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Immunology: Research + Clinic > HC2: Mechanisms of inflammation and transendothelial cell migration > Flashcards

HC2: Mechanisms of inflammation and transendothelial cell migration Flashcards

HC2

1
Q

How can the migration of leukocytes go against the current of the blood flow?

A

By adhering to the vessel wall

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2
Q

Neutrophil response

A

Innate cells, quick response
> other innate cells: macrophages, eosinophils, basophils, mast cells

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3
Q

How do innate cells reach infection site?

A

Injury > release alarm signals > extravasation of phagocytes
» bacteria release alarm signals to trigger the blood vessel
» signalling to phagocytes

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4
Q

Steps of leukocyte transendothelial migration

A

Rolling > adhesion > crawling > diapedesis
(increase in inflammation throughout)

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5
Q

Rolling adhesion and crawling to diapedesis

A

s-Lex of leukocyte binds to E-selectin (and other) of endotheial cells, than from rolling adhesion to tight binding with LFA-1 to ICAM-1 of EC and then diapedesis

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6
Q

ICAM regulation in endothelial cells

A

Upregulated and expressed when endothelium activated > strong adhesion leukocytes

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7
Q

Impaired endothelial activation in patients leads to:

A
  • Always inactive: chronic infections
  • Always activated: autoimmune reactions
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8
Q

Where do leukocytes extravasate (immune cells)

A

Microcirculation

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9
Q

Extravasation research in oncology

A

Of T-cells near tumor site
> research focus: efficiency in extravasation

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10
Q

How do neutrophils know when to phagocytose?

A

By chemokine gradient > find bacterium by signals to kill it.

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11
Q

In vivo transendothelial migration model

A

Mouse model
> highly coordinated movement of lymphocytes and innate cells to infection heard is observed
> Specific places of extravasation observed
> Cremaster (ball sack) pulled out: inflammation induced and leukocyte migration
> fluids stay in circulaiton!

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12
Q

Immune surveillance

A

Immune cells regularly extravasate and go back

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13
Q

Why in vitro model for transendothelial migration (TEM)?

A

In vivo model observation lacks detail, more controlled system needed to check which proteins involved

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14
Q

In vitro TEM model: how to get Endothelial cells (ECs)

A

Endothelial cells needed : gets inflamed and then extravasation
> from umbilical cord: 2 arteries and 1 vein
» needed in research: vein can be used to take endothelial cells for culture
» plasticity of endothelial cells

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15
Q

Plasticity endothelial cells

A

can migrate
> dynamic blood vessels
» but fluid remains inside, barrier retains integrity

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16
Q

2 methods for TEM in lab

A
  • Transwell system (chemotaxis assay, Boyden chamber assay)
  • Physiological Flow assay
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17
Q

Transwell system assay

A

Transwell inside the well with own compartment
> filter placed with endothelial cells that are cultured
> chemokines / plasma put in lower well
> put immune cells in transwell
> measure minimal migration concentration

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18
Q

Downside of Transwell migration assay

A

No current of flow inside system, which is normally present

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19
Q

Flow chamber assay

A

Flowing fluid from tubes across plate with endothelial cell barrier
Put neutrophils in it and endothelial cell barrier
Look at the flow
> no inflammation: some cells appear and disappear: immune surveillance
> inflammation: cells (orbs) stick around

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20
Q

Upon inflammation: first reaction of ECs

A

Quick upregulation (within 30 min) of selectins: induce rolling adhesion
> later: ICAMs and VCAM upregulated: firm stop of neutrophils or T-cells or other leukocyte
> then: paracellular or transcellular diapedesis

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21
Q

To which molecules of the leukocyte do the ICAM/VCAM adhesins of EC bind?

A

Integrins
> Beta2 and Beta1 integrins
Beta2: Mac-1/LFA-1
Beta1: VLA3/4

22
Q

Role ICAM-1 / CD54 and integrins in binding leukocyte to ECs

A

Integrins of leukocyte are folded in, but folded out when activated by chemokine/attractant signalling
> integrins bind adhesion molecules of ECs like ICAM-1
> Expression of integrins is less important than the activation of integrins

23
Q

What if transwell migration assay with expression of ICAM-1, but also specific antibodies for it

A

Cells do not know where to go, cannot transmigrate, because adhesion molecule ICAM-1 is blocked

24
Q

What components makes leukocytes adhere to vessel wall

A
  • s-Lex to selectins of ECs
  • Integrins to ICAM-1, VCAM-1 adhesion molecules
25
Interaction ECs involved for paracellular diapedesis
Flexible cadherins of the EC lay between ECs and maintain strong interaction to prevent leaking > but, they need to move for paracellular diapedesis (between cells of layer) > in transcellular diapesis, the junctions between ECs stay intact
26
What prevents leakage during paracellular diapedesis
Actin dynamics during leukocyte TEM > the endothelial cells form protrusions (extensions) which go around the leukocyte to engulf it and fill the potential gap made for the leukocyte > The actin skeleton works elastic around the migrating leukocyte, the pressure is around the leukocyte and when passing the membrane closes by the elastic force around the leukocyte which moves with it: minimalize the gap > limited leakage (always tiny bit) > elastic tension > endothelium IS NOT a flat layer, especially when transmigration of leukocyte
27
Imaging of TEM with flow chamber assay
Grey image > white cell: in front of EC layer > grey cell: behind the EC layer
28
Actin dynamics imaging in 3d (volume rendering)
LifeAct marker for actin and other markers for leukocyte
29
Transcellular diapedesis and actin dynamics
Actin is risen around the leukocyte when migrating through the EC > leukocyte goes right through EC > for specific situations > EC supports migration by plasticity and reforming around transmigrating cell
30
Why does the endothelium not leak when TEM?
Endothelial cells make elastic membranes in actin to create elastic tension around the leukocyte and prevent and limit the leakage when the leukocyte transmigrates
31
Choice paracellular or transcellular diapedesis
Preference between leukocyte subsets > neutrophils and monocytes prefer paracellular > T-cells prefer transcellular
32
T-cell generic markers
All T-cells: CD3+ Tc cells: CD8+ Th cells: CD4+ Interest in TEM: CD8+ T-cells: needed in site of infection
33
CD8+ T-cell subsets
- Naive CD8+ T-cell - Memory cell: >> central memory cells (CM) >> effector memory cells (EM) - Effector cells
34
T-cell and endothelium form a synapse: for what?
To let the EC engulf the cell for transmigration > grab the cells > the T-cells stay (while neutrophils are flatter and can crawl further)
35
Which CD8+ T-cell subset uses more paracellular TEM
CM: central memory cells
36
Which CD8+ T-cell subset uses transcellular more
Effector cells
37
Preference CD8+ T-cell in TEM in duration inflammation
- Shorter inflammation: more paracellular - Longer inflammation: more transcellular
38
T-cell TEM depends on ICAM-1: how is this shown? (what drives TEM?)
Beta2 or ICAM-1 block decrease CTL transmigration rate
39
Why difference between CTL subsets in TEM?
- Same integrin profile of subsets, but different chemokine preference - Most CX3CR1 expression of Effector cells
40
CX3CR1 is also known as ..1.. and reacts to ..2..., also known as ..3..
1: Fractalkine receptor 2: CX3CL1 3: Fractalkine
41
Where does the Fractalkine chemokine come from?
From ECs > after long (!) TNFa stimulation of endothelium: expression Fractalkine > bound by the Fractalkine receptor of effector CTLs > longer inflammation, more transcellular migration > Fractalkine determines route of TEM
42
Block Fractalkine
Transcellular TEM blocked for CTLs > role ICAM-1 > because when no ICAM-1, no transcellular TEM
43
ICAM-1 and transcellular migration
ICAM-1 clustering when binding interins > clustering recruits SNAP23 > Proteomics of ICAM-1 adhesome > SNAP23 as candidate: perform western blot > SNAP23 higher expressed > positive control in western blot: TCL > when ICAM-1 clustered with help of beads: a lot of SNAP23
44
SNAP23 role
Involved in vesicle fusion > role in secretion of chemokines like CX3CL1 / Fractalkine
45
Brevaldin-1 in TEM assay
Less transcellular TEM > Brevaldin-1 blocks vesicle transport: no vesicle fusion and Fractalkine release by ECs. > shift towards paracellular TEM > Essential role of ICAM-1 through SNAP23 and fractalkine proven.
46
Shift in chemokine release when SNAP23 knockdown
Less CX3CL1 release (and overall chemokine release, vesicle fusion important for all chemokines) > less transcellular TEM (only chemokine release upon ICAM-1 clustering) >> impaired exocytosis
47
Vessel-on-a-chip technology: why and how
Because: transwell and flow chamber are very artificial > on a chip: based on real situation > very small chip Space on chip filled with substrate like collagen which hardens (resemble ECM) > tube within tissue is endotheliased with ECs > make endothelial tube > create flow through tube
48
Vessel-on-a-chip response to TNFa
Inflammatory cytokine TNFa decreases the vessel diameter: vasoconstriction > why? To rise the blood pressure a bit (important for organism: more inflammatory condition and outflow immune cells) > number of cells per vessel stays the same (no cell death induced) > more cell adhesion
49
Advantage vessel-on-a-chip beyond extravasation
Possible to track cells beyond extravasation: what do they do next (not possible in mice (lacks detail) or transwell and flow chamber (no tissue beyond ECs) >> substrate on chip can be adjusted (change collagen concentration: stiffen tissue when more) >> substrate is tissue: substance that fills around the tube
50
1 What makes leukocytes adhere to vessel wall 2 Why do vessels not leak during extravasation 3 How do leukocytes cross vasculature 4 Methods to study extravasation in lab
1: Adhesion molecules, filopodia, membrane structures 2: Actin-rich contractile ring that works like elastic strap 3: Paracellular and transcellular route with help of local chemokine release 4: physiological flow chambers, Transwells, Vessel-on-a-chip
51
Both the immunological and diapedesis synapses are mediated by interactions:/...
Beta2 integrins and ICAM > prevent leaking

Immunology: Research + Clinic (7 decks)

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