Complement-Mediated Stimulation of Inflammation
Three major roles of complement:
1.
Opsonize particles for
phagocytosis (need only reach
the C3b stage)
2.
Elicit inflammatory reaction by
acting on leukocytes, mast cells,
endothelium
3.
Complement-mediated cytolysis
Neutrophils
Leukocytes
• aka. polymorphonuclear leukocytes
• most abundant circulating leukocyte
myeloid progenitor (bone marrow)
• 2 types of intracellular granules
• common progenitor with monocyte
• adult human produces average of 1011 per day
• short life span (6 hours then apoptosis)
granulocyte-monocyte CFU
Monocytes (macrophages)
• phylogenetically the oldest cell of immune system
• circulate as inactive monocytes, enter tissue and
become activated macrophages (increased
vacuoles, cytoplasmic organelles)
neutrophil
monocyte
(polymorphonuclear leukocyte) (mononuclear phagocyte)
• occupy sites of microbe entry
• subepithelial connective tissue
• interstitia of parenchymal organs
• linings of vascular sinusoids in liver and
spleen
• lymphatic sinuses of lymph nodes
• response is usually later than neutrophils at sites of
injury/infection
• primary effectors of innate immune system
circulating monocyte
activated macrophage
Leukocytes Respond to Inflammatory Stimuli
• primary “purpose” of phagocytes -> clear invading organisms, foreign non-self materials
•Mechanisms of phagocyte-mediated wound clearing:
RECRUITMENT: adhesion proteins that facilitate attachment to endothelium
MIGRATION: receptors that mediate chemotaxis to target site
RECOGNITION AND PHAGOCYTOSIS: specific receptors for microbes and “opsonized”
materials – phagocytosis – Fc receptors and C3 receptors are major mediators of attachment
RELEASE OF CYTOTOXIC COMPOUNDS: reactive oxygen and nitrogen species
CYTOKINE PRODUCTION secretion of numerous cytokines and chemokines with local and
systemic activity
• positive factors – increase macrophage activation, recruitment, stimulate adaptive
immune system
• negative factors – inhibit activation and proliferation
• secretion of factors that facilitate wound remodeling, matrix production, angiogenesis
•Macrophages dominate biomaterial interfaces in tissue, often present chronically
Initiating Inflammatory Events
assume prototypical bacterial invasion in a wound
1.
Coagulation and complement cascades
•
C3a and C5a release, fibrinogen, thrombin
•
macrophage activation
2.
Tissue macrophage binds deposited complement, fibrinogen, bacteria
3.
Resident macrophage activation
4.
Cytokine release + complement fragments
5.
Mast cells activate, histamine release – increased vascular
permeability
6.
TNF-alpha mediated increased endothelial adhesivity to monocytes
7.
Recruitment of more macrophages to wound site
Leukocyte Recruitment
• secreted TNF-alpha, IL-1 increase by local macrophages
• increases endothelial VCAM, ICAM-1, and E-selectin
• circulating monocytes bind but affinity not sufficient to resist blood flow -> results in rolling along vessel
wall
• chemokines secreted by local activated macrophages bind to heparin sulfate proteoglycan on
endothelial cells, increased local concentration
•Chemokines increase leukocyte integrin receptor affinity for endothelial cells
• monocytes flatten in response to chemokine signaling, migrate through endothelium (extravasation) into
wound site by chemotaxis and become activated
• Vascular permeability increased by degranulation and histamine release by nearby mast cells (mediated
by C3a and C5a)
Leukocyte Receptors
1.
Receptors to bacterial mannose
2.
Opsonin receptors – engagement causes increased cytokine production, increase in oxidative
enzymes
3.
4.
5.
6.
1.
FcR
2.
Complement receptors (CR1-CR4)
Integrin receptors
1.
Fibrinogen receptor (integrin-mediated) -> key in response to many biomaterial surfaces
2.
Fibronectin
Toll-Like receptors
•
Family of receptors, each specific to one product (LPS, bacterial sugars, unmethylated CpG)
•
Signal cascade ends in NF-kB activation -> increased TNFa, increased E-selectin
G protein-coupled receptors – stimulate motility, neutrophil degranulation, release of proteolytic
enzymes
•
C5a receptor, C3a receptor
•
Prostaglandin receptors, leukotriene B4 receptor (products of COX and LOX pathways
stimulated NF-kB)
Cytokine Receptors (TNFa, IFNg, IL1b, and others)
•
Major effect is activation of NFkB
Leukocyte Complement Receptors
Common theme: binding to C3b or iC3b, mediate phagocytosis of opsonized particles
Phagocytosis stimulates cytokine production, increase in oxidase enzymes
Mac-1/CD11b in Rat Cerebral Cortex
100 mm
Mac-1+ Microglia on Retrieved Microelectrodes
Mac-1
Mac-1
Phagocytosis and Destruction
Toll-Receptor signals
IFNg
phagocyte oxidase enzymes
• engaged opsonized particles ingested into
lysosome
NADPH
NADP
• phagocytic vacuoles fuse with lysosome to yield
a phagolysosome
• Phagolysosome is site of toxic oxidative systems
• oxidative enzyme synthesis increased by IFNg
(produced by T-cells in response to inflammation,
and TLR signals
molecular oxygen
reactive oxygen intermediate (ROI)
superoxide dismutase
H2O2
• reactive species oxidize proteins and lipids
• These products often are released to the
extracellular space
myeloperoxidase
halide ions
• damage nearby tissue
• impact on biomaterials?
hypohalous acid
Toll-Receptor signals
IFNg
iNOS
arginine
citrulline + nitric oxide gas (NO)
superoxides, H2O2
peroxynitrite
Macrophages Release Cytokines
• proficient cytokine producers, unlike neutrophils
• Cytokine release critical to mount inflammatory response and to resolve inflammation
• General properties of cytokines
• secretion is brief, mRNA unstable, de novo synthesized, rapidly secreted
• cytokines are often redundant and actions are pleiotropic
• cytokines often affect the production and action of other cytokines (sometimes self)
– positive feedback
• action is often local, but at high doses can be systemic (TNFa, prostaglandins)
• bind receptors with very high affinity (Kd 10-10 to 10-12 M) – only small amounts are
necessary
• other signals regulate sensitivity to cytokines
• receptor # on cell surface
• often cytokines increase production of their own receptor – positive feedback
TNFa
• principal mediator of acute inflammation
• major source: macrophages
• LPS is a potent stimulator (through TLR)
• major effect: activation of pro-inflamm NFkB
transcription factor
LPS-Toll-Like Receptors
• Major actions:
• increase endothelial expression of
selectins and integrins
• increase chemokine production in
endothelial cells, macrophages
TRAF
• increases cycloxygenase and
lipoxygenase
• Increased arichodinate metabolism
• Production of prostaglandins and
leukotrienes (pyrogens, chemotactic)
• induces prostacyclin expression in
endothelium – increases local blood flow
• increase IL-1 production in macrophages
• induces apoptosis in some cells (through
p55 receptor – activates caspase)
• at high concentrations in blood, can
induce septic shock, organ failure,
thrombosis, high mortality
INFLAMMATORY
GENES
PHENOTYPIC PREDICTIONS OF TNF-alpha KNOCKOUT MOUSE?
Major Macrophage Cytokines and Growth Factors
IL-1b
• similar action as TNFa
• major source: macrophages, but also
neutrophils, endothelial cells, epithelial
cells
• activates NFkB
• Does not induce apoptosis or septic
shock at high concentrations
PDGF and FGF
• mitogens for fibroblasts and endothelial
cells
• important for fibroblast wound
remodeling at later stages and
angiogenesis
Chemokines
• chemotactic cytokines
• many cell sources during inflammation
• 8-12 Kda receptors: G Protein coupled
• bind to heparin sulfate proteoglycan on
endothelium and become concentrated
• Negative feedback – many chemokine
receptors are rapidly down-regulated when
stimulated
Interleukin-12 (IL-12)
• macrophages are major source
• binds to helper T-cells, stimulates IFNg
production
• IFNg activates macrophages, increases
chemokine production (including IL-12),
stimulates phagocytosis
Interleukin-10 (IL-10)
•
• Major actions:
• recruit leukocytes
• increase integrin affinity
• induce cytoskeletal dynamics –
actin-mediated motility
• Negative regulator of inflammation
• inhibits macrophage activation
• inhibits IL-12 production, shuts down
major source of IFNg production
Transforming Growth Factor Beta (TGFb)
• major inhibitor of inflammation
• secreted by T-cells and activated macrophages (especially under LPS stimulation)
• inhibits macrophage division
• induces expression of matrix synthetic enzymes and associated genes
• collagen, fibronectin, connective tissue growth factor
• begins positive feed loop for wound remodeling and fibrosis
Coagulation Factors also Stimulate Inflammation
Fibrinogen
• substrate for Mac-1
• in presence of TNFa, induces oxidative burst, IL-1b release
• Major contributor to macrophage attachment to biomaterial surfaces
• Fibrin clot functions as a scaffold for macrophage migration
Major Activities of Leukocyte Secreted Factors
Putting it Together
Factor H, Factor I – complement inhibition
penetrating wound
bacteria
some biomaterials
mast cell
TNFa
helper T-cell
bacterial sugars, LPS,
complement, fibrinogen
IFNg
Increase phagocytosis
oxidation enzymes
Increases IL-12
neutrophil activation
IL-12
TNFa
C3a, C5a
complement cascade
C3a, C5a – chemotaxis, macrophage activation
C5a
degranulation
ACTIVATON
TNFa
Increase ICAM-1, selectins
chemokine production, prostacyclin
MCP-1, IL1, IL10
neutrophil
resident tissue macrophage
blood vessel endothelium
FGF, PDGF, TGFb
histamine
increases vascular permeability
fibroblast
CTGF, collagen, fibronectin
circulating monocyte
When Inflammation Will Not Resolve
• Around some device implants
• continuous cytokine production
• high degree of fibrosis often seen around high inflammation (FGF,
PDGF?)
• fusion into foreign body giant cells
• chronic release of cytotoxic reactive species
• granulomatous tissue
• clusters of chronically active macrophages surrounded by
high fibrosis
• necrosis at center of tissue
• often interferes with tissue function and can impact
biomaterial performance
Inflammation at Biomaterial Interfaces
WHAT WE KNOW:
• Much current data is anecdotal, little mechanism understood
• Inflammation appears to occur around a variety of artificial materials (but not
so around biologics) even thought the materials are considered inert
• Deposition of plasma proteins
• Coagulation
• Leukocyte attachment and persistence
• Cytokine production
• Connective tissue fibrosis (sometimes granulomas)
• Inflammation represents unique problems for different applications
• But a common denominator appears to be the persistence of leukocytes at
interface – (why do they persist over time?)
• A few examples of common problems:
TISSUE
DEVICE
MATERIALS
PHENOMENA
BONE
articulating prostheses
polyethylene, titanium
osteolysis, loosening of implant
BLOOD
hemodialyis membranes
cellulose acetate and others
complement deposition, neutropenia, thrombosis
SUBCUTANEOUS
breast implants
silicone
fibrosis, calcification, contraction
BRAIN
electrodes
silicon, various metals
encapsulation, loss of chronic recording
Important Contributing Factors: Plasma Protein Adsorption
ADHESIVE FOR MACROPHAGES, MAY
STIMULATE PHAGOCYTOSIS
Greco, Implantation Biology, 1994
Important Contributing Factors: COMPLEMENT
Q-CMD shows mass increase
iC3b around PE debris in capsular synovium
• Complement activation by alternative pathway
• Appears independent of “tick-over” pathway
*
• non-specficic C3 adsorption alone can trigger
activation by factor B to generate a functional C3
convertase
• Adsorbed C3 is resistant to factor H and I
• Conformational change upon adsorption is likely
cause
• C5a release also detected, potential to initiate
leukocyte chemotaxis
Greco, Implantation Biology, 1994
release of C5a
Important Contributing Factors: FIBRINOGEN
• fibrinogen deposition on biomaterial surfaces occurs
rapidly
• conformational changes upon adsorption reveal
adhesive domains (mimics thrombin mediated conversion
to fibrin)
peritoneal PET disc model
# attached macrophages
• extent varies with material identity
• mediates macrophage attachment and increased
cytokine production
• in this model (PET disc), macrophage attachment was
normal in SCID mice (no IgG) and complement depleted
mice (cobra venom factor)
• severe hypofibrinogenemic mice do not mount
inflammatory response to PET unless fibrinogen is preadsorbed
• Hence, fibrinogen adsorption may be more influential in
macrophage attachment than complement or antibodies
Mac-1 inhibitor
Macrophage attachment increases with amount of adhesive epitope
exposure in fibrinogen
Important Contributing Factors:
MAST CELLS / HISTAMINE
Reduced neutrophils and macrophages
on PET discs when H1 and H2 histamine
receptor antagonists applied
Reduced neutrophils and macrophages
in mast cell deficient mice; phenotype
“rescued” by addition of mast cells
Important Contributing Factors:
PARTICULATE SIZE AND CONCENTRATION
• Problems in joint prostheses
• periprosthetic osteolysis
• chronic inflammation
• release of wear particles
• loosening of implant
• 30,000 revision surgeries/year in U.S.
• debris activates macrophages
• TNF-alpha release recruits osteoclasts
• Stimulates NO production -> PGE release
• Osteoclasts degrade bone
Important Contributing Factors: TNF-alpha
Macrophages
consume wear
debris and express
TNF-alpha
surrounding a
spinal implant
no recruitment of
osteoclasts (red) in
TNF receptor
double KO mouse
in response to
PMMA particles
Important Contributing Factors: Motion
• prostaglandins are
products of cycloxegenase
pathway
• induced by TNF-alpha
• potent pro-inflammatory
mediator
• this is still a subject of
debate – no definitive in
vivo data
Elastic membrane, 4% stretch, 1
Hz strain, 1 hour