Week 5 Lecture 7 - Cell Migration

Question 1

Roles of Lymphoid System

Answer 1

1. Production of large numbers of lymphocytes carrying clonally determined antigen receptors for the detection of foreign antigen
2. The bringing together of antigen and lymphocytes carrying the appropriate antigen receptors → antigen driven cell proliferation and differentiation
3. Dispersal of effector and memory lymphocyte populations to sites appropriate for the elimination of infection

Question 2

Primary and Secondary Lymphoid Tissue

Answer 2

Primary lymphoid tissue - sites producing mature naïve lymphocytes:
- bone marrow and foetal liver (B lymphocytes);
- bone marrow and thymus (T lymphocytes)
Secondary lymphoid tissues - drain defined tissues/compartments collecting antigen, and are the main sites of lymphocyte differentiation and of recirculating lymphocyte traffic
- where antigen is presented to naïve lymphocytes

Question 3

Other Areas of the Lymphoid System

Answer 3

Lymph nodes - located at points of convergence of vessels of lymphatic system, collect antigen from lymph (extracellular tissue fluid)
Spleen - collects blood antigens
Gut-associated lymphoid tissue (GALT): tonsils, adenoids, appendix and Payer’s patches
- collect gut antigens
More diffuse lymphoid tissue collects antigens from: the respiratory epithelium (bronchial associated lymphoid tissue/BALT), and other mucosa (mucosal associated lymphoid tissue/MALT)

Question 4

Cell Migration Basics

Answer 4

Lymphocytes develop from bone marrow stem cells, mature in the bone marrow (B cells) or thymus (T cells), then circulate via the blood to secondary lymphoid organs
- B cells mature in secondary lymphoid organs, but T cells mature in the thymus and mature naïve T cells leave the thymus for secondary lymphoid tissues
- if naïve lymphocytes do not encounter Ag in these lymphoid tissues, they return via lymphatic drainage to the blood & recirculate through other lymphoid organs

Question 5

T Cell Migration

Answer 5

Necessary for maturation
T cells develop from bone marrow stem cells, mature in the thymus and mature but naïve T cells leave the thymus for secondary lymphoid tissues
If Naïve T cells encounter antigen from dendritic cells, they develop into effector cells and migrate to the site of infection

Question 6

Main Patterns of Lymphocyte Traffic

Answer 6

1. Migration of naïve lymphocytes (both B and T lymphocytes) from blood into secondary lymphoid tissue
2. Migration of naïve T lymphocytes between different secondary lymphoid tissues
   - naïve lymphocytes migrate continuously between the secondary tissues until they respond to antigen or die
   - when naïve lymphocytes encounter antigen -> differentiate into effector or memory cells -> migrate to sites of inflammation or infection
3. Migration of activated memory or effector lymphocytes from lymphoid tissue to sites of inflammation
4. Migration of memory T cells from sites of inflammation back through lymphoid tissues

Question 7

What are High Endothelial Venues (HEVs)?

Answer 7

Specialised regions of the endothelium where lymphocytes migrate from blood into lymphoid tissue

Question 8

Pathway of T Cell Recirculation

Answer 8

Naïve T cells preferentially leave the blood and enter lymph nodes across HEVs
DCs carrying Ag enter the lymph nodes via lymphatic vessels
If the T cells recognise the Ag they are activated, & they return to the circulation via the efferent lymphatics
Effector & memory T cells preferentially leave the blood and enter peripheral tissues through venules at sites of inflammation

Question 9

Lymphocyte Recirculation

Answer 9

Lymphocyte recirculation is not random - it is regulated by lymphocyte-endothelial cell recognition events
Different trafficking patterns:
1. Naïve lymphocytes recirculate through secondary lymphoid tissue
2. Memory & effector lymphocytes recirculate through extra lymphoid tissue (e.g. inflamed skin & joints) and back to secondary lymphoid tissue
3. Memory lymphocytes display tissue-selective patterns of recirculation -> recirculating preferentially to those tissues where they first encountered antigen e.g. skin or mucosa

Question 10

Cell Migration - Inflammatory Response

Answer 10

The recruitment of activated phagocytes during an acute inflammatory response is sequential:
First Neutrophils accumulate in < 6 hours:
- release cytotoxic mediators that can cause tissue damage
Short time later ~6 hours activated macrophages enter:
- activated macrophages exhibit increased phagocytosis and increased release of mediators and cytokines that contribute to inflammation
Later activated lymphocytes enter:
- lymphocytes activated by antigen presenting DCs in draining lymph nodes
- this sequence is regulated by:
- 1. adhesion molecules expressed on the endothelia
- 2. adhesion molecules expressed on the leukocytes
- 3. the numbers of available leukocytes

Question 11

Different Types of Cell-Cell Adhesion

Answer 11

Very transient - inital cell interaction with blood vessel walls
Stable/firm - resists shear forces but is reversible in minutes
Firm/but reversible - allows cell migration
Very stable - long lasting (cells in tissue)

Question 12

Types of Selectins

Answer 12

E-selectin - found exclusively on endothelia
L-selectin - found on all circulating leukocytes except activated T-lymphocytes
P-selectin - found in secretory granules of platelets and endothelial cells

Question 13

Selectin Ligands/Mucins

Answer 13

Selectin ligands are cell surface, transmembrane, mucins which present glycosylation structures to the selectins
P-selectin ligands: PSGL-1 (P-selectin mucin ligand) is most important for leukocyte homing and CD24 is important for tumour cell binding
L-selectin ligands: GlyCAM-1 (glycosylation dependent cell adhesion molecule) & CD34
- in Peyer’s patches the ligand is MAdCAM-1 (mucosal addressin cell adhesion molecule).
Ligands for E-selectin include PSGL-1 and ESL-1 (E-selectin ligand-1)

Question 14

Ig Super Family Cell Adhesion Molecules

Answer 14

All possess one or more Ig-like domain
Ig domains resistant to proteases -> adaptable for the presentation of recognition domains
They recognise both homophilic and heterophilic ligands
- example of homophilic recognition - the same molecules on different cells bind each other
Vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), platelet endothelial cell adhesion molecule (PECAM)
PECAM-1, ICAM-1 -2 & -3, VCAM-1 are important for leukocyte extravasation
Integrins are frequently heterophilic ligands for Ig-superfamily members

Question 15

Integrins

Answer 15

Involved in cell-extracellular matrix adhesion and cell-cell adhesion
Structure: heterodimer consisting of two glycoprotein subunits (α and β), which are non-covalently bound
Functional integrins always have: one α subunit and one β subunit
Both subunits contribute to ligand binding
About 18 α subunits and 8 β subunits have been identified, giving ~24 unique integrins
Common ligands are: ECM proteins fibronectin, vitronectin, collagen and laminin or members of the Ig superfamily

Question 16

Leukocytes Crossing the Endothelium - Stages of an Inflammatory Response

Answer 16

1. Dilation of capillaries ↑ blood volume & ↓ blood flow
2. Microvascular structural changes and leakage of plasma proteins
3. Leukocytes migrate through the endothelium and accumulate at site of injury - the migration process has 5 steps each of which must be completed

Question 17

Crossing Endothelium - Rolling

Answer 17

P-selectin is most important selectin for rolling
It is rapidly expressed on endothelial cell surfaces upon stimulation by trauma or inflammatory cytokines (e.g. TNF)
- its ligand, PSGL-1 is on all lymphocytes, monocytes, neutrophils, eosinophils
- mice lacking P-selection → no rolling
L-selectin captures leukocytes and initiates rolling, but is less effective at rolling than P-selectin
E-selectin is involved in slow rolling and appears on the endothelial surface a few hours after P-selectin, and acts towards initiating the firm adhesion step

Question 18

Crossing the Endothelium - Slow Rolling -> Adhesion

Answer 18

The velocity of leukocyte rolling drops after a few hours → expression of E-selectin on endothelial cells and CD18 integrins (β2 integrins) on rolling leukocytes
- this process is called “slow rolling”
Contact time with the endothelium is important for determining whether the next step is successful
- slow rolling is better
The importance of rolling time is related to activation of the leukocyte and of leukocyte CD18 integrins by chemokines (e.g. IL-8) that are presented on the endothelial cell surface
Activated CD18 integrins (e.g. LFA-1) bind members of the Ig-SF, e.g. ICAM-1

Question 19

Crossing the Endothelium - From Rolling to Adhesion

Answer 19

The adhesive capability of CD18 integrins is dependent upon their activation status, normally expressed in an inactive state
Factors important for integrin activation and adhesion are cations & conformational changes (↑ affinity) & clustering on cell surface (↑ avidity)
Lymphocytes rolling on endothelium do not stop unless their CD18 integrins are triggered into their high affinity conformation
Chemokines are important for activating integrins
- chemokine up-regulated integrins induce arrest and firm adhesion of rolling cells

Question 20

Chemokines

Answer 20

Chemokines are secreted proteins that are potent attractors of various leukocyte subsets
4 Sub families
Subfamilies are grouped according to the spacing of their N-terminal cysteine residues: most chemokines belong to either the CXC or CC subfamilies, with the XC and CX3C being minor subfamilies
- “X” is a non-conserved amino acid
Around 19 chemokine receptors have been identified - proteins with seven transmembrane domains (7 TMR), and transmit intracellular signals through G proteins

Question 21

Inflammatory vs Lymphoid Chemokines

Answer 21

Inflammatory chemokines primarily attract neutrophils, monocytes and other innate immune cells
- main sources are activated endothelial cells, epithelial cells and leukocytes
Lymphoid chemokines are primarily produced in lymphoid tissue - they guide cell movement within these organs

Question 22

Chemokines and Triggering of Adhesion

Answer 22

Different chemokines trigger the adhesion of particular leukocyte subsets, and this depends upon expression of the appropriate chemokine receptor
- IL-8 will induce adhesion of neutrophils which express CXCR1 and CXCR2
- MCP-1 will trigger the adhesion of monocytes through CCR2
- Eotaxin will trigger adhesion of eosinophils expressing CCR3
- different memory lymphocyte subsets express different chemokine receptors and respond to chemokines accordingly, e.g if express CCR1 and CCR5 they will respond to RANTES

Question 23

Crossing Endothelium - Macrophages

Answer 23

At sites of infection macrophages stimulated by microbes release cytokines and chemokines -> activates endothelial cells to express adhesion molecules and chemokines
Chemokines activate integrins to a high affinity state -> adhesion and diapedesis in response to the chemokine gradient

Question 24

Other Attractants

Answer 24

Bacteria produce a peptide, fMet-Leu-Phe
- a potent chemoattractant for inflammatory cells
Complement fragments C3a, C4a & particularly C5a can play a role.
Platelet activating factor (PAF) and leukotriene B4 are chemoattractants during allergic inflammation

Question 25

Specificity & Combinations of Adhesion Molecules

Answer 25

The engagement and activation of multiple different sets of adhesion molecules operating in concert is important for favouring one cell type over another
- L-selectin is important for lymphocyte recruitment
- a 50% ↓ in L-selectin expression causes a 70% ↓ in lymphocyte migration
- a low density of P-selectin causes the selective recruitment of eosinophils over neutrophils

Question 26

Chemokine-Receptor Pairs

Answer 26

Lymph node chemokine-receptor pairs:
- receptor: CCR7
- chemokine: CCL19/CCL21
Peripheral tissue chemokine-receptor pairs:
- receptor: CXCR3 CCR5
- chemokine: CXCL10 CCL4/CCL5

Question 27

Directed Movement within Tissues

Answer 27

B cells express CXCR5, the lymph node follicles express a ligand for CXCR5, CXCL13
- CXCL13-CXCR5 ligand-receptor pair mediates compartmental homing of B cells in lymph nodes
The CCR7 ligand, CCL21 is expressed by stromal cells within T cell areas of lymph nodes, spleen and Peyers patches
A second CCR7 ligand, CCL19 is also expressed in the same areas
Both act to mediate homing of T cells in secondary lymphoid tissue