Leukocyte trafficking in rheumatoid arthritis Flashcards
(99 cards)
1
Q
what is the leukocyte adhesion cascade?
A
- how leukocytes exit blood and enter tissue
- Occurs as a protective inflammatory response to pathogens and damage
- Leukocytes roll along endothelium and migrate into tissue
2
Q
how is the adhesion cascade implicated in disease?
A
This process underpins pathology
- In CVD, RA, liver disease and IBD – aberrant trafficking of leukocytes from blood into tissues – chronic inflammation in tissues
- Associated with ageing - changes with immune system and leukocyte recruitment profiles
3
Q
what occurs during the adhesion cascade?
A
- Margination of leukocytes from centre of blood to vessel wall
- This happens in absence of inflammation
- Upon inflammation, endothelial cells begin to express receptors to mediate capture, rolling, firm adhesion and migration of leukocytes
- In resting state/healthy – endothelial cells act as barrier to keep blood and leukocytes within vessel
- Components within subendothelial space (basement membrane, fibroblasts and ECM) can influence endothelial phenotype and regulate this cascade
4
Q
how is the recruitment cascade controlled?
A
- endothelial phenotype
- endothelial activation
- leukocyte activation once they have bound to the endothelial surface
5
Q
what influences the endothelial phenotype?
A
Haemodynamic forces of flowing blood – this determines anatomical location where recruitment can occur:
- Shear stress within arteries are too high for leukocytes to form stable interactions with inflamed endothelium
- Leukocyte recruitment tends to occur in postcapillary venules and at vessel bifurcations (link to CVD)
Stromal microenvironment e.g. ECM, fibroblasts
- cytokines derived from leukocytes e.g. macrophages, or damaged cells (DAMPs)
Hypoxia
6
Q
what influences endothelial activation?
A
- Endothelial response to cytokines, inflammatory mediators, endotoxin, hypoxia/reperfusion, damage induced by hypoxia
- Upon activation, endothelial cells modify expression of adhesion receptors
7
Q
what happens when the endothelium is activated?
A
It upregulates capture and adhesion receptors
- Endothelial cells present chemokines and lipids
- Anything released from endothelium into blood will be washed away, so molecules need to be presented on surface to interact with leukocytes
8
Q
what influences leukocyte activation and what does this induce?
A
- become activated once bound to endothelial surface
- Response to activating stimuli (cytokines, chemokines, lipids)
- Integrin activation of leukocytes – changes to a high affinity state to stabilise binding
- Cytoskeletal rearrangement of leukocyte – spherical to flattened on endothelial surface to reduce frictional forces from flowing blood
9
Q
how does the phenotype of leukocytes change?
A
Shape change via cytoskeleton
Metabolism of T cells can affect how migratory they are
10
Q
what is margination?
A
Margination: red blood cells form route down centre of blood vessel
- they become the largest item within flowing blood
- This pushes platelets and leukocytes towards the vessel wall
– allows leukocytes and platelets to contact vessel wall
- Under inflammation, leukocytes are now close enough to interact with receptors on endothelium
11
Q
why do leukocytes roll along the vessel wall?
A
Endothelial activation causes upregulation of capture receptors e.g selectins
- Density of selectins dictates speed of cell rolling
- High density = slower rolling, less dense = faster rolling
- Intermittent density = stop-go interactions, cell binds selectin and bounces off over and over again
- Rolling occurs because kinetics of selectin interaction with receptors have fast on and off rates
- The interaction isn’t weak, but the dynamics of binding are rapid
- Physical forces of blood flow cause interactions to form and dissociate quickly, enabling cells to roll to next interaction
12
Q
how are neutrophils captured?
A
Neutrophils are captured from flow by selectin coated surfaces, not ICAM-1 coated surfaces
- ICAM-1 is an immunoglobulin superfamily members
- If you coat glass coverslips with ICAM and perfuse over neutrophils, there is no capture from flow
- If neutrophils are perfused over a surface coated with CD62, there is capture from flow
- There is decrease in capture as wall shear stress increases
13
Q
what is the nomenclature for selectins?
A
- CD62 is the CD number for selectins
P-selectin = CD62p, same for E and L
14
Q
how are T cells captured?
A
Occurs under TNFa+IFNy-induced inflammation:
- T cells can be captured from flow mainly through E-selectin on endothelial cells
- They can also be captured via a4b1 integrins – acts as capture receptor of T cells, which binds VCAM on the endothelium
15
Q
how can T cell capture be studied?
A
by inhibiting capture receptors on the endothelium
by inhibiting capture receptors on the lymphocyte
16
Q
what happens to T cell capture when endothelial capture receptors are blocked?
A
T cell binding to endothelial cells in response to inflammation – antibodies added to block endothelial cell receptors
- In absence of antibody, there is 100% binding (normalised to no antibody control)
- Blockage of VCAM domain 4, or 1 and 4 = there is inhibition of T cell binding
- Blockage of E-sel in conjunction with VCAM inhibition also impairs T cell binding
- Both VCAM and E-sel are required for T cell capture
17
Q
what happens to T cell capture when lymphocyte capture receptors are blocked?
A
Antibodies blocking lymphocyte adhesion receptors:
- Treat with a4 integrin antibody = reduced T cell binding
- Reduction not seen when b2 integrin is blocked
a4 bound to b1 integrin subunit is the ligand for VCAM
18
Q
why don’t leukocytes continue to roll?
A
due to endothelial activation
- upregulation of IgSF adhesion receptors - density and strength
- presentation of chemokines and lipids e.g. PGD2
due to leukocyte activation
- high affinity integrin activation
- cytoskeletal rearrangement
19
Q
how do endothelial cells become activated to stop leukocyte rolling for stabilisation?
A
Endothelium not only upregulates selectin capture receptors, but also upregulates adhesion molecules – IgG superfamily members ICAM and VCAM
- ICAM and VCAM form bonds and detach bonds slowly with their ligands – slow kinetics
– not stronger interactions, but slower dynamics
- As kinetics are slower, cells can no longer roll
- Endothelial cells also express chemokines and lipids
- Chemokines signal to the leukocyte to induce integrin activation
20
Q
how do leukocytes become activated to stop rolling for stabilisation?
A
Integrin activation of leukocytes changes their affinity state
- Low affinity to high affinity state
- There is activation of the leukocyte, causing cytoskeletal rearrangement, becoming flattened against endothelium
21
Q
how do neutrophils become stabilised during the adhesion cascade?
A
CXCR2-chemokine induces b2-integrin activation to support stable adhesion
- CXCR2 on the neutrophil binds CXCL1 and CXCL5 on the endothelium
- that neutrophils receive signal via CXCR2 – this causes activation of b2 integrins, changing affinity state to stabilise adhesion
22
Q
how can neutrophil stabilisation be studied?
A
- Blocking CXCR2 = reduction in neutrophil binding, measured by reduction in neutrophil transmigration - inhibits stability of neutrophil adhesion, leading to rolling
- Blocking b2 integrins on neutrophils reduces adhesion
23
Q
what are the two chemokine receptors typically expressed on neutrophils?
A
2 chemokine receptors on neutrophils:
- CXCR1 which binds IL-8 (CXCL)
- CXCR2 which binds CXCL1 and CXCL5
24
Q
what is chemokine inside-out signalling?
A
Integrins on leukocyte are inactive until they encounter chemokines expressed on surface of endothelium
- When chemokine binds leukocyte chemokine receptor, it upregulates integrin expression to then bind ICAM and VCAM on endothelium, enabling flattening
- Endothelium chemokines activate migration of leukocytes – stabilised adhesion and cytoskeletal changes
25
how do T cells become stabilised during the adhesion cascade?
T cell stabilisation requires interaction with CXCR3
- This causes a4b1 integrin to become activated, leading to firm adhesion via VCAM1
- Also requires prostaglandin signal via DP2 receptor to support onward migration of adherent T cells along endothelium
26
how can T cell stabilisation be studied?
CXCR3 blocking antibody reduces T cell adhesion
Inhibition of DP2 receptor (receptor for prostaglandin D2) blocks subsequent transendothelial migration of T cells
27
what adhesion molecules are important for leukocyte migration?
- ICAM1 and VCAM1 (Ig superfamily members)
- Junctional molecules e.g. CD31 and JAMs (junctional adhesion molecules)
- Lipids e.g. prostaglandin D2
28
what are the main stages of leukocyte migration?
1. Migration across endothelial surface (abluminally)
2. Transendothelial
3. Subluminal/subendothelial migration
4. Trans-basement membrane
Once cells have penetrated basement membrane, they are now within tissue and have left vascular barrier
29
what controls neutrophil migration?
- Need signal via DP1 receptor for PGD2
- Need signal via b2 integrins for across and through endothelial
- Need signal via b1 integrins for migration through and beneath endothelium
30
how can neutrophil migration be studied?
Inhibitor to DP1 receptor = reduction in neutrophil migration
Block b2 integrin = reduction in neutrophil transendothelial migration
Block b1 integrin = reduction in neutrophil subendothelial migration
31
how do neutrophils and T cells differ in their prostaglandin receptors?
DP1 receptor on neutrophils is different to the DP2 receptor that T cells require to migrate
- But both mediate PGD2 signal
32
how are prostaglandins generated?
Prostaglandins generated by arachidonic acid via COX1 and 2 from endothelium and leukocytes
- When in tissue, leukocytes become activated by PAMPs/DAMPs
33
what controls T cell migration?
T cell migration mediated by ICAM1 and b2 integrins
- In addition to prostaglandin, integrin and ICAM signals, increase in sphingosine-1-phosphate (S1P) dose causes inhibition of T cell migration across endothelial cells
- T cell migration can be negatively regulated by different lipids
34
how can T cell migration be studied?
Blockage of ICAM1 or LFA-1 (alphaL-b2 integrin/CD11a/CD18) reduces T cell migration
- blocking of VCAM1 has no effect
35
what is leukocyte transendothelial migration?
migration through the endothelium
- complex
- involves junctional molecules, basement membrane
36
what junctional molecules control migration?
Junctional molecules control paracellular migration – ICAM-2, JAM-A, and PECAM-1 (CD31) act sequentially to control neutrophil migration across postcapillary venules into inflamed tissue in vivo
- Paracellular = migration of leukocyte through junctions
37
what must leukocytes do once they have migrated through the endothelium?
Once through endothelium, leukocytes have to traverse the basement membrane
- Not much is known about the molecules involved in T cell migration across basement membrane, so this data is mostly on neutrophils
- Basement membrane has regions of low expression of proteins
- These are mirrored in the gaps in the pericyte coverage and basement membrane
- In the gap is a neutrophil
- These low expression regions in the basement membrane are where leukocytes preferentially migrate through
38
what markers show gaps in the basement membrane?
LNa5 = laminin
A-SMA = smooth muscle actin
CD11b = neutrophil marker
39
what proteins do neutrophils use to migrate through the gaps of the basement membrane?
Neutrophils use integrins in a series:
- b2 integrins to migrate through endothelium
- b1-integrins for migration through and underneath endothelium
- They start using b1 integin to start migrating through basement membrane
- b3-integrins for migration across the basement membrane – take over from b1
40
what is the role of chemotactic gradients in leukocyte migration?
Once within tissue, there are haptotactic and chemotactic gradients to allow neutrophils to migrate to site of injury/infection, across ECM proteins or along stromal cells
- Evidence along ECM proteins shows this occurs in an integrin-independent manner
41
how do leukocytes move within the tissue?
Chemokines expressed in ECM
- When cells are attached to endothelium and migrate, they move on ECM and stromal cells
42
how do T cells exit tissues via lymphatic endothelial cells?
Migration of T cells through blood vascular endothelial cells provides them with a signal to enable efficient migration through lymphatic endothelial cells
43
how can T cell exit via lymphatic endothelial cells be studied?
- Allow T cells to rest or to migrate through blood vascular endothelial cells
- Then put untreated or migrated T cells onto a lymphatic endothelial barrier
- T cells that have already migrated though endothelial cells can then migrate more efficiently through lymphatic endothelium
44
what signal is provided to enable T cell migration through lymphatic endothelial cells?
Giving a PGD2 signal can mimic signal received from blood vascular endothelial cells, allowing T cells to migrate efficiently through lymphatic endothelial cells
- Migration through blood vascular endothelial cells provides a PGD2 signal to allow T cells to migrate through lymphatic endotheilal cells
45
what does migration through lymphatic endothelial cells depend on? how is this shown in studies?
CCR7:
Inhibition of CCR7 impairs lymphatic endothelium migration
B2 integrins (CD18)
Also to a lesser extent, on b1 integrins (CD29)
- Blocking either of these reduces lymphocyte migration through lymphatics
also S1P
46
what are the key features of lymphatic endothelial cells?
Lymphatic endothelial cells are polarised – apical and basal surfaces with different molecule expression
47
how does the polarity of lymphatic endothelial cells affect migration?
- Coated lymphatic endothelial cells so that T cells migrate across basal surface first, and then into apical surface on the side of the lumen of the lymphatics
- This depends on CCL19, ligand for CCR7
- Also depends on LFA-1 (aLb2 integrin)
48
what is S1P?
S1P is a negative regulator of T cell migration across blood vascular ECs
But S1P is a positive regulator of trafficking across lymphatic endothelial cells
49
how was it shown that S1P is important for trafficking across lymphatic endothelial cells?
Block ability of S1P to signal using FTY720 – this inhibits migration across lymphatics
- S1P-dependent signal is required for exit from tissues into lymphatics
50
what happens to lymphatic migration when CCL19 is inhibited?
Inhibition of CCL19 causes log-jamming at basal surface – this is where cells are stuck on basal surface of lymphatic ECs and cannot traffic
51
How do cells know that they’re in joint and not lung or gut?
Chemokines generated by stromal cells dictate this, as well as the ECM
- Types of fibroblasts can differ between fingers and legs due to mechanical loading – less weight in fingers
- Hox genes changed depending on joints and dexterity
- Differences in types of arthritis in those joints
52
what genetic predispositions can affect adhesion and migration in RA?
HLA-DRB1
PTPN22
PAD enzymes
53
how is the PTPN22 variant shown to affect leukocyte migration into tissues in pre-RA?
- treated endothelial cells with different doses of TNF
- increase in neutrophil transmigration in healthy people with the PTPN22 variant compared to healthy controls
- these PTPN22 SNP individuals already have a greater propensity for their neutrophils to migrate into tissues
- this may drive damage to the joint as more cells infiltrate
54
how does smoking affect leukocyte trafficking in pre-RA?
Treat leukocytes with cigarette smoke extract:
- Altered neutrophil phenotype – increasing β2-integrin expression;
- Stimulates endothelial cell activation and selectin expression - increased capture and migration
- Triggers influx of neutrophils, monocytes and lymphocytes into non-inflamed lungs;
- Amplifies trafficking into the lung in response to inflammatory cues (LPS);
- Increases transcripts associated with Th17 cells in the lungs - generates pathogenic T cells
55
how does smoking affect RA pathology?
This can cause trafficking of leukocytes into lungs before developing joint inflammation
- leads to autoantibody production before symptoms
56
how does leukocyte infiltration in the lung change with ACPA positivity and smoking?
ACPA+RA = higher lymphocyte infiltrate in lungs when not treated with DMARDS
ACPA-RA = lower lymphocyte infiltrate, similar to that of healthy controls
presence of autoantibodies and smoking can drive infiltration into the lungs
57
how does RA change from asymptomatic to symptomatic?
- asymptomatic - lack joint symptoms but have risk
- loss of tolerance and autoantibodies
- this causes epitope spreading and increased autoantibody titer
- this leads to joint swelling and presentation to clinic
- this is the inflammatory disease
58
what are the key cells in RA?
Fibroblasts – epigenetic change of fibroblasts in RA
- Take on new phenotypes
- In RA joint, there are 5 types of fibroblast
- Some drive inflammation – deletion of these can resolve inflammation
- Some drive tissue damage – damage to cartilage
Macrophages
T cells
B cells
59
how can the endothelium differ in acute vs chronic inflammation?
Endothelium can exhibit transformed phenotype in RA sites due to inflammation
- Can see difference between acutely inflamed joint and chronically inflamed joint
60
how does altered leukocyte metabolism in RA affect migration?
- T cells become hypermotile in RA due to metabolic rewiring
- reduced glycolytic flux
- upregulates TKS5 (podosome scaffold adaptor protein)
- TKS5 involved in membrane protrusions of migrating cells - enables motility
- pyruvate kinase activation can reduce T cell accumulation in RA synovium of mouse models
61
what is PEPITEM?
PEPITEM is an endogenous 14 aa peptide – cleavage product of parent protein 14-3-3z (adaptor protein expressed in most cell types)
62
how do B cells regulate T cell migration?
In response to adiponectin binding to its receptor on the B cell surface, B-cells release PEPITEM
- PEPITEM binds its receptor of endothelial cells – upregulates cadherin 15 (CDH15)
- Triggering formation of S1P and its secretion through SPSN2
- S1P binds S1PR1 on T-cells inhibiting their transendothelial migration into tissue
- S1P dampens affinity status of integrins, preventing T cell entry into tissues
63
how is PEPITEM altered in RA?
Patients with RA have a defect in the adiponectin-PEPITEM
- defect in signalling downstream of adiponectin receptors
- reduction in adiponectin receptors expressed on B cells
- more T cell migration across inflamed epithelium
- Synthetic PEPITEM restores the endogenous regulation of T-cell recruitment in RA
64
what is the adiponectin signalling pathway? how is it affected in early RA?
- adiponectin signals through its receptors by APPL1
- this leads to transcription of the 14-3-3z parent protein for PEPITEM
- in RA patients, there is downregulation of APPL1, so less PEPITEM production
both receptor levels and downstream signalling are reduced
- less PEPITEM so less inhibition of T cell trafficking
65
what are the 4 phenotypes in RA joint based on leukocytes within the tissue?
- myeloid
- follicle - lymphoid-driven - tertiary lymphoid structure
- fibroids with leukocytes present
- poor-sign immune - few leukocytes within the tissue
differences suggest changes in adhesion molecules on endothelial cells lining entry into tissue
66
how are synovial endothelial cells affected in RA?
Synovial endothelial cells are stably imprinted with a pathogenic phenotype: - Heterogeneity in the joint
- Selectins and VCAM-1 expressed on subpopulation of vessels
- upregulation of ICAM-1 and ICAM-2 constitutively
- EC near tertiary lymphoid structures acquire HEV-like phenotype – express PNAd
- Express vascular adhesion protein 1 (VAP1): normally found on mucosal endothelial cells, allows recruitment of gut-derived T-cells (receptor for a4b7, a gut homing marker for T cells) - gut-joint cross talk
- enables more neutrophil recruitment
67
how do adhesion molecules and expression on endothelium change in RA?
constitutive expression of ICAM and VCAM, upregulation of PNAD, allows leukocytes to enter
- Antigen presentation by endothelium of autoantibodies, enables autoreactive T cells to bind and migrate across into the tissue
68
how are endothelial cells altered by their microenvironment?
they change their transcriptional response depending on shear stress
- where they are in vasculature e.g. arteries, tissues or veins
also affected by stromal environment
endothelial cells can act as a postcode/reporter for leukocytes and tell them where they are in the body
69
how does the stromal microenvironment dictate the phenotype of endothelial cells?
- cultured endothelial cells with fibroblasts
- transcriptional responses change depending on fibroblasts from skin vs synovial joint in the same patient
70
what cells are found in the rheumatoid joint?
- expansion of CD45+ leukocyte infiltration
- increase vascularisation of the joint - mature and immature
- synovial hyperplasia of stromal cells (fibroblasts)
- 2 different types of fibroblasts in the RA joint
71
what are the different stages of RA?
resolving/early RA
- unclassified arthritis
- treatment-naive
- >12 week symptom duration
early RA = joint inflammation that doesn't fulfill criteria for diagnosis
resolving RA = presents to clinic with joint inflammation of undefined origin which resolves from 12 weeks to 6 months
- may be due to molecular mimicry during infection
established RA - patients may undergo joint replacement surgery
- automatically fulfill criteria for diagnosis
72
How do fibroblasts from different stages of RA influence endothelial function?
- in absence of cytokines, the fibroblasts from established RA activate the resting endothelium to recruit more lymphocytes
- early and resolving RA fibroblast cause low lymphocyte adhesion to endothelium
73
how do fibroblasts increase adhesion of leukocytes in established RA?
Neutrophils: capture is mediated by CD62P and CD62E
- adhesion stabilised by fibroblast-derived CXCL5 being presented on EC surface binding CXCR2 on neutrophils
Lymphocytes: capture is mediated by a4b1-integrin
- adhesion stabilised by fibroblast-derived CXCL12 being presented on EC surface binding CXCR4 on lymphocytes
Both: IL-6 is the bioactive agent in the EC-FB cross-talk
74
how are RA fibroblasts altered in early disease?
RA fibroblasts lose their the ability to regulate cytokine-induced recruitment in early disease
- immunosuppressive effects of fibroblasts are lost with RA - leads to amplified levels of leukocyte infiltration
75
what is IL-6 and how implicated in early RA?
IL-6 is an acute-phase pro-inflammatory cytokine
- it is hijacked in chronic inflammation as it isn't switched off
- inhibition of IL-6 reduces leukocyte binding and trafficking across the endothelium
- anti-IL-6R treatment is used therapeutically
76
how is TGFb implicated in early RA?
usually upregulated in wound repair:
- but in chronic inflammation, TGFb exacerbates damage
- TGFb treatment exacerbates RA in vivo
- TGFb can inhibit the inhibitor of IL-6 (SOCS3), leading to prolonged IL-6 signalling and Th17 differentiation
77
what happens when both TGFb and IL-6 are blocked in RA?
combined blocking of IL-6 and TGFb limits leukocyte binding to the endothelium
- Aberrant recruitment to inflamed EC-very early RA fibroblast co-cultures is dependent on IL-6 and TGFb
78
how are the effects of IL-6 and TGFb different in resolving RA?
IL-6 is also considered anti-inflammatory:
- In EC-healthy stromal cell co-cultures, IL-6 responsible for limiting leukocyte adhesion
- anti-IL-6 and anti-TGFb amplify binding - these are responsible for immunosuppression in resolving RA
- TGF-β1 treatment alleviates arthritis in vivo
but:
- Blocking IL-6 and TGFβ inhibits neutrophil recruitment to inflamed lungs in vivo
-IL-6 can activate SMAD7 (TGFb inhibitor) to prevent TGFb-induced Treg differentiation
79
why do some cytokines have dual functions?
some cytokines can be pro or anti-inflammatory based on their environment
- context-dependent
- presence of other mediators in microenvironment
- temporally differs between neutrophil vs lymphocyte infiltration
80
why does IL-6 have a dual role?
In resolving RA:
- IL-6 induces phosphorylation of STAT3 or STAT1
- Triggers SOCS3 production in endothelial cells
- SOCS3 inhibits EC response to TNF-alpha, so there is reduced endothelial activation and reduced leukocyte trafficking across endothelium
- there is worse arthritis in mice where endothelial cells are deficient in SOCS3
81
overall, how are IL-6 and TGFb implicated in resolving RA?
IL-6 and TGFb are inhibitory to leukocyte trafficking
- they are pro-resolving/anti-inflammatory in resolving synovitis
- limits leukocytes entering joint
82
overall, how are IL-6 and TGFb implicated in early RA?
IL-6 and TGFb become stimulators of migration and inflammation
- increase leukocytes within joint
- driven by stromal postcode altering the vascular postcode
83
overall, how is IL-6 implicated in established RA?
IL-6 is activatory
- activates endothelial cells to a pathogenic phenotype
- induces leukocyte migration into joint
84
how does PTPN22 variant affect leukocyte exit from tissues?
it may increase the stickiness of PTPN22-expressing T cells, so they have slower migration through the tissue and have increased residency periods
- these slow-moving CD4 T cells may interact with DCs for longer, amplifying neoantigen-specific responses
- more TCR engagement in the joint
85
why else do T cells remain in the joint tissue for longer in RA?
- heightened TCR engagement in the joint upregulates LSP-1 which reduces migration efficiency
- expression of SPHK1 (S1P kinase) is upregulated in the RA synovium - may lead to T cells being retained in the joint
86
what are current RA therapies targeting trafficking?
Adalimumab (anti-TNF) reduces influx of neutrophils in established RA
Tocilizumab (anti-IL-6R) impacts EC-FB crosstalk
JAK1-JAK2 inhibitor blocks RA neutrophil migration to IL-8
PEPITEM halts onset of RA and reduces joint swelling and erosion - enhances migration regulatory pathway, so reduces leukocyte infiltration
- also switches off production of inflammatory cytokines in the joint
- dampens down osteoblast stromal cells
87
how can a 2D culture be useful for studying the endothelium?
2D culture seeds cells onto plastic, and other side is available to liquid
- Recapitulates endothelium exposed to blood flow, whilst anchored to basement membrane
88
how can a 3D culture be useful for studying the endothelium?
Stromal cells are in 3D environment where they are contacted by ECM or other cells on all sides
- Organoids or organ-on-a-chip
- Allow stroma to self-assemble and avoid just a monolayer
- Culture cells either side and enable cross-talk
- enables the stroma to alter the endothelial phenotype
89
how can circadium rhythms influence RA?
immune system has circadian clock
- High numbers of leukocytes in circulation in morning, lower at night
- Vaccinating an elderly population should occur in morning to get better response – more leukocyte trafficking and higher expression of VCAM/ICAM
- Mice are awake overnight, so opposite – mice have reversed circadian clock
- Patients may feel pain more in the morning due to higher levels of trafficking and inflammation based on circadian rhythm
- Important for physiological response
90
why is sex a risk factor for RA?
Oestrogen receptors change throughout life of female on leukocytes – changes following menopause which can affect immune responses
91
how is inflammation kept controlled?
- Cytokines have short half-life – lower levels prevent endothelium from excessively upregulating adhesion molecules
- Endogenous leukocyte and EC inhibitors e.g. annexins, resolvins, PEPITEM (negatively regulates T cell trafficking, and this is lost in RA, leading to excessive T cell recruitment)
- Can supply patients with PEPITEM to reduce trafficking
92
how are RA patients determined to be in remission following therapy?
Disease activity score less than 2.5 = remission
- These patients are kept on therapy as they may have some inflammation remaining
- These patients have resolving macrophages – therapy has altered stromal microenvironment sufficiently to produce proliferation of resolving macrophages
93
what is bioflare?
BioFlare – remising individuals are taken off drugs to see if they relapse
- Downside of this study – anybody that does flare becomes non-responsive to the previous therapy – ethical issue
94
what regulates endothelial cells?
Inflammatory cytokines and mediators from:
- Tissue-resident macrophages & mast cells
- T-cells
- Bacteria
- Damaged tissue cells
+ local environment
95
haemodynamic summary:
Adhesion to endothelium influenced by haemodynamic parameters:
- presence of other flowing cells affects collision with the wall
- formation of adhesive bonds depends on how fast cells are
moving and how rapidly adhesion molecules can interact
- formed bonds must withstand flow forces
- selectin-bonds able to act rapidly for capture
- activated-integrin bonds stable enough to resist flow
96
how is adhesion to the endothelium influenced by stromal cells?
- expression of stromal derived chemokines on endothelial surface
- activation of endothelium and up-regulation of adhesion molecules (e.g. see in disease – RA/CVD)
- involvement of IL-6 and TGF-b (anti inflammatory response in health tissues, tends to be pro-inflammatory in diseased tissues)
- regulates the transcriptional response of EC and varies across the vascular beds given some level of tissue specificity
97
how are endothelial adhesion molecules regulated?
Expression modified in response to:
- cytokines (IL-1b, TNF-a, IL-4, INFy)
- inflammatory mediators (histamine, thrombin, C5a)
- endotoxin
- hypoxia/reoxygenation
- May only appear after stimulation e.g., E- or P-selectin
- May be constitutive but increased after stimulation e.g., ICAM-1
98
what happens when leukocytes are stimulated?
Cause stabilisation of adhesion (integrin activation) and cell migration (cytoskeletal changes)
Chemokines made (or transferred) and presented:
- Neutrophils (interleukin-8, IL-8)
- Lymphocytes (stromal cell-derived factor-1α, SDF; CXCL9-11)
- Monocytes (monocyte chemoattractant protein, MCP-1)
Lipid-derived chemoattractants
- platelet-activating factor (PAF)
- prostaglandin D2
99
how is endothelial trafficking kept local and controlled?
Localised endothelial activation by stimulatory agents
- Multi-step process
- roll-signal-stop-signal-migrate-signal-through endogenous leukocyte/EC inhibitors - Annexin, lipoxins, resolvins, PEPITEM
