Homing and Inflammation
Micro 204. Molecular and Cellular Immunology
Lecturer: Jason Cyster
jason.cyster@ucsf.edu
• How do cells migrate from blood into tissue?
– the four step model
– role of selectins, chemokines and integrins
• What controls recruitment of appropriate cell
types (neutrophils, monocytes, lymphocytes)
to the site of inflammation?
• How do cells exit from tissue into circulation?
• Are homing defects involved in human
disease?
Lymphocyte Homing
• Constitutive trafficking of naive T and B lymphocytes
to secondary lymphoid organs
• in lymph nodes, Peyer’s patches, tonsil this requires
active migration across blood vessels
• Entry into secondary lymphoid organs is highly
selective for lymphocytes
• Egress from lymphoid organs involves distinct
molecular mechanisms from entry
Lymphocyte homing and recirculation
(PP)
Adapted from
Anderson, artnscience.us
Inflammation
• involves local release of cytokines and chemokines by
tissue cells in response to pathogen products or
damage
• cytokines cause increase in vascular permeability
leading to local swelling, increased entry of antibody,
complement, etc.
• cytokines cause increased expression of adhesion
molecules on vascular endothelium and these work
together with chemokines to recruit cells - neutrophils,
monocytes, NK cells and, later, effector lymphocytes
The cascade (multistep) model of leukocyte
extravasation
Selectins are calcium-dependent (C-type) lectins
(carbohydrate binding proteins)
• L-selectin - entry to LNs, PPs
– on lymphocytes (neutrophils)
– binds specialized sulfated mucins (‘peripheral node addressins’ or
PNAd) made by high endothelial venules (HEV)
• P-selectin - early role in entry to site of inflammation
– in Weibel-Palade bodies in endothelial cells and a-granules of platelets
– translocates to membrane in response to thrombin, histamine, C5a, etc
– binds PSGL-1, a tyrosine sulfated mucin present on neutrophils, some
effector T cells
• E-selectin - delayed role in entry to site of inflammation
– cytokine inducible on endothelial cells (especially cutaneous)
– binds carbohydrate ligand (sialyl-Lex) on neutrophil and effector T cell
glycoproteins /glycolipids
Selectin ligands
Lymphocytes traverse high endothelial venules (HEVs)
to enter lymph nodes
Follicle or B zone
HEV
T cell area (paracortex)
LN section stained with:
B220 (pan B cell marker)
PNAd (‘peripheral node
address’ – sulfated glycans
recognized by mAb
MECA79)
Key property of selectins is fast binding kinetics
• The rapid kon and koff of selectin–carbohydrate ligand
interaction allows flowing leukocytes to tether and roll along
endothelial cells under shear flow
• Rolling slows down flowing leukocytes and places them in
proximity to endothelial cells where chemokines are transported
and expressed
Leukocyte rolling immediately after mild trauma
depends on P-selectin - PSGL1 interactions
wild-type
PSGL1-deficient
- both movies are of cremaster muscle venules within 30 minutes of
surgery (selectins induced by inflammatory trauma)
PSGL-1 = P-selectin glycoprotein ligand-1
Yang et al. 2000, JEM 190, 1769
The cascade (multistep) model of leukocyte
extravasation
Chemokines
>40, structurally related basic proteins of ~10kD
Four families: C, CC, CXC, CX3C
chemokine
NH2
S-S
receptor
Ga  
Lymphoid chemokines – help direct the homeostatic trafficking of cells into
and through peripheral lymphoid tissues (e.g. CCR7-CCL21 and CCL19;
CXCR5-CXCL13)
Inflammatory chemokines – help recruit cells to sites of inflammation
(e.g. CXCR2 / IL-8; CCR2 / MCP1; CCR5 / MIP1a)
Chemokine (IL8)-mediated firm adhesion
of rolling neutrophils
Lymphocyte adhesion to HEV depends on
chemokines
normal lymph node
CCL21/CCL19-deficient lymph node
Stein et al., (2000)
J. Exp. Med. 191, 61
The cascade (multistep) model of leukocyte
extravasation
Questions / Experiments for group discussion
1. What mechanisms might allow chemokines to act
locally on endothelium?
2. Does the chemokine have to be expressed by the
endothelial cell?
3. Could other classes of chemoattractants work at this
step?
The Integrin
Family
integrins are dimers of
a and  chains
Most bind extracellular matrix
proteins (e.g. fibronectin,
laminin) but some have
transmembrane ligands
Integrins involved in leukocyte
attachment to endothelium
Integrin
aL2
Ligands
ICAM-1, ICAM-2 (ICAM-3)
(CD11a/CD18, LFA1)
a41
a47
VCAM-1, MADCAM-1, FN
MAdCAM-1, VCAM-1, FN
ICAM1 and VCAM1 are constitutively expressed on lymph node HEV
and are upregulated on inflammed endothelium
MAdCAM-1 is expressed by HEV in mucosal lymphoid tissues
Integrin Activation
•
Integrins can be in resting (low affinity for ligand) and
active states
•
intracellular signals cause ‘inside-out’ signaling in the
integrin, converting it from an inactive to an active
state
•
chemokine signaling can cause ‘inside-out’ signaling
and activate integrins
•
ligand binding can cause ‘outside-in’ signaling that
also promotes formation of the active state
switchblade-like activation
Inside-out signaling occurs by separation
of the integrin cytoplasmic domains
Kim, Carman & Springer 2003 Science 301, 1720
Conformational change of integrin
during activation
Multistep Model of Lymphocyte Transmigration
into LNs
Girard JP, Moussion C, Förster R. Nat Rev Immunol. 2012
Diapedesis / transendothelial migration
• The forward migration of leukocytes across endothelium
• Integrins may be involved in migration from point of attachment to
endothelial cell junctions
• Additional molecules involved – e.g. monocyte transmigration
involves Junctional Adhesion Molecules (JAMs) and PECAM-1
(CD31)
• Signaling occurs into the endothelial cell
• May be promoted by shear stress (via mechanoreceptors?)
Questions / Experiments for group discussion
1. What mechanisms might allow chemokines to act
locally on endothelium?
2. Does the chemokine have to be expressed by the
endothelial cell?
3. Could other classes of chemoattractants work at this
step?
4. Do leukocytes only cross endothelium at cell-cell
junctions?
Questions / Experiments for group discussion
1. What mechanisms might allow chemokines to act
locally on endothelium?
2. Does the chemokine have to be expressed by the
endothelial cell?
3. Could other classes of chemoattractants work at this
step?
4. Do leukocytes only cross endothelium at cell-cell
junctions?
5. What mechanism might underly tissue-specific
homing of lymphocytes? And other cell types?
Homing specificity provided by the selectin/chemokine/integrin code
Tissue selective chemokine and adhesion molecule
expression in the organization of the immune system
Kunkel & Butcher (2002) Immunity 16, 1
Multistep cascade of lymphocyte migration to site of infection/inflammation:
same logic, different ‘area code’
Lipid Mediators
(Prostaglandins &
leukotrienes) can
function as
chemoattractants
• Activated components of complement (C5a, C3a) also act as chemoattractants
• Leukocyte adhesion deficiency (LAD) type I:
– defects in 2 integrin -> defective neutrophil migration to
inflammed skin, peritoneum; lymphocytes less affected
due to continued use of a41, a47
– LAD patients have recurrent bacterial infections
Experiment: You have a mutant mouse with symptoms
of LAD but its integrins are intact. What other classes of
molecules might you test as being potentially defective?
MEDULLARY
SINUSES
Diagram courtesy of Ted Yednock
How do cells exit from lymphoid tissues?
• Sphingosine-1-phosphate receptor-1 (S1PR1) is required
intrinsically in lymphocytes for:
- T cell egress from the thymus
Lymphoid Organ
- T and B cell egress from
HEV
peripheral lymphoid organs
% of co-transferred wt
control
1 day after i.v. transfer
180
140
S1PR1 wt
S1PR1 ko
CD4 T
wt
ko
Efferent lymphatic
100
60
20
Mesenteric
Blood
Peripheral LNs Peyer’sLymph
Spleen LNs
Patches
Matloubian, Lo, Cinamon,
Cyster Nature 2004
Sphingosine 1-phosphate (S1P)
Sph kinases
Sphingosine
S1P S1P lyase
Lipid phosphatases
S1PR1
(Edg1)
S1PR2
S1PR3
Ethanolamine
+
Hexadecanal
S1PR4
S1PR5
•
Abundant in plasma (~1uM) and lymph (~0.1uM)
•
Made intracellulary by all cell types during sphingolipid degradation but only
secreted by some cell types
•
Ligand for a family of G-protein coupled receptors (S1PR1-5)
•
S1P receptors have roles in blood vessel and heart development
Lymphocytes express S1PR1 and exit lymphoid
organs in response to S1P
thymus, spleen
lymph node, Peyer’s patch
S1PR1
S1PR1
S1P lyase
radiation
resistant
cells
RBC
S1P lyase
radiation
Lymphatic
resistant
Endothelial
cells
cells
S1P
blood
S1P
lymph
• S1P is abundant in blood and lymph
• RBC major source of blood S1P
• Lymphatic endothelial cells are source of lymph S1P
• Extracellular S1P is kept low inside lymphoid tissue
• S1PR1 is a point of egress control
Lymph Node Egress Pathway?
Cortex
T cell accumulation within cortical sinusoids
requires S1PR1
LYVE-1 is a lymphatic marker
Note: the sinsuses are packed with cells
Pham et al. 2008 Immunity 28, 122
T cells extend processes into cortical sinuses during
egress decision-making
Grigorova et al. 2009 Nature Immunol. 10, 58
Multi-step model of T cell egress
1.
Cortical sinus probing
1.
S1PR1 dependent entry
3.
Capture in a region of
flow and exit from lymph
node
Grigorova et al. 2009 Nature Immunol. 10, 58
Grigorova et al. 2009 Nature Immunol. 10, 58
Innate Immune stimuli cause a generalized
lymph node egress shutdown
Lymph
Node
Infection
(e.g. dsRNA)
6h
IFNa
Non-specific
Ag-specific
Lymphocytes
12 h
Model to account for IFNa-mediated
block in egress
Innate Immune Stimuli
IFNa etc.
S1P
IFNa
receptor
CD69
S1P1
Internalize
CD69
Egress
promoting
signal
Egress Blocked
CD69 gates migration of activated lymphocytes
into cortical sinuses and egress from LN
Grigorova et al., 2014 PNAS 107, 20447
Prolonged retention of antigenactivated lymphocytes
S1PR1
• Antigen activated T cells downregulate
S1PR1 mRNA and are retained
Naïve
• Recovery of S1PR1 after ~4 cell
divisions associated with egress
> Retention may help ensure appropriate
clonal expansion and receipt of instruction
signals before exiting
• Activated B cells downregulate S1PR1
• A subset of plasma cells upregulate S1PR1 and
exit spleen, LN to travel to BM
Activated
Effector
Translational Impact of Homing
Research
• a41 blocking antibody (Natalizumab/Tysabri®) in use for
treatment of Multiple Sclerosis and Crohn’s disease
 a47 blocking antibody (Vedolizumab/Entyvio®) approved May
2014 for ulcerative colitis and Crohn’s disease
• CCR9 antagonist in phase III trial for Crohn’s disease
• FTY720 (Fingolimod) first oral treatment for relapsing/remitting
multiple sclerosis; in trial for ulcerative colitis
• Inhibitors of prostaglandin and leukotriene synthesis have
mutliple anti-inflammatory effects, including antagonizing cell
recruitment
• CXCR4 antagonists used to mobilize HSC from bone marrow
• Led to advanced understanding of causes of Leukocyte
Adhesion Deficiency (LAD)
Recommended reading:
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Girard JP, Moussion C, Förster R. HEVs, lymphatics and homeostatic immune cell trafficking
in lymph nodes. Nat Rev Immunol. 2012. 12:762-73
Cyster JG, Schwab SR. Sphingosine-1-phosphate and lymphocyte egress from lymphoid
organs. Annu Rev Immunol. 2012. 30:69-94.
Sperandio M, Gleissner CA, Ley K. Glycosylation in immune cell trafficking. Immunol Rev.
2009 230:97-113
Muller WA. Mechanisms of transendothelial migration of leukocytes. Circ Res. 2009 105:22330
Carman CV, Springer TA. Trans-cellular migration: cell-cell contacts get intimate. Curr Opin
Cell Biol. 2008 20:533-40
Auffray C, Fogg D, Garfa M, Elain G, Join-Lambert O, Kayal S, Sarnacki S, Cumano A,
Lauvau G, Geissmann F. Monitoring of blood vessels and tissues by a population of
monocytes with patrolling behavior. Science. 2007 317:666-70.
Shamri R, Grabovsky V, Gauguet JM, Feigelson S, Manevich E, Kolanus W, Robinson MK,
Staunton DE, von Andrian UH, Alon R. Lymphocyte arrest requires instantaneous induction
of an extended LFA-1 conformation mediated by endothelium-bound chemokines. Nat
Immunol. 2005 6:497-506.
von Andrian, U. H. and Mempel, T. R. 2003. Homing and cellular traffic in lymph nodes. Nat.
Rev. Immunol. 3, 867
Lowe JB. Glycan-dependent leukocyte adhesion and recruitment in inflammation. Curr Opin
Cell Biol. 2003 15:531-8
Carman CV, Springer TA. Integrin avidity regulation: are changes in affinity and conformation
underemphasized? Curr Opin Cell Biol. 2003
Kunkel & Butcher (2002) Chemokines and the tissue-specific migration of lymphocytes.
Immunity 16, 1