofluorescence using a custom made animal holder. Corneas were then harvested, fixed and analyzed by histology. Time lapse imaging demonstrated the first in vivo evidence of immune cell migration into lymphatic vessels and luminal transport of individual cells. Cells immigrated within 1-5.5 min into the vessel lumen. Mean velocities of intrastromal corneal immune cells were around 9 µm/min and therefore comparable to those of T-cells and macrophages in other mucosal surfaces.
CONCLUSIONS: To our knowledge we here demonstrate for the first time the intravital real-time transmigration of immune cells into lymphatic vessels. Overall this study demonstrates the valuable use of intravital autofluorescence two-photon microscopy in the model of suture-induced corneal vascularizations to study interactions of immune and subsequently tumor cells with lymphatic vessels under close as possible physiological conditions.

PMID: 22028842 [PubMed - in process]

[In vivo imaging of mature osteoclasts and RANKL signaling].

Clin Calcium. 2011 Aug;21(8):1181-5

Authors: Kikuta J, Ishii M

Abstract
Osteoclasts are 'bone-resorbing' giant polykaryons that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursors. However how the activity of mature osteoclasts is regulated in vivo remains unclear. To answer the question, we utilized an advanced imaging system for visualizing live bone tissues with intravital multiphoton microscopy that we have recently established. By means of the system we have recently succeeded in visualization of mature osteoclasts in live bones and revealed that RANKL regulates bone-resorptive functions of mature osteoclasts in vivo . Here we show the latest data and the detailed methodology of intravital imaging of bone tissues, and also discuss its further application.

PMID: 21814023 [PubMed - indexed for MEDLINE]

In vivo imaging of lymphocytes in the CNS reveals different behaviour of naive T cells in health and autoimmunity.

J Neuroinflammation. 2011 Oct 6;8(1):131

Authors: Herz J, Paterka M, Niesner RA, Brandt AU, Siffrin V, Leuenberger T, Birkenstock J, Mossakowski A, Glumm R, Zipp F, Radbruch H

Abstract
ABSTRACT: BACKGROUND: Two-photon laser scanning microscopy (TPLSM) has become a powerful tool in the visualization of immune cell dynamics and cellular communication within the complex biological networks of the inflamed central nervous system (CNS). Whereas many previous studies mainly focused on the role of effector or effector memory T cells, the role of naive T cells as possible key players in immune regulation directly in the CNS is still highly debated. METHODS: We applied ex vivo and intravital TPLSM to investigate migratory pathways of naive T cells in the inflamed and non-inflamed CNS. MACS-sorted naive CD4+ T cells were either applied on healthy CNS slices or intravenously injected into RAG1 -/- mice, which were affected by experimental autoimmune encephalomyelitis (EAE). We further checked for the generation of second harmonic generation (SHG) signals produced by extracellular matrix (ECM) structures. RESULTS: By applying TPLSM on living brain slices we could show that the migratory capacity of activated CD4+ T cells is not strongly influenced by antigen specificity and is independent of regulatory or effector T cell phenotype. Naive T cells, however, cannot find sufficient migratory signals in healthy, non-inflamed CNS parenchyma since they only showed stationary behaviour in this context. This is in contrast to the high motility of naive CD4+ T cells in lymphoid organs. We observed a highly motile migration pattern for naive T cells as compared to effector CD4+ T cells in inflamed brain tissue of living EAE-affected mice. Interestingly, in the inflamed CNS we could detect reticular structures by their SHG signal which partially co-localises with naive CD4+ T cell tracks. CONCLUSIONS: The activation status rather than antigen specificity or regulatory phenotype is the central requirement for CD4+ T cell migration within healthy CNS tissue. However, under inflammatory conditions naive CD4+ T cells can get access to CNS parenchyma and partially migrate along inflammation-induced extracellular SHG structures, which are similar to those seen in lymphoid organs. These SHG structures apparently provide essential migratory signals for naive CD4+ T cells within the diseased CNS.

PMID: 21978405 [PubMed - as supplied by publisher]