![destruction, and/or debilitating illness. Better understanding of
the mechanisms underlying T-cell motility will facilitate the tuning
of T-cell migratory activity for the therapeutic management of
immune-mediated disorders.
2 Mechanisms of T-Cell Migration
The past two decades have witnessed significant advances in reveal-
ing the complexities and intricacies underlying T-cell-mediated
immune response regulation. T-lymphocytes navigate to effector
sites through the generation of precisely regulated cell-intrinsic
migratory events involving adhesion receptors and a number of
signaling pathway mediators. The motility of T-cells requires phys-
ical guidance through receptor–ligand interactions, several inde-
pendent but coordinated signaling cascades, and dynamic
cytoskeletal rearrangements [1–4]. The lymphocyte function-
associated antigen 1 (LFA-1) integrin receptor present on T-cells
binds to the ligand intercellular adhesion molecule 1 (ICAM-1)
expressed on the endothelium and this LFA-1/ICAM-1 contact
acts as a trigger for T-cell motility.
A typical T-cell motility process requires an initial transient
attachment to the endothelium, which is followed by rolling and
firm adhesion of activated T-cells on the endothelial surface, and
finally migration through the blood vessel wall to sites of infection,
tissue insult, or injury [5]. The ability of T-lymphocytes to enter
lymph nodes also depends on adhesion molecules and chemoat-
tractants on the high endothelial venules [5]. As T-cells move
through distinct tissue compartments, they interact with endothe-
lial cells and this interaction regulates the process of T-cell migra-
tion. Typically, a motile T-cell moves at a speed of 10–12 μm/min.
Migrating T-cells undergo a series of molecular change in the
expression of signaling proteins and surface receptors. The require-
ment and relative importance of these molecular changes varies
between tissues and is dictated by many factors such as activation
state of T-cells and tissue microenvironment. A gain in the expres-
sion of a set of homing molecules on activated T-cells enables them
to migrate to the tissue sites of inflammation or injury. For example,
ligands for E-selectin and P-selectin facilitate tethering and rolling
of T-cells on the endothelium [5]. The engagement of chemokine
receptors activates integrins, such as the LFA-1 and the very late
antigen 4 (VLA-4) by inducing conformational changes in the
integrins. Activated LFA-1 and VLA-4 bind to their corresponding
ligands, mainly ICAM-1 and the vascular cell adhesion molecule
1 (VCAM-1), respectively, expressed on inflamed/infected tissues.
The adherent T-cells acquire an asymmetric characteristic polarized
morphology with a distinct leading edge and the trailing uropod,
and crawl along the luminal surface on the endothelium.
2 Navin Kumar Verma and Dermot Kelleher](https://image.slidesharecdn.com/4980883-250525115114-1f875670/85/Tcell-Motility-Methods-And-Protocols-1st-Ed-Navin-Kumar-Verma-15-320.jpg)
![The leading edge points toward the direction of movement, which
is driven by cytoskeleton-mediated protrusions and dynamic adhe-
sion turnover at the front and uropod retraction at the back
(Fig. 1).
T-cell migration can be viewed as a multistep process incorpor-
ating the following four sequential stages:
2.1 Induction of Cell
Polarization
Upon receptor engagement, a multitude of signaling cascades is
initiated causing strong asymmetry within the cell, which results in
a polarized phenotype. T-cell polarization involves the formation of
a ‘front–back’ asymmetry in which cell surface adhesion receptors,
such as LFA-1 integrin, are accumulated at the front of the cell
[6]. Lipid rafts are also implicated in the polarization of migrating
T-cells [6]. Asymmetrical arrangement of surface receptors pro-
duces an asymmetry of intracellular signaling components. A func-
tional cytoskeleton and its reorganization are necessary for these
cell-intrinsic processes.
2.2 Generation
of Membrane
Protrusions at
the Leading Edge
Once a T-cell has been polarized, it generates actin-enriched mem-
brane protrusions (filopodia and lamellipodia) at the leading edge.
The forward move of membrane protrusions is facilitated by actin
polymerization at the front followed by stabilization of the new
architecture of the resulting membrane extensions [7, 8].
2.3 Formation
of Cell–Substratum
Contact Sites
After membrane extension has been formed, new points of contact
are established by the T-cell surface receptors with their specific
ligands. This involves adhesion molecules of the integrin family [9].
In rapidly migrating T-cells, integrin binding to specific ligand leads
to integrin clustering and the recruitment of cytoskeletal proteins
to the cytoplasmic domain of the integrin. This prevents retraction
of the newly formed membrane protrusions and provides ‘grip’ for
the tractional force to assist T-cell motility.
Uropod
Leading edge
Main cell body
ICAM-1
LFA-1
Fig. 1 A cartoon illustrating a typical motile T-cell phenotype displaying leading edge at the front and the
trailing uropod at the back
T-Cell Motility 3](https://image.slidesharecdn.com/4980883-250525115114-1f875670/85/Tcell-Motility-Methods-And-Protocols-1st-Ed-Navin-Kumar-Verma-16-320.jpg)
![2.4 Retraction
of the Uropod
and Forward
Movement
of the Cell Body
The next stage of T-cell movement involves the flux of intracellular
contents into the newly extended cell body, and the retraction of
the trailing edge. Besides extension of membrane protrusions at the
front by cytoskeletal remodeling following the engagement of
integrins, a contractile force is generated to move the body of the
T-cell forward [10].
3 The LFA-1 Integrin
The integrins are a large family of transmembrane receptors
expressed at the cell surface and are composed of two
non-covalently associated α- and β-glycoprotein subunits that
form a heterodimer. T-lymphocytes express at least 12 of the
24 known integrins and the expression pattern of integrins on
T-cells depends on the subset and effector state of the cell
[8, 11]. In human T-cells, the integrin αLβ2 (also known as
CD11a/CD18 or LFA-1) is the most abundant in expression
[8, 9]. LFA-1 contact to the β2-integrin ligands, mainly ICAM-1,
causes high-affinity adhesion and plays a prominent role in T-cell
motility. In addition, LFA-1 forms the peripheral ring of the immu-
nological synapse [12].
Under normal conditions, the LFA-1 receptor remains inactive
or non-ligand-binding conformation when peripheral blood
T-lymphocytes are circulating in the bloodstream, which ensures
no inappropriate binding to their ligands. Expression of the inactive
form of LFA-1 in circulating T-cells is crucial in maintaining
homeostasis. Constitutively active LFA-1 causes rapid aggregation
of circulating cells and clogging of the vessels [13]. Exposure to
cytokines or chemokines and engagement with adhesion receptors
results in instantaneous activation of LFA-1 precisely where its
function is needed [9, 14, 15]. Following activation, the cytoplas-
mic domains of the LFA-1 integrin associate with adaptor proteins
(e.g. Talin 1, Kindlin 3) [16], connect the cytoskeletal systems, and
recruit several signaling molecules, including cytoplasmic kinases
and other proteins, such as protein kinase C [17, 18]. Notably, the
distribution of the LFA-1 on the cell surface is altered once the
T-cell begins to migrate. The amount of LFA-1 at the leading edge
of a motile T-cell is relatively low in comparison to that in the
non-attached uropod at the rear. This distribution of LFA-1 has
been correlated with the dynamic changes in cell morphology dis-
played by migrating T-cells [8].
4 Cytoskeletal Remodeling in Motile T-cells
The importance of cytoskeletal structure in cell migration was first
recognized about 50 years ago [19]. Further experimental advances
established a direct link between the lymphocyte-specific signal
4 Navin Kumar Verma and Dermot Kelleher](https://image.slidesharecdn.com/4980883-250525115114-1f875670/85/Tcell-Motility-Methods-And-Protocols-1st-Ed-Navin-Kumar-Verma-17-320.jpg)
![transduction pathways that trigger cytoskeletal reorganization
[20, 21]. It is now evident that the LFA-1/ICAM-1 contact trig-
gers dynamic rearrangements in the cytoskeleton, which facilitates
T-cell motility [22, 23]. LFA-1 connects to the cytoskeleton
through adaptor proteins, such as Talins and Kindlins, which
binds to the β-subunit of LFA-1 cytoplasmic domain [16]. This
connection prompts an array of downstream signal transduction
cascades in addition to transmitting force between the extracellular
ligand and the cytoskeleton [24], which ultimately influences cyto-
skeletal remodeling implicated in T-cell motility. Although signifi-
cant advances have been made to understand the molecular
mechanism that dynamically rearrange T-cell cytoskeleton in migra-
tory processes, we are still far away from complete information
about the complex interplay of all the functional elements involved.
Increasing evidence has confirmed that cytoskeletal proteins are
important for receptor-mediated intracellular signal transduction
events.
4.1 The T-Cell Actin
Filaments
Actin filaments are a cytoskeletal component that provide structural
support and generate cellular movements involved in cell migration
and other functions, such as endocytosis and cytokinesis. The
importance of cytoskeleton remodeling in motile T-cells was initi-
ally recognized based on the observation that the formation of a
receptor cap and a uropod were inhibited by an inhibitor of actin
polymerization cytochalasins [25]. Subsequent studies established
importance of actin involvement and underlying molecular
mechanisms in T-cell migration [26]. At the leading edge of the
polarized and migrating T-cell, actin is organized in parallel bundles
which form filopodia and in dense meshworks which form lamelli-
podia [20, 27]. In the cell body and at the cell trailing edge, actin
filaments form contractile force responsible for the contraction of
the cell body and retraction of the trailing edge during T-cell
motility [20, 27].
4.2 The T-Cell
Microtubule
Cytoskeleton
The microtubule cytoskeleton is mainly composed of α- and
β-tubulin heterodimers. This dynamic structure enables
T-lymphocytes to change their cell shape during migration in addi-
tion to the regulation of other cellular processes, such as division,
intracellular transport, organization and positioning of organelles,
anchoring and guiding for protein complexes, etc. Polymerized
microtubule networks exist in equilibrium with free tubulin. They
assemble around microtubule organizing center (MTOC) and
show polarity, requiring GTP to grow at the plus-end. Microtubule
plus-end-associated proteins are also crucial players in this process
and are responsible for the generation of a microtubule network
and consecutive organization of the actin cytoskeleton. We have
recently discovered a role of an adaptor protein AKAP450, also
called centrosome- and Golgi-localized protein kinase N-associated
protein (CG-NAP), in the nucleation of microtubule [28].
T-Cell Motility 5](https://image.slidesharecdn.com/4980883-250525115114-1f875670/85/Tcell-Motility-Methods-And-Protocols-1st-Ed-Navin-Kumar-Verma-18-320.jpg)
![Following migratory signals triggered by LFA-1/ICAM-1 contact,
microtubules undergo massive reorganization in motile T-cells,
which is often accompanied by the movement of organelles such
as Golgi apparatus as well as signaling molecules [28]. In migrating
T-cells, microtubules have been shown to directly interact with the
LFA-1 integrin [18, 29]. Moreover, disruption of the microtubules
cytoskeleton alters T-cell motility [30].
4.3 Microtubules
and Actin Actively
Cooperate During
the Process of T-Cell
Motility
T-cell motility relies on the force-generation machinery that forms
a polarized cell asymmetry, which is dependent on the two distinct
processes. First, a motile T-cell organizes its cytoskeletal elements
in a polarized manner with concomitant protrusive and contractile
activities at opposite cellular locations. Actin polymerization and
turnover are essential for the traction force generation needed for
T-cell motility, and this protrusion–contraction cycle is regulated by
a dynamic cross-talk between both actin and microtubules
[31]. Second, the migrating T-cell orients its intrinsic polarity axis
in response to the extracellular cues, such as chemoattractant gra-
dient. Membrane fluctuations facilitated by the dynamics actin and
microtubule networks allows sensing of the cellular environment.
The asymmetry in shape of migrating T-cells is determined by an
ongoing cooperation between the actin filament arrays and the
growing microtubule cytoskeleton, which influence each other’s
polymerization/depolymerization dynamics directly or through
regulation of signaling molecules. Both actin fibers and microtu-
bules are polarized during cell migration at the leading edge and
retracting trailing edges and are linked together either directly or
indirectly through adaptor proteins or signaling molecules. Micro-
tubules also modulate actin stress fiber formation, thus regulate
actin dynamics and force generation in motile T-cells [31, 32]. In
addition, Vimentin, the only known intermediate filament protein
in leukocytes [33], plays a crucial role in maintaining cellular integ-
rity and in regulating T-cell adhesion and transmigration [34].
5 T-Cell Motility in Health and Diseases
The orchestration of an immune response is crucially dependent on
the inherent ability of T-cells to migrate. The recruitment of T-cells
from the bloodstream into tissues is a necessary component of the
inflammatory response to tissue injury caused by infection or
wounding. Failure of T-cells to migrate or an inappropriate T-cell
motility can result in chronic inflammation, so-called immunopa-
thology, and may have life-threatening consequences.
Following clearance of infection or resolution of injury, T-cell
infiltration subsides and inflammation resolves without overt tissue
damage. However, inappropriate inflammatory responses fre-
quently associated with persistent T-cell infiltration may lead to
6 Navin Kumar Verma and Dermot Kelleher](https://image.slidesharecdn.com/4980883-250525115114-1f875670/85/Tcell-Motility-Methods-And-Protocols-1st-Ed-Navin-Kumar-Verma-19-320.jpg)
![autoimmune disease conditions such as asthma, rheumatoid arthri-
tis, and psoriasis [35–38]. Asthma, a chronic inflammatory disease
of the airways, results from an immune response to inhaled aller-
gens. The constant presence and prolong activation of T-cells in
the airways causes tissue damage, leading to airway hyper-reactivity
against otherwise harmless stimuli such as cold air [39]. In rheu-
matoid arthritis, an autoimmune response by overactive infiltrating
T-cells causes destruction of joint tissue resulting in compromised
limb function and crippling pain. Psoriasis is an inflammatory dis-
ease of the skin, which is characterized by T-cell infiltration, kera-
tinocyte proliferation, and the production of several
pro-inflammatory cytokines and chemokines. The central role of
T-cell migration in the pathogenesis of psoriasis is well-
documented [40].
Despite the advances in molecular and imaging technologies,
the complex mechanistic process that operates the migratory
behavior of T-cells at various stages of development and activation
is not fully understood. Since T-cell migration is a promising target
for an anti-inflammatory therapy, the development of antagonists
for integrin receptors, adhesion molecules, and cytoskeletal dynam-
ics is among the most actively pursued areas in drug discovery
research. Indeed, drugs that inhibit the interaction between the
T-cell integrin receptors and their ligands have been applied in
clinically. For example, a humanized antibody-based drug Efalizu-
mab, which specifically binds to the α-subunit of LFA-1, was devel-
oped for the treatment of the moderate-to-severe psoriasis and
approved by the FDA in 2003 [41, 42]. However, despite proof-
of-concept and initial success in treating psoriasis, the drug was
withdrawn from the market in 2009 due to an increased risk of the
development of progressive multifocal leukoencephalopathy, which
is a fatal brain infection caused by reactivation of latent JC virus
[43, 44]. New drugs that inhibit organ-specific homing cascades of
populations of pathogenic T-lymphocytes would permit the use of
tissue-selective anti-inflammatory interventions, which would pro-
vide selective regimes of safe and effective immunosuppressive
therapy for chronic inflammatory diseases. The paucity of studies
that effectively link receptor-mediated signaling, cytoskeleton, and
T-cell motility highlights the need of further experiments to eluci-
date the exact mechanism. In this context, the purpose of this book
series is to bring together a collection of methodologies and pro-
tocols that are useful for researchers studying T-cell motility in
physiological and pathological conditions.
Acknowledgments
This work was supported by the Lee Kong Chian School of Medi-
cine, Nanyang Technological University Singapore Start-Up Grant
T-Cell Motility 7](https://image.slidesharecdn.com/4980883-250525115114-1f875670/85/Tcell-Motility-Methods-And-Protocols-1st-Ed-Navin-Kumar-Verma-20-320.jpg)
Tcell Motility Methods And Protocols 1st Ed Navin Kumar Verma
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- 5. T-Cell Motility Navin KumarVerma Editor Methods and Protocols Methods in Molecular Biology 1930
- 6. M E T H O D S I N M O L E C U L A R B I O L O G Y Series Editor John M. Walker School of Life and Medical Sciences, University of Hertfordshire, Hatfield, Hertfordshire AL10 9AB, UK For further volumes: http://www.springer.com/series/7651
- 7. T-Cell Motility Methods and Protocols Edited by Navin Kumar Verma Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Singapore, Singapore
- 8. Editor Navin Kumar Verma Lee Kong Chian School of Medicine Nanyang Technological University Singapore Singapore, Singapore ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-9035-1 ISBN 978-1-4939-9036-8 (eBook) https://doi.org/10.1007/978-1-4939-9036-8 Library of Congress Control Number: 2018965599 © Springer Science+Business Media, LLC, part of Springer Nature 2019 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Cover Caption: Human T-cells migrating via LFA-1/ICAM-1 contact: Human T-cells were stimulated to migrate by incubating on rICAM-1-coated surface for 2 h. Cells were fixed and fluorescently stained for tubulin (green), actin (red) and nuclei (blue). The image was captured by an automated microscope using 20 magnification objective. This Humana Press imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.
- 9. Preface The mobilization of T-cells into lymph nodes and tissue sites of infection or inflammation is crucial for the initiation and maintenance of an effective immune response. On the other hand, uncontrolled and persistent tissue infiltration of T-cells can lead to the development of autoimmune diseases, such as psoriasis, multiple sclerosis, and rheumatoid arthritis. This complex process of T-cell motility is precisely regulated by a set of adhesion receptors, an array of highly integrated molecular networks, and a concordant series of signaling path- ways. Thus, to understand T-cell motility in health and diseases as well to develop an approach to potentially target T-cell migration for therapeutic purposes, it is important to perform a wide range of T-cell migration assays under various experimental conditions. This volume of the Methods in Molecular Biology series provides a collection of protocols for commonly used experimental procedures and advanced methodologies in T-cells biol- ogy. The main aim of this book is to provide researchers with state-of-the-art cellular, molecular, biochemical, and imaging assays necessary for carrying out a wide range of experiments to study the biology and functions of T-cells. Each chapter describes the basic principle, lists chemicals, reagents, buffers, and other materials necessary for carrying out the experiment followed by detailed step-by-step descriptions of the protocol. It also discusses how best to deal with potential difficulties that might arise in a particular step. The book chapters are selected for practical use in laboratories for graduate students, postdoctoral fellows, and principal investigators in the area of immunology, T-cell biology, biochemistry, and molecular biology. In addition, this book will be useful to researchers from other fields, such as imaging. We hope that this book will be an essential part of many laboratories and libraries and will benefit graduate students, postdoctoral fellows, and principal investigators studying T-cell motility in physiological or pathological conditions. Singapore, Singapore Navin Kumar Verma v
- 10. Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix 1 An Introduction to LFA-1/ICAM-1 Interactions in T-Cell Motility . . . . . . . . . . 1 Navin Kumar Verma and Dermot Kelleher 2 Isolation of Human Peripheral Blood T-Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . 11 Atish Kizhakeyil, Seow Theng Ong, Mobashar Hussain Urf Turabe Fazil, Madhavi Latha Somaraju Chalasani, Praseetha Prasannan, and Navin Kumar Verma 3 A Laboratory Model to Study T-Cell Motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Navin Kumar Verma, Shyam Mohan Palapetta, Seow Theng Ong, Mobashar Hussain Urf Turabe Fazil, Madhavi Latha Somaraju Chalasani, Praseetha Prasannan, Atish Kizhakeyil, and Dermot Kelleher 4 Quantification of T-Cell Migratory Phenotypes Using High-Content Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Aik Seng Ng, Seow Theng Ong, Dermot Kelleher, and Navin Kumar Verma 5 Live Cell Imaging and Analysis to Capture T-Cell Motility in Real-Time. . . . . . . 33 Seow Theng Ong and Navin Kumar Verma 6 Three-Dimensional Structured Illumination Microscopy (3D-SIM) to Dissect Signaling Cross-Talks in Motile T-Cells. . . . . . . . . . . . . . . . . 41 Seow Theng Ong, Graham D. Wright, and Navin Kumar Verma 7 Real-Time Impedance-Based Detection of LFA-1-Stimulated T-Cell Transwell Chemotaxis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Praseetha Prasannan and Navin Kumar Verma 8 Quantitative Real-Time PCR for Evaluating Transcriptional Changes in T-Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Atish Kizhakeyil and Navin Kumar Verma 9 GapmeR-Mediated Gene Silencing in Motile T-Cells . . . . . . . . . . . . . . . . . . . . . . . . 67 Mobashar Hussain Urf Turabe Fazil, Seow Theng Ong, Madhavi Latha Somaraju Chalasani, Atish Kizhakeyil, and Navin Kumar Verma 10 Live Imaging of Resident T-Cell Migration in Human Lymphoid Tissue Slices Using Confocal Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Emmanuel Donnadieu, Yvonne Michel, and Martin-Leo Hansmann 11 Phosphoprotein Enrichment for Protein Analysis in Motile T-Lymphocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Praseetha Prasannan, Rathi Saravanan, and Navin Kumar Verma 12 Immunometabolomic Phenotyping of Motile T-Cells . . . . . . . . . . . . . . . . . . . . . . . 91 Madhavi Latha Somaraju Chalasani, Gaelin Looi Wen Dong, and Navin Kumar Verma vii
- 11. 13 Profiling Activity of Cellular Kinases in Migrating T-Cells. . . . . . . . . . . . . . . . . . . . 99 Chandra Sekhar Chirumamilla, Mobashar Hussain Urf Turabe Fazil, Claudina Perez-Novo, Savithri Rangarajan, Rik de Wijn, Padma Ramireddy, Navin Kumar Verma, and WimVanden Berghe 14 Biotinylation Assay to Determine LFA-1 Recycling in Motile T-Lymphocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 Malin Samuelsson and Lena M. Svensson 15 A Protocol to Study T-Cell Signaling in an Immune Synapse by Microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 Mobashar Hussain Urf Turabe Fazil, Pankaj Kumar, and Navin Kumar Verma 16 Enzyme-Linked Immunosorbent Assay for T-Cell Dependent Immunogenicity Assessment of Therapeutic Peptides . . . . . . . . . . . . . . . . . . . . . . . 129 Madhavi Latha Somaraju Chalasani, Rajamani Lakshminarayanan, and Navin Kumar Verma 17 Modified Intravital Microscopy to Assess Vascular Health and T-Cell Motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Geoffrey W. Payne, Kevin Mitchell, and Stephanie L. Sellers 18 Computational Analysis of Protein–Protein Interactions in Motile T-Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 Sunil Kumar, Mobashar Hussain Urf Turabe Fazil, Khurshid Ahmad, Minaketan Tripathy, Jagath Chandana Rajapakse, and Navin Kumar Verma Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 viii Contents
- 12. Contributors KHURSHID AHMAD ICAR-NBAIM, Mau, Uttar Pradesh, India MADHAVI LATHA SOMARAJU CHALASANI Autoimmunity and Inflammation Program, Hospital for Special Surgery, New York, NY, USA; Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Singapore, Singapore CHANDRA SEKHAR CHIRUMAMILLA Laboratory of Protein Chemistry, Proteomics and Epigenetic Signalling (PPES), Department of Biomedical Sciences, University of Antwerp (UA), Antwerpen, Belgium RIK DE WIJN PamGene International B.V., 5200 BJ ‘s-Hertogenbosch, The Netherlands GAELIN LOOI WEN DONG Singapore Phenome Center, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Singapore, Singapore EMMANUEL DONNADIEU Département Immunologie, Inflammation, et Infection, INSERM, U1016, Institut Cochin, Paris, France; CNRS, UMR8104, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France MOBASHAR HUSSAIN URF TURABE FAZIL Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Singapore, Singapore MARTIN-LEO HANSMANN Dr. Senckenbergisches Institut für Pathologie, Klinikum der Johann Wolfgang Goethe-Universit€ at, Frankfurt am Main, Germany; Frankfurt Institute for Advanced Studies (FIAS), Frankfurt am Main, Germany DERMOT KELLEHER Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Singapore, Singapore; Departments of Medicine and Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada ATISH KIZHAKEYIL Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Singapore, Singapore PANKAJ KUMAR Lee Kong Chain School of Medicine, Nanyang Technological University Singapore, Singapore, Singapore SUNIL KUMAR ICAR-NBAIM, Mau, Uttar Pradesh, India; Institute of Life Sciences, Bhubaneswar, Odisha, India RAJAMANI LAKSHMINARAYANAN Singapore Eye Research Institute, Singapore, Singapore YVONNE MICHEL Dr. Senckenbergisches Institut für Pathologie, Klinikum der Johann Wolfgang Goethe-Universit€ at, Frankfurt am Main, Germany KEVIN MITCHELL Northern Medical Program, University of Northern British Columbia, Prince George, BC, Canada AIK SENG NG Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Singapore, Singapore; School of Biological Sciences, Nanyang Technological University Singapore, Singapore, Singapore SEOW THENG ONG Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Singapore, Singapore SHYAM MOHAN PALAPETTA Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Singapore, Singapore; ix
- 13. Nanyang Institute of Technology in Health and Medicine, Interdisciplinary Graduate School, Nanyang Technological University Singapore, Singapore, Singapore GEOFFREY W. PAYNE Northern Medical Program, University of Northern British Columbia, Prince George, BC, Canada CLAUDINA PEREZ-NOVO Laboratory of Protein Chemistry, Proteomics and Epigenetic Signalling (PPES), Department of Biomedical Sciences, University of Antwerp (UA), Antwerpen, Belgium PRASEETHA PRASANNAN Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Singapore, Singapore JAGATH CHANDANA RAJAPAKSE School of Computer Science and Engineering, Nanyang Technological University Singapore, Singapore, Singapore PADMA RAMIREDDY Laboratory of Protein Chemistry, Proteomics and Epigenetic Signalling (PPES), Department of Biomedical Sciences, University of Antwerp (UA), Antwerpen, Belgium SAVITHRI RANGARAJAN PamGene International B.V., 5200 BJ ‘s-Hertogenbosch, The Netherlands MALIN SAMUELSSON Department of Experimental Medical Science, Lund University, Lund, Sweden RATHI SARAVANAN Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Singapore, Singapore STEPHANIE L. SELLERS Centre for Heart Lung Innovation, St. Paul’s Hospital, University of British Columbia, Vancouver, BC, Canada; Department of Radiology, St. Paul’s Hospital, University of British Columbia, Vancouver, BC, Canada LENA M. SVENSSON Department of Experimental Medical Science, Lund University, Lund, Sweden; The School of Medical Sciences, Örebro University, Örebro, Sweden MINAKETAN TRIPATHY Laboratory of Fundamentals of Pharmaceutics, Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), Bandar Puncak Alam, Selangor, Malaysia WIM VANDEN BERGHE Laboratory of Protein Chemistry, Proteomics and Epigenetic Signalling (PPES), Department of Biomedical Sciences, University of Antwerp (UA), Antwerpen, Belgium NAVIN KUMAR VERMA Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Singapore, Singapore GRAHAM D. WRIGHT A*STAR Microscopy Platform, Skin Research Institute of Singapore, Singapore, Singapore x Contributors
- 14. Chapter 1 An Introduction to LFA-1/ICAM-1 Interactions in T-Cell Motility Navin Kumar Verma and Dermot Kelleher Abstract The inherent ability of T-cells to migrate is critical for a fully functional immune system, both in normal immune surveillance and for mounting an adaptive immune response. At the same time, inappropriate trafficking of T-cells can be a pathological factor for immune-mediated or chronic inflammatory diseases. T-cell motility is critically dependent on a series of ligand–receptor interactions, a precisely regulated intracellular signaling, an involvement of adaptor proteins, and dynamic remodeling of the cytoskeletal systems. The leukocyte integrin LFA-1 receptor present on T-cells binds to the ligand intercellular adhesion molecule 1 (ICAM-1) and this LFA-1/ICAM-1 contact acts as a trigger for T-cell motility. In this book, we present a collection of methods and protocols that are frequently used by researchers to better understand T-cell motility in health and diseases. Key words T-cell contact, Integrin, Signal transduction, Inflammation, Autoimmunity 1 T-Cell Motility Is a Fundamental Immunological Process T-lymphocytes play a key role in the immune response. In order to perform their immunosurveillance duties, T-cells continuously recirculate in the bloodstream and move through tissues to scan for specific antigens. The varying requirement for T-cells to migrate from blood to lymphatic organs and peripheral non-lymphoid tis- sues to quickly detect their cognate antigens is appreciated for eliminating pathogens and tumors, while maintaining tolerance against self. However, T-cell migration must be regulated and T-cells must tune their intrinsic mobility strategies depending on the tissue microenvironment in order to mount an effective immune response. Uncontrolled migration of T-cells, either by aberrant signaling or tissue-specific impairments, can have catastrophic consequences including unwanted autoimmune responses causing chronic inflammation, immune-mediated tissue Navin Kumar Verma (ed.), T-Cell Motility: Methods and Protocols, Methods in Molecular Biology, vol. 1930, https://doi.org/10.1007/978-1-4939-9036-8_1, © Springer Science+Business Media, LLC, part of Springer Nature 2019 1
- 15. destruction, and/or debilitating illness. Better understanding of the mechanisms underlying T-cell motility will facilitate the tuning of T-cell migratory activity for the therapeutic management of immune-mediated disorders. 2 Mechanisms of T-Cell Migration The past two decades have witnessed significant advances in reveal- ing the complexities and intricacies underlying T-cell-mediated immune response regulation. T-lymphocytes navigate to effector sites through the generation of precisely regulated cell-intrinsic migratory events involving adhesion receptors and a number of signaling pathway mediators. The motility of T-cells requires phys- ical guidance through receptor–ligand interactions, several inde- pendent but coordinated signaling cascades, and dynamic cytoskeletal rearrangements [1–4]. The lymphocyte function- associated antigen 1 (LFA-1) integrin receptor present on T-cells binds to the ligand intercellular adhesion molecule 1 (ICAM-1) expressed on the endothelium and this LFA-1/ICAM-1 contact acts as a trigger for T-cell motility. A typical T-cell motility process requires an initial transient attachment to the endothelium, which is followed by rolling and firm adhesion of activated T-cells on the endothelial surface, and finally migration through the blood vessel wall to sites of infection, tissue insult, or injury [5]. The ability of T-lymphocytes to enter lymph nodes also depends on adhesion molecules and chemoat- tractants on the high endothelial venules [5]. As T-cells move through distinct tissue compartments, they interact with endothe- lial cells and this interaction regulates the process of T-cell migra- tion. Typically, a motile T-cell moves at a speed of 10–12 μm/min. Migrating T-cells undergo a series of molecular change in the expression of signaling proteins and surface receptors. The require- ment and relative importance of these molecular changes varies between tissues and is dictated by many factors such as activation state of T-cells and tissue microenvironment. A gain in the expres- sion of a set of homing molecules on activated T-cells enables them to migrate to the tissue sites of inflammation or injury. For example, ligands for E-selectin and P-selectin facilitate tethering and rolling of T-cells on the endothelium [5]. The engagement of chemokine receptors activates integrins, such as the LFA-1 and the very late antigen 4 (VLA-4) by inducing conformational changes in the integrins. Activated LFA-1 and VLA-4 bind to their corresponding ligands, mainly ICAM-1 and the vascular cell adhesion molecule 1 (VCAM-1), respectively, expressed on inflamed/infected tissues. The adherent T-cells acquire an asymmetric characteristic polarized morphology with a distinct leading edge and the trailing uropod, and crawl along the luminal surface on the endothelium. 2 Navin Kumar Verma and Dermot Kelleher
- 16. The leading edge points toward the direction of movement, which is driven by cytoskeleton-mediated protrusions and dynamic adhe- sion turnover at the front and uropod retraction at the back (Fig. 1). T-cell migration can be viewed as a multistep process incorpor- ating the following four sequential stages: 2.1 Induction of Cell Polarization Upon receptor engagement, a multitude of signaling cascades is initiated causing strong asymmetry within the cell, which results in a polarized phenotype. T-cell polarization involves the formation of a ‘front–back’ asymmetry in which cell surface adhesion receptors, such as LFA-1 integrin, are accumulated at the front of the cell [6]. Lipid rafts are also implicated in the polarization of migrating T-cells [6]. Asymmetrical arrangement of surface receptors pro- duces an asymmetry of intracellular signaling components. A func- tional cytoskeleton and its reorganization are necessary for these cell-intrinsic processes. 2.2 Generation of Membrane Protrusions at the Leading Edge Once a T-cell has been polarized, it generates actin-enriched mem- brane protrusions (filopodia and lamellipodia) at the leading edge. The forward move of membrane protrusions is facilitated by actin polymerization at the front followed by stabilization of the new architecture of the resulting membrane extensions [7, 8]. 2.3 Formation of Cell–Substratum Contact Sites After membrane extension has been formed, new points of contact are established by the T-cell surface receptors with their specific ligands. This involves adhesion molecules of the integrin family [9]. In rapidly migrating T-cells, integrin binding to specific ligand leads to integrin clustering and the recruitment of cytoskeletal proteins to the cytoplasmic domain of the integrin. This prevents retraction of the newly formed membrane protrusions and provides ‘grip’ for the tractional force to assist T-cell motility. Uropod Leading edge Main cell body ICAM-1 LFA-1 Fig. 1 A cartoon illustrating a typical motile T-cell phenotype displaying leading edge at the front and the trailing uropod at the back T-Cell Motility 3
- 17. 2.4 Retraction of the Uropod and Forward Movement of the Cell Body The next stage of T-cell movement involves the flux of intracellular contents into the newly extended cell body, and the retraction of the trailing edge. Besides extension of membrane protrusions at the front by cytoskeletal remodeling following the engagement of integrins, a contractile force is generated to move the body of the T-cell forward [10]. 3 The LFA-1 Integrin The integrins are a large family of transmembrane receptors expressed at the cell surface and are composed of two non-covalently associated α- and β-glycoprotein subunits that form a heterodimer. T-lymphocytes express at least 12 of the 24 known integrins and the expression pattern of integrins on T-cells depends on the subset and effector state of the cell [8, 11]. In human T-cells, the integrin αLβ2 (also known as CD11a/CD18 or LFA-1) is the most abundant in expression [8, 9]. LFA-1 contact to the β2-integrin ligands, mainly ICAM-1, causes high-affinity adhesion and plays a prominent role in T-cell motility. In addition, LFA-1 forms the peripheral ring of the immu- nological synapse [12]. Under normal conditions, the LFA-1 receptor remains inactive or non-ligand-binding conformation when peripheral blood T-lymphocytes are circulating in the bloodstream, which ensures no inappropriate binding to their ligands. Expression of the inactive form of LFA-1 in circulating T-cells is crucial in maintaining homeostasis. Constitutively active LFA-1 causes rapid aggregation of circulating cells and clogging of the vessels [13]. Exposure to cytokines or chemokines and engagement with adhesion receptors results in instantaneous activation of LFA-1 precisely where its function is needed [9, 14, 15]. Following activation, the cytoplas- mic domains of the LFA-1 integrin associate with adaptor proteins (e.g. Talin 1, Kindlin 3) [16], connect the cytoskeletal systems, and recruit several signaling molecules, including cytoplasmic kinases and other proteins, such as protein kinase C [17, 18]. Notably, the distribution of the LFA-1 on the cell surface is altered once the T-cell begins to migrate. The amount of LFA-1 at the leading edge of a motile T-cell is relatively low in comparison to that in the non-attached uropod at the rear. This distribution of LFA-1 has been correlated with the dynamic changes in cell morphology dis- played by migrating T-cells [8]. 4 Cytoskeletal Remodeling in Motile T-cells The importance of cytoskeletal structure in cell migration was first recognized about 50 years ago [19]. Further experimental advances established a direct link between the lymphocyte-specific signal 4 Navin Kumar Verma and Dermot Kelleher
- 18. transduction pathways that trigger cytoskeletal reorganization [20, 21]. It is now evident that the LFA-1/ICAM-1 contact trig- gers dynamic rearrangements in the cytoskeleton, which facilitates T-cell motility [22, 23]. LFA-1 connects to the cytoskeleton through adaptor proteins, such as Talins and Kindlins, which binds to the β-subunit of LFA-1 cytoplasmic domain [16]. This connection prompts an array of downstream signal transduction cascades in addition to transmitting force between the extracellular ligand and the cytoskeleton [24], which ultimately influences cyto- skeletal remodeling implicated in T-cell motility. Although signifi- cant advances have been made to understand the molecular mechanism that dynamically rearrange T-cell cytoskeleton in migra- tory processes, we are still far away from complete information about the complex interplay of all the functional elements involved. Increasing evidence has confirmed that cytoskeletal proteins are important for receptor-mediated intracellular signal transduction events. 4.1 The T-Cell Actin Filaments Actin filaments are a cytoskeletal component that provide structural support and generate cellular movements involved in cell migration and other functions, such as endocytosis and cytokinesis. The importance of cytoskeleton remodeling in motile T-cells was initi- ally recognized based on the observation that the formation of a receptor cap and a uropod were inhibited by an inhibitor of actin polymerization cytochalasins [25]. Subsequent studies established importance of actin involvement and underlying molecular mechanisms in T-cell migration [26]. At the leading edge of the polarized and migrating T-cell, actin is organized in parallel bundles which form filopodia and in dense meshworks which form lamelli- podia [20, 27]. In the cell body and at the cell trailing edge, actin filaments form contractile force responsible for the contraction of the cell body and retraction of the trailing edge during T-cell motility [20, 27]. 4.2 The T-Cell Microtubule Cytoskeleton The microtubule cytoskeleton is mainly composed of α- and β-tubulin heterodimers. This dynamic structure enables T-lymphocytes to change their cell shape during migration in addi- tion to the regulation of other cellular processes, such as division, intracellular transport, organization and positioning of organelles, anchoring and guiding for protein complexes, etc. Polymerized microtubule networks exist in equilibrium with free tubulin. They assemble around microtubule organizing center (MTOC) and show polarity, requiring GTP to grow at the plus-end. Microtubule plus-end-associated proteins are also crucial players in this process and are responsible for the generation of a microtubule network and consecutive organization of the actin cytoskeleton. We have recently discovered a role of an adaptor protein AKAP450, also called centrosome- and Golgi-localized protein kinase N-associated protein (CG-NAP), in the nucleation of microtubule [28]. T-Cell Motility 5
- 19. Following migratory signals triggered by LFA-1/ICAM-1 contact, microtubules undergo massive reorganization in motile T-cells, which is often accompanied by the movement of organelles such as Golgi apparatus as well as signaling molecules [28]. In migrating T-cells, microtubules have been shown to directly interact with the LFA-1 integrin [18, 29]. Moreover, disruption of the microtubules cytoskeleton alters T-cell motility [30]. 4.3 Microtubules and Actin Actively Cooperate During the Process of T-Cell Motility T-cell motility relies on the force-generation machinery that forms a polarized cell asymmetry, which is dependent on the two distinct processes. First, a motile T-cell organizes its cytoskeletal elements in a polarized manner with concomitant protrusive and contractile activities at opposite cellular locations. Actin polymerization and turnover are essential for the traction force generation needed for T-cell motility, and this protrusion–contraction cycle is regulated by a dynamic cross-talk between both actin and microtubules [31]. Second, the migrating T-cell orients its intrinsic polarity axis in response to the extracellular cues, such as chemoattractant gra- dient. Membrane fluctuations facilitated by the dynamics actin and microtubule networks allows sensing of the cellular environment. The asymmetry in shape of migrating T-cells is determined by an ongoing cooperation between the actin filament arrays and the growing microtubule cytoskeleton, which influence each other’s polymerization/depolymerization dynamics directly or through regulation of signaling molecules. Both actin fibers and microtu- bules are polarized during cell migration at the leading edge and retracting trailing edges and are linked together either directly or indirectly through adaptor proteins or signaling molecules. Micro- tubules also modulate actin stress fiber formation, thus regulate actin dynamics and force generation in motile T-cells [31, 32]. In addition, Vimentin, the only known intermediate filament protein in leukocytes [33], plays a crucial role in maintaining cellular integ- rity and in regulating T-cell adhesion and transmigration [34]. 5 T-Cell Motility in Health and Diseases The orchestration of an immune response is crucially dependent on the inherent ability of T-cells to migrate. The recruitment of T-cells from the bloodstream into tissues is a necessary component of the inflammatory response to tissue injury caused by infection or wounding. Failure of T-cells to migrate or an inappropriate T-cell motility can result in chronic inflammation, so-called immunopa- thology, and may have life-threatening consequences. Following clearance of infection or resolution of injury, T-cell infiltration subsides and inflammation resolves without overt tissue damage. However, inappropriate inflammatory responses fre- quently associated with persistent T-cell infiltration may lead to 6 Navin Kumar Verma and Dermot Kelleher
- 20. autoimmune disease conditions such as asthma, rheumatoid arthri- tis, and psoriasis [35–38]. Asthma, a chronic inflammatory disease of the airways, results from an immune response to inhaled aller- gens. The constant presence and prolong activation of T-cells in the airways causes tissue damage, leading to airway hyper-reactivity against otherwise harmless stimuli such as cold air [39]. In rheu- matoid arthritis, an autoimmune response by overactive infiltrating T-cells causes destruction of joint tissue resulting in compromised limb function and crippling pain. Psoriasis is an inflammatory dis- ease of the skin, which is characterized by T-cell infiltration, kera- tinocyte proliferation, and the production of several pro-inflammatory cytokines and chemokines. The central role of T-cell migration in the pathogenesis of psoriasis is well- documented [40]. Despite the advances in molecular and imaging technologies, the complex mechanistic process that operates the migratory behavior of T-cells at various stages of development and activation is not fully understood. Since T-cell migration is a promising target for an anti-inflammatory therapy, the development of antagonists for integrin receptors, adhesion molecules, and cytoskeletal dynam- ics is among the most actively pursued areas in drug discovery research. Indeed, drugs that inhibit the interaction between the T-cell integrin receptors and their ligands have been applied in clinically. For example, a humanized antibody-based drug Efalizu- mab, which specifically binds to the α-subunit of LFA-1, was devel- oped for the treatment of the moderate-to-severe psoriasis and approved by the FDA in 2003 [41, 42]. However, despite proof- of-concept and initial success in treating psoriasis, the drug was withdrawn from the market in 2009 due to an increased risk of the development of progressive multifocal leukoencephalopathy, which is a fatal brain infection caused by reactivation of latent JC virus [43, 44]. New drugs that inhibit organ-specific homing cascades of populations of pathogenic T-lymphocytes would permit the use of tissue-selective anti-inflammatory interventions, which would pro- vide selective regimes of safe and effective immunosuppressive therapy for chronic inflammatory diseases. The paucity of studies that effectively link receptor-mediated signaling, cytoskeleton, and T-cell motility highlights the need of further experiments to eluci- date the exact mechanism. In this context, the purpose of this book series is to bring together a collection of methodologies and pro- tocols that are useful for researchers studying T-cell motility in physiological and pathological conditions. Acknowledgments This work was supported by the Lee Kong Chian School of Medi- cine, Nanyang Technological University Singapore Start-Up Grant T-Cell Motility 7
- 21. Exploring the Variety of Random Documents with Different Content
- 22. A better man. A better man—if not, So shall he go again in that same grade Where like a laggard half-asleep in school, He wakes to find himself a scholar still, With all the vexing problems yet unsolved, Which, in his idleness and lust of life, Were left until the morrow, and the sun To usher in another dreamless day. So manhood comes—and so it surely goes, Till those who here have studied to become Proficient in the lessons of this life, Shall be excused from school, and left to play By running brooks and hills that shout for joy, And living waters wild in their delight. So is it meet that all should labor now To learn these lessons well, so, when the day Of graduation comes, a Voice will say:— Well-done; perfect in life, perfect in death; Receive thy rich reward, for thou hast found— Perfection is the only key to Heaven.
- 23. A VISIT FROM THE CRICKET I. hou shrill-voiced cricket there In yonder corner, Thou remindest me Of joys departed, and of fair And fallen summer. O little mourner, Cease thy pensive fluting, Lest a flood of melancholy, Sad as thine, That to my heart is suiting, Encompass me—it is unholy Thus to pine For fallen joys or days departed, E'en though thou art so broken-hearted, For moments are divine. II. Silent art thou?—thanks to thee, O little cricket Underneath my chair; Thanks to thee—yet would I see Thy shadow less—out to yon thicket! There let thy dull repining Drive where the winds are driven, Nor deign to bring Thy sorrows back—let such be given To those in shades reclining
- 24. Who love to sing, With thee, of dear departed Summer, And hear again her sad funereal drummer, Thou little, mournful thing. III. One moment stay—why comest thou With doleful ditty Unbidden to my room; Wee, dusky mourner, do not go, But say—what is it claims thy pity, And sets thee telling, telling Such a solemn story So to me, As if there knelling, knelling Of some departed glory Dear to thee? O sad musician, put aside thy fiddle, And admit life is a riddle, And Heaven holds the key. IV. Thou mindest not; for hark!—again Resounds thy racket Shriller than before; Singst thou this sad strain As if befitting to thy ebon jacket, With carvings curious, And a color glossy, Like old wine— Tiny thing, be not so furious And uneedful noisy; Cease to pine
- 25. For something fled—for joys or hopes departed, Or thou wilt make the angels broken-hearted, O mourner most divine.
- 26. IN PRAISE OF INEZ. weet Inez, would that I might pledge My thoughts to thee with line on line, And prove, if tender words can prove, That all my tender thoughts are thine. Would that my feeble pen might pluck From the green fields of poetry, Some flower, sweet girl, wherewith to deck Thy name so near, so dear to me. Would that my hand might gather here From the sweet fields of tender thought, Some blossom, fragrant as the rose, Some lily, lovely as I ought. But why should I commit a sin By wishing any flower for thee; Thou art more beautiful, I know, Than all the flowers of poetry. What shall I then with thee compare, To make a true comparison— The dawning day, the dying light, The rising or the setting sun? At morn I see the early sun Appear with glory in her eye, But looking there, I think of thee, And thinking of thee, for thee sigh. At noon I see that fervid orb
- 27. Proclaim the sultry hour of day, But looking there, I think of thee, And thinking of thee, turn away. At length I see that same bright sun Descend below the western blue, Yet looking there, I think of thee, And thinking of thee love thee, too. Fade then, ye flowers of the field, And sink, ye dying beams of light, But let, O let my Inez be Forever present to my sight.
- 28. THE CRIME OF CHRISTMASTIME. I. wo thousand years!—two thousand years Since Mary, with a mother's fears, Brought forth for all humanities The Christian of the centuries; And now men turn from toil away To celebrate his natal day By feasting happy hours away And giving gifts with lavish hand, Throughout the length of every land;— A noble custom nobly born In Bethlehem one holy morn, But intermingling with the good, A pagan custom long has stood, As you and I and all may see— This war against the greenwood tree, This robbing of posterity,— Until the burden of my rhyme Is of this crime of Christmastime. II. The skies are white with soft moonlight; In Christian lands the lamps burn bright, In splendor gleaming from the walls Of parlors and of festive halls; Or yet, amid some snow-white choir, Sweet maidens sing the world's desire,
- 29. Till, answering in low refrain, The people all repeat the strain Of peace on earth, to men good-will, When sudden all the hall is still. Then tender music, soft and low, Heavenward seems to float and flow, But—mid these glittering lights, O see The stately form of greenwood tree! Whose graceful arms are drooping wide As grieving this fair Christmastide. III. The hills are white with lovely light, And everywhere the stars burn bright In splendor gleaming on the wood, Where once, in loyal familyhood, The evergreens together stood, But—now no vespers, sweet or low, In happy measures upward flow, For there—by Heaven's lights, O see The absence of the greenwood tree! Whose noble form once waiving wide, This melancholy waste did hide. IV. Yet here and there a lonely tree Still sounds a mournful melody, And answering, in low refrain, The winds repeat the solemn strain, Until the hills conscious of harm, Awaken in a wild alarm, Until, with trumpets to the sky,
- 30. They echo up to Heaven the cry:— Ye Forests, rouse—shake off thy shroud, And sound a protest, long and loud; Ye Mountains, speak, and Heaven, chide This carelessness of Christmastide— And Man, thou prodigal of Time, Bestir thyself—and heed my rhyme, And curb this crime of Christmastime.
- 31. THE MINER. eyond the beams of brightening day A lonely miner, moving slow Along a darkly winding way, Is daily seen to go, Where shines no sun or cheerful ray To make those gloomy caverns gay. For there no glorious morning light Is burning in a cloudless sky And there no banners flaming bright, Are lifted heaven-high, But that lone miner, far from sight, Treads boundless realms of boundless night. There neither brook nor lovely lawn Allures the miner's weary eye, For, having caught one glimpse of dawn, With many an anxious sigh, Those precious lights are left in pawn To be by fainter hearts withdrawn. Nor tender leaf nor fragrant flower Dare penetrate that fearful gloom, Where, low beneath a crumbling tower, Or dark, resounding room, Yon miner, in some evil hour, A ruined prisoner may cower. Yet, while the day is speeding on, Far from those skies that shine so clear, Far from the glory of the sun
- 32. And happy birds that cheer— Hark!—through those echoing caves, anon The hammer's merry monotone. There, far from every happy sound Of blithesome bird or cheerful song, In yonder solitudes profound, The miner, all day long, Hears his own music echo round Those deep-voiced caverns underground. There, in that gloom which doth affright Faint-hearted, sky-enamoured men, The miner, with his little light, Hews out a hollow den, And seems to find some keen delight Where others see but noisesome night. Thus many a heart, along life's way, Must labor where no cheerful sun Of golden hopes or pleasures gay, Shines till the day is done, For where the deepest shadows play The purest hearts are led astray. Yet some, unseen by careless Fate, Know naught of gloom or sorrow here. But happily, with hearts elate, They walk a charmed sphere, And lightly laugh, or lightly prate Of lonely souls left desolate. So are we miners, great and small, By sunny slope or lower gloom, And day by day we hear a call As from the distant tomb,
- 33. But, when the evening shadows fall, The lights of home will gleam for all.
- 34. LOVE OF COUNTRY. ove of country is the life of war; Love not your country then, If loving it should lead you into war; Oh do not be deceived—Love is broader,— Love is broader than a wheatfield, Love is broader than a landscape; Do not be misled—love the world; Begin at home—love your birthplace, Then your county, then your state, Then your country, then the countries Of your brothers and sisters, who look So much like you—like hands, like feet, Like ears, like eyes, like lips; like sorrows, Like hopes, like joys; like body, mind And spirit, for the spirit of one man Differeth not from the spirit of another, Or high or low, or rich or poor, being The same yesterday, to-day and forever. Love of country is the life of war; Love not your country then, If loving it should lead you into war— Should lead you into hatred Of your neighbor's country—lead you To strike down even unto death Your brother who so resembles you, Made in your image, and in the likeness Of the living God.
- 35. THE SINKING OF THE TITANIC itanic!—rightly named, sir—says the captain of the ship, And the safest of all vessels—now mark her maiden trip, And all think as the captain thinks—all her two thousand souls As steadily out o'er the sea the stately vessel rolls. For she is shod with iron and her frame is built of oak, And stout hearts man the vessel, wherefore the captain spoke; And with naught for pleasure lacking, so stately and so fair, She seems a floating palace—fit for angels living there. So farewell, says merry England, farewell says each green isle, And blessings for this noble ship on her initial trial, And praise be to her makers, and good-will to her crew, And safety to her passengers—take this as our adieu. O there were pleasant partings as the vessel sail'd away, And there was joy in every heart that pleasant April day, And there were happy thoughts of home—of meeting kith and kin, For the stately vessel soon would be her harbor safe within.
- 36. And so blue the sky above them and so blue the wave beneath, That all,—all thought of living and no one thought of death, As, hour by hour, the vessel left England far behind, And, hour by hour, the ship sped on as speeds an ocean wind. And when night came, with fond good-nights the floating city slept, Yet ever o'er the rolling waves the mighty vessel swept, And no one thought of danger—until with thunderous roar, The great ship struck the rock-like ice, and shook from floor to floor. Then there was breaking timbers, and bulging plates of steel, And noise of great commotion along that vessel's keel — Then there were cries of anguish, and curses from rough men, And earnest prayers for safety—O prayers for safety then. For women wept in terror, and stout men drop'd a tear, And the shouting and the tumult was maddening to hear, Yet there amidst that seething the life-boats, one by one, Were set adrift at midnight—where cold sea-rivers run. Then, on that fated vessel, the thousand waited there In hope some sea-born sister would snatch them from despair,
- 37. But no ship came to aid her, and, in the dead of night, The noble ship Titanic sank suddenly from sight. O midway in old ocean, in her darkest, deepest gloom, A thousand brave hearts bravely went down to meet their doom,— And what a tragic picture!—Oh, what a solemn sight Upon that fated vessel with the stars still shining bright! Then there was time for thinking—O time enough to spare, And there was time for cursing and time enough for pray'r,— Time,—time for retrospection, and time enough to die, Time, time for life's great tragedy—and time to reason why. That was the greatest battle that ever yet was fought; That was the greatest picture on any canvas wrought; That was the greatest lesson that mortal man can teach; That was the greatest sermon that priests of earth can preach. Yet no one fought that battle with saber or with gun, And no one saw that picture, save those brave hearts alone, And no one read that lesson there written in the dark, And no one heard that sermon that went straight to its mark. Nor shall we know their story, the saddest of the sea, Or shall we learn the sequel, the sorrow yet to be, But long shall we remember how brave men bravely died
- 38. For some poor, lowly woman with a baby at her side. And when the world gets scorning the greatest of the great, When poverty sits cursing the man of large estate, O then let men remember, how, in that awful hour, The wealth of all the world was powerless in its power.
- 39. WAR AND PEACE. ar is hell!—war is hell!— This is what the war-men yell Yet they love to be in hell, Love to hear the iron hail Strike, till even strong men quail; Love the dying soldier's knell, Ringing shot and shrieking shell, Love to hear the battle-cry, Love to see men fight and die With the struggle in their eye— War is hell—war is hell,— This is what the war-men yell. War is wrong—war is wrong; This the burden of my song: War is wrong—war is wrong— Sound the pean, human tongue; Let the message far be flung— Sound it, sound it heaven-high, Sound it to the starry sky, And Heaven, repeat the echoing, Till all the earth of peace shall sing. Peace loves day, but war loves night; Peace loves calmness, war—to fight In the wrong or in the right; Peace the hungry man gives bread, War would give a stone instead; Peace is honest—not so war, Crying—any way is fair;
- 40. Peace loves life—War loves the dead With a halo overhead; Peace pleads justice—War cries might In the wrong or in the right; Peace pleads—love your fellow-man, War cries—kill him if you can; Peace no evil thing would slight, Yet while daring dares not fight, Knowing might makes nothing right; Peace means liberty and life, War means enmity and strife; Peace means plenty, peace means power, War means—hell, and would devour All who do not trust its power; Peace means joy and love tomorrow, War means hatred, death and sorrow; Peace says—Bless you—men are brothers, War says—Damn you, and all others. War is hell, war is hell!— This is what the war-men yell; War is wrong, war is wrong— This the burden of my song; War is wrong, war is wrong, There never was a just one, Never; There never was a just one, Never. True as two from two leaves none, True as days are never done, True as rivers downward run, True as heaven holds the sun,— War is wrong, war is wrong, There never was a just one, Never; There never was a just one,
- 41. Never— Sound the message, human tongue, Sound it, sound it heaven-high, Sound it to the starry sky, And Heaven, repeat the echoing Till all the earth of peace shall sing.
- 42. PEACE AND WAR. Blest is that man who first cries peace, But curst is he who first cries war, For war is murder. It must cease Forever and from everywhere.
- 43. TO ANDREW CARNEGIE. hilanthropist, far-sighted millionaire, Lover of prose and friend of poetry, What needs my pen in furtherance declare Thou art also a friend of liberty,— Thou art, indeed, a very Prince of Peace, Who, conscious of the uselessness of war, Believest man's red carnage soon should cease, And nations now for nobler things prepare: What needs my pen in furtherance recite Thy kindly interest in the weal of man— Yet, lacking need, I nothing lose to write, But rather gain in praising as I can, For, if thy wealth the world sweet peace may give, Perhaps my lines in praise of peace may live. Transcriber's notes: The index entries for The Miner and Love of Country have been moved from after The Sinking of the Titanic. In The Miner a stanza break was inserted before the line Nor tender leaf nor fragrant flower. The following is a list of other changes made to the original. The first line is the original line, the second the corrected one.
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