Homing and Inflammation

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Homing and Inflammation
Micro 204. Molecular and Cellular
Immunology Lecturer Jason Cyster Jason.Cyster_at_ucs
f.edu

• How do cells exit 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 lymphoid organs?

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Lymphocyte Homing

• Constitutive trafficking of naive T and B
  lymphocytes to secondary lymphoid organs
• in lymph nodes, Peyers 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

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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

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The cascade (multistep) model of leukocyte
extravasation
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Important leukocyte adhesion molecules
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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 cells (HEVs)
• 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 - 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 glycoproteins /glycolipids and
  cutaneous leukocyte antigen (CLA) on effector T
  cells

L C-type lectin domain E EGF-like domain C
complement regulatory domain
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Selectin ligands are complex carbohydrates

• E- and P- selectin ligands contain fucose as
  well as galactose, N-acetylglucosamine and sialic
  acid
• Glycosyl transferases are essential to generate
  selectin ligands, including fucosyl transferases,
  GlcNac, Gal and Sialyl transferases.
• L-selectin ligands are sulfated and this
  requires the additional action of Glycosyl
  Sulfo-Transferases (GSTs)

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Key property of selectins is fast binding kinetics

• The rapid kon and koff of selectincarbohydrate
  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
• a4 integrins can also support rolling
  interactions

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Glyco-Sulfotransferase deficiency leads to faster
L-selectin mediated rolling and reduced ability
to undergo the rolling-to-sticking transition
Rolling fraction
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Tethering and rolling

• Localization of L-selectin and selectin ligands
  to microvilli favors encounters between these
  molecules
• Experimentally change localization of L-selectin
  from microvilli to
• cell body -- no rolling

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Chemokines
gt40, structurally related basic proteins of
10kD Four families C, CC, CXC, CX3C
chemokine
receptor
NH2
S-S
G?
?
?
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)
Cyster (1999) Science 286, 2098
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• Chemokines in Inflammation
• The large number of chemokines and chemokine
  receptors allows for a significant amount of
  homing specificity to be imparted by these
  molecules
• Examples (PARTIAL LIST)
• Cell type Chemokine Receptors Ligands
• Neutrophils CXCR1, CXCR2 IL-8, GCP-2, Gro-a
• Eosinophils CCR1, CCR3 Eotaxin, MIP-1a, MCP-3
• Monocytes CCR1, CCR2, CCR5 MCP-1, 2, 3, 5,
  RANTES, MIP-1a
• Naïve T CCR7, CXCR4 SLC, SDF-1
• Naïve B CXCR5, CXCR4, CCR7 BLC, SDF-1, SLC
• Th1 effector CCR2, CCR5, CXCR3 MIP-1a, MCP1,
  RANTES, IP10
• Th2 effector CCR3, CCR4, CCR8 Eotaxin, MDC, TARC,
  I309
• CD8 effector CCR2, CCR5, CXCR3 MCP1, MIP1a,
  RANTES, IP10
• Immature DC CCR1,2,3,4,5,6 MCP-1, 2, 3, 5,
  RANTES, MIP-1a
• (see Zlotnik and Yoshie, Immunity 12, 121
  (2000) for standardized chemokine nomenclature)
• Other chemoattractants
• - monocytes, neutrophils are attracted by C5a,
  fMLP, PAF
• - Th2 cells, eosinophils, basophils express
  CRTH2, a receptor for prostaglandin D2

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Chemokine code required for T and B cell entry to
lymph nodes and Peyers patches
HEV in LN or PP T zone
HEV in PP follicle
CCR7
CXCR5
T
CXCL13
CCL21
B
blood flow
B
SDF1
T
T
CXCR4

• Note
• chemokines are highly basic and bind to
  positively charged proteoglycans on the lumenal
  surface of endothelial cells
• there may also be specialized scaffolding
  molecules for chemokine retention and display

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The Integrin Family
Most bind extracellular matrix proteins (e.g.
fibronectin, laminin) but some have transmembrane
ligands
integrins are dimers of a and b chains
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Integrins involved in leukocyte attachment to
endothelium
Integrin Ligands aLb2
ICAM-1, ICAM-2 (ICAM-3) (CD11a/CD18, LFA1) ?4b1
VCAM-1, MADCAM-1, FN ?4b7? 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
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A switchblade-like model for integrin activation
Resting
Active
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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

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• Leukocyte adhesion deficiency (LAD) type I
• defects in b2 integrin -gt defective neutrophil
  migration to inflammed skin, peritoneum
  lymphocytes less affected due to continued use of
  a4b1, a4b7
• LAD patients have recurrent bacterial infections
• Other types of LAD involve defects in expression
  of glycosyltransferases needed to make selectin
  ligands and defects in intracellular signaling
  molecules needed for chemokine-mediated integrin
  activation

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Inside-out signaling occurs by separation of the
integrin cytoplasmic domains
Kim, Carman and Springer (2003). Science 301, 1720
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Lymphocyte arrest requires instantaneous
induction of an extended LFA-1 conformation
mediated by endothelium-bound chemokines
Shamri, Alon et al. Nature Immunology 6, 497 -
506 (2005)
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Homing specificity provided by the
selectin/chemokine/integrin code
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Diapedesis / transendothelial migration

• The forward migration of leukocytes through
  endothelial junctions
• Integrins may be involved in migration of cells
  from point of attachment to EC junctions
• monocyte transmigration regulated by JAMs, PECAM1
  (CD31), CD99
• Shear stress (via mechanoreceptors?) may be
  involved in promoting diapedesis
• Some cells may migrate through the body of the
  endothelial cell
• after diapedesis further molecular steps govern
  migration across the subendothelial basal lamina
  and through interstitial tissues

Aurrand-Lions et al., (2002) Nature Immunol 3, 116
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Integrins function during transmigration

• Additional molecules are likely to be involved
• Monocyte transmigration involves Junctional
  Adhesion Molecules (JAMs) and PECAM-1 (CD31)
• Signaling occurs into the Endothelial Cell

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Para- and Trans-cellular migration of monocytes
ICAM1 and VE-Cadherin (on endothelial cell),
LFA-1 (on monocyte
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Matrix metalloproteases - mediators of
extracellular proteolysis

• Once in the tissue, cells have to digest
  extracellular matrix to move
• protease release induced by activation e.g. IL8
  induces gelatinase release from neutrophils
• Types of metalloproteases
• 1. collagenases
• 2. gelatinases
• 3. stromelysins
• 4. membrane-type metalloproteases

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(No Transcript)
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Tissue selective chemokine and adhesion molecule
expression in the systemic organization of the
immune system
Kunkel Butcher (2002) Immunity 16, 1
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Receptor decoys help down-regulate the
inflammatory response

• D6 is a non-signaling chemokine receptor
  expressed by lymphatic endothelium that mops up
  chemokines and delivers them to degradative
  enodosomes
• D6 deficiency leads to a defective ability to
  downregulate acute inflammation in the skin,
  resulting in prolonged inflammation

Nat Immunol. 2005 Apr6(4)403-11
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How do cells exit from lymphoid tissues?
MEDULLARY SINUSES
Diagram courtesy of Ted Yednock
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Sphingosine 1-phosphate (S1P)
SphK
Ethanolamine Hexadecanal
S1P
Sphingosine
S1P lyase
S1P phosphatase

• 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 (S1P1-5, formerly known as EDG
  receptors)
• S1P receptors have roles in blood vessel and
  heart development

S1P1 (edg1)
S1P2 (edg5)
S1P4 (edg6)
S1P5 (edg8)
S1P3 (edg3)
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Lymphocytes express S1P1 and exit lymphoid organs
in response to S1P
spleen, thymus
lymph node
S1P1
S1P1
S1P lyase
S1P lyase
radiation resistant cells
radiation resistant cells
RBC
S1P
S1P
blood
efferent lymph

• S1P is supplied to blood by RBCs (95) and to
  blood (95) and lymph by a radiation resistant
  source (perhaps endothelial cells)

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• S1P1 is required intrinsically in lymphocytes
  for
• T cell egress from the thymus
• T and B cell egress from peripheral lymphoid
  organs

Lymphoid Organ
S1P1 wt
CD4 T
180
wt
ko
140
of co-transferred wt control
100
60
20
Mesenteric LNs
Peripheral LNs
Peyers Patches
Blood
Spleen
Lymph
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S1P1 is a point of egress control during
lymphocyte recirculation
2 lymphoid organ draining infection
thymus days
CD69
IFNa/b signal
double- positive
2 lymphoid organ quiescent hours
circulation minutes
circulation minutes
circulation minutes
DC
S1P
S1P
S1P
S1P
S1P
immature single- positive
S1P
S1P
TCR signal
S1P
S1P1
3 days
naïve T cell
S1P1
S1P
S1P
S1P
mature single- positive
effector T cells

• S1P1 upregulation during last step in thymocyte
  maturation
• S1P1 down-regulated in blood and lymph by S1P
  time needed for upregulation after entering
  lymphoid tissue may contribute to dwell time
• Egress shut-down during inflammation promotes
  local cell accumulation -gt CD69 induced by e.g.
  IFNa/b, physically interacts with and inhibits
  S1P1
• TCR activation causes transcriptional
  down-regulation of S1P1

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Recommended reading

• Required Reading
• 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 Aug
  3317(5838)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 6497-506.
• von Andrian, U. H. and Mempel, T. R. 2003. Homing
  and cellular traffic in lymph nodes. Nat. Rev.
  Immunol. 3, 867
• Additional Reviews
• Cyster, JG. Chemokines, sphingosine-1-phosphate,
  and cell migration in secondary lymphoid organs.
  Annu Rev Immunol. 200523127-59
• Lowe JB. Glycan-dependent leukocyte adhesion and
  recruitment in inflammation. Curr Opin Cell Biol.
  2003 15531-8
• Rosen SD. Ligands for L-selectin homing,
  inflammation, and beyond. Annu Rev Immunol.
  200422129-56
• Carman CV, Springer TA. Integrin avidity
  regulation are changes in affinity and
  conformation underemphasized? Curr Opin Cell
  Biol. 2003
• Aurrand-Lions et al. (2002) The last molecular
  fortress in leukocyte trans-endothelial
  migration. Nature Immunol 3, 116
• Kunkel Butcher (2002) Chemokines and the
  tissue-specific migration of lymphocytes.
  Immunity 16, 1