Download Inflammation

Inflammation
Acute
Marked by
1) changes in vessels
a. flow and caliber
b. Permeability
2) Cellular events
a. Accumulation of neutrophils and macrophages
i. Margination – Adhesion – Chemotaxis – Phagocytosis
3) Chemical Mediators
a. From cells
i. Preformed – histamine, serotonin
ii. Synthesised
1. PAF
2. cytokines, growth factors
3. arachidonic acid derivatives
a. cyclooxygenase pathway – Prostaglandins
b. lipoxygenase pathway – leukotrienes
b. from plasma
i. Complement system (classic, alternate)
ii. Kinin system
iii. Clotting/fibrinolytic system
c. From injury
i. O2 radicals
Chronic
Marked by
1)
infiltration by macrophages, lymphocytes and plasma cells
2)
proliferation of fibroblasts and small blood vessels
3)
increased connective tissue – fibrosis
4)
tissue destruction
Granulation Tissue
Formed during the proliferative phase of inflammation
Fragile tissue composed of matrix of fibrin, fibronectin, GAGs with proliferating
endothelial cells and fibroblasts mixed with a population of macrophages and neutrophils.
The ability to release these molecules gives rise to the concept that the
macrophage plays a pivotal role in regulation of healing (Clark, 1996). This is
supported by the demonstration that depletion of macrophages from the wound
site inhibits healing (Liebovich & Ross, 1975), that defective healing in aged mice
can be restored by transfer of macrophages from young mice (Danon et al, 1989)
and that the proliferation of fibroblasts and endothelial cells during granulation
tissue formation follows on from the increase in macrophages during the late
inflammatory phase of healing.
CYTOKINES AND GROWTH FACTORS IN HEALING
Cytokines and growth factors are polypeptide molecules that regulate
migration, proliferation, differentiation and metabolism of cells. They may act
in:
1) paracrine manner on cells adjacent to the secreting cell
2) autocrine factors on the secreting cell and
3) bound to carrier proteins as endocrine factors.
A diverse range of these factors have been identified in wound interstitial fluid
and a number have been identified as potentially playing a key role in regulating
healing. Platelet derived growth factor (PDGF) and Transforming Growth Factor
(TGF) are released by platelets and are important in initiating healing. The
inflammatory phase of healing is modulated by cytokines such as Tumour
Necrosis Factor alpha (TNFa), Interleukin-1 (IL-1), Interleukin-4 (IL-4) and the
peptide chemokine Interleukin-8 (IL-8). The sequential phases of granulation
tissue formation, re-epithelialisation and extracellular matrix formation are
regulated by Fibroblast Growth Factors, Transforming Growth Factors and
Epidermal Growth Factor amongst many others.
Platelet Derived Growth Factor
PDGF is a family of three isoforms, composed of dimers of the PDGF- or
PDGF- chains, which have overlapping but distinct biological properties
generated by interaction with two types of receptor. It is produced by platelets,
macrophages, endothelial cells and keratinocytes. Release of PDGF by platelets
is important in initiating healing. It stimulates chemotaxis of fibroblasts,
neutrophils and macrophages. Once these cells are attracted to the wound site
PDGF can then activate macrophages and induce proliferation of fibroblasts.
Additionally it can stimulate the production of the extracellular matrix components
fibronectin and hyaluronan although not as effectively as other factors such as
TGF.
Recombinant PDGF has been evaluated in several clinical trials to treat nonhealing chronic wounds and has been demonstrated to be of benefit in treatment
of wounds where healing is impaired by diabetes (Brown and Breeden,1994).
Transforming Growth Factor
The three isoforms of TGF (1,2 and3) have a broad range of activity within
healing. TGFb1 is the most abundant in all tissues and is the form found in
platelets.
All cells involved in healing can produce and/or respond to TGF.
Release of TGF by platelets at the same time as PDGF is important in initiating
healing as, at low concentrations, it is chemotactic for monocytes, lymphocytes,
and fibroblasts. Its role in angiogenesis is controversial as in some experimental
systems it stimulates endothelial cell proliferation and tubule formation whilst in
others it is inhibitory. Its actions may therefore be contextual and concentration
dependent.
TGF plays a central role in regulating maturation and strength of wounds.
It regulates many matrix proteins including collagen, proteoglycans, fibronectin,
matrix degrading proteases and their inhibitors. Topical application of TGF
increases the strength of experimental incisional wounds (Mustoe et al 1987).
Manipulation of the TGF isoforms during healing can modify scarring. Antibody
neutralisation of TGF1 and 2 or increasing TGF3 concentrations by
exogenous application decreases post surgical scarring (Shah et al 1995).
Fibroblast Growth Factor
Although at least ten fibroblast growth factors (FGFs) family members are known
FGF-1 (acidic FGF or FGF) and FGF-2 (basic FGF or bFGF) are most widely
characterised with respect to wound healing. They are weakly soluble with a
strong affinity for heparan sulphate leading to them being bound in the wound
extracellular matrix. FGF-2 has been detected at the wound site early in healing
and its rapid appearance after injury suggests that pre-existing tissue FGF-2 may
be important in healing rather than that synthesised de novo by inflammatory
macrophages. It stimulates angiogenesis leading to accelerated reepithelialisation after experimental application(Mustoe TA et al, 1991).
Epidermal Growth Factor
Epidermal growth factor (EGF) is a small molecule which exhibits homology with
regions of the TGF molecule. It is produced by macrophages and epidermal
cells with the keratinocyte and fibroblast as targets. Its primary role is to stimulate
keratinocytes to migrate across the wound provisional matrix and induce
epidermal regeneration(Brown et al 1991)
Cytokines
The term cytokine generally refers to molecules such as the Interleukins which
have initially been investigated for their role in regulation of the immune
response. Because of the inflammatory response that is associated with
wounded tissue they also have a potential role to play in regulation of healing
either directly by their effect on the structural cells in wounded tissue such as
fibroblasts, endothelial cells or keratinocytes, or indirectly by their modulation of
growth factor production by macrophages.
IL-1 and TNF are both pro-inflammatory cytokines that will induce expression of
adhesion molecules such as ICAM-1 and E-Selectin by endothelial cells allowing
leukocytes to adhere to the lumen of capilleries and extravasate into the wound
site. These cytokines activate macrophages and initiate production of growth
factors required for healing and more pro-inflammatory mediators to prolong the
inflammatory response. Overproduction of TNF may lead to a persisting chronic
inflammatory response that is involved in the pathogenesis of chronic wounds.
However there is a requirement for TNFa to allow normal healing (Lee et al 1999)
and induction of a transient TNF response may re-initiate healing in non-healing
chronic wounds (Moore 1999).
In order for monocytes to differentiate into activated macrophages they need to
pass through a priming stage of differentiation. During this stage they are
programmed for responsiveness to subsequent stimuli by exposure to the
cytokine microenvironment. For example interferon-g will give a positive priming
signal whilst exposure to interleukin-4 acts to down regulate the inflammatory
response by inhibition of priming.
Other Factors
Many other factors are likely to be involved in the regulation of healing including
Vascular Endothelial Cell Growth Factor (VEGF), Insulin Like Growth Factor
(IGF-1), Granulocyte Monocyte Colony Stimulating Factor (GM-CSF).
THE EXTRACELLULAR MATRIX
Collagen is the major protein component of the extracellular matrix (ECM) of skin
and composes 60-80% of its dry weight. Wound ECM is composed of a mixture
of collagen and elastin fibrils interspersed with glycosaminoglycans, long
unbranched polysaccharides and proteoglycans, glycosaminoglycans combined
with protein. Whilst the ECM fulfills an important structural role it also plays a
bioregulatory role in modifying the behaviour of cells that come into contact with it
and also by acting as a reservoir of bound growth factors and enzymes.
Collagens are composed of 3 alpha chains which have a common repeating GlyX-Y motif that allows folding into a triple helix. Thirty two distinct alpha chains are
known allowing the existence of at least nineteen different collagen types in
vertebrates. Of these only 7 are found in significant quantities in skin. These are
Collagens I, III, V, XII and XIV which form structural fibrils, Collagen VI which
forms microfilaments, Collagen V which forms reticular fibres and Collagen VII
which forms fibrils anchoring the epidermis to the dermis.
Collagen is synthesised primarily by wound fibroblasts. It is released at the
ribosome as a three chain molecule which then undergoes post translational
modification to form procollagen. These modifications include prolyl and lysyl
hydroxylation and glucosyslation and galactosylation of lysyl and hydroxylysyl
residues (Kivirikko & Myllyla, 1985). The trimeric molecule is then secreted into
the extracellular space where mature collagen is formed by the proteolytic
cleavage of procollagen peptides. The collagen molecules are then able to
associate into fibrils which are stabilized by intermolecular cross link formation.
The cross linkages acquire greater stability as the healing process continues.
Collagen synthesis is regulated by the co-ordinated actions of a number of
cytokines. TGF-1 mRNA co-expresses with Collagen1 mRNA early in healing
and IL-4 and low concentrations of IL-1 stimulate production of Collagen I by
fibroblasts in vitro. Counter regulation has been demonstrated by high
concentrations of IL-1, Interferon-g and TNF. (Eckes et al,1996).
The other major ECM components are the proteoglycans. These are proteincarbohydrate complexes characterised by their glycosaminoglycan (GAG)
component. GAGs are highly charged sulphated and carboxylated polyanionic
linear polysaccharides. Those most commonly present within the ECM are
Hyaluronan, Chondroitin Sulphate, Dermatan Sulphate, Heparan Sulphate and
Keratan Sulphate.
Hyaluronan (HA), previously known as hyaluronic acid, consists of alternating
glucuronic acid and N-acetylglucosamine units. Each repeating disaccharide unit
has one carboxyl, four hydroxyl and an acetamido group. It differs from the other
major GAGs in that it does not possess any sulphate groups and is not covalently linked to proteins to form proteoglycans although it can non-covalently
bind proteins and cell surface receptors such as the CD44 molecule. It is
synthesised by fibroblasts and is a major component of wound ECM being highly
hydrated and conferring viscosity to tissues and fluids.
HA is a component of normal skin and is present throughout the entire healing
process. It has the potential to modulate cell function by its physicochemical
properties, by acting as a hygroscopic osmotic buffer, its viscoelasticity, its
chemical properties such as free radical scavenging, anti-oxidant effects and
ability to exclude enzymes from the local cellular environment. Additionally it may
interact directly with cells via the RHAMM receptor (Receptor for HA Mediated
Motility), the CD44 receptor and the ICAM-1 (Inter-Cellular Adhesion Molecule 1) receptor. Receptor interaction may be via a ligand receptor type interaction
(CD44) or via a receptor blockade (ICAM-1).
HA has the potential to interact in each phase of healing. During the inflammatory
phase of healing wound tissue is rich in HA. It may play multiple roles promoting
inflammation early in healing by enhancing leucocyte infiltration and also
moderating the inflammatory response as healing progresses towards
granulation tissue formation.
Granulation tissue is also rich in HA and here its role includes facilitation of cell
migration (fibroblasts and endothelial cells), into the provisional matrix, Although
HA has not been demonstrated to be mitogenic its oligosaccharide derivatives do
stimulate endothelial cell proliferation (West & Kumar, 1989).
HA is present in high concentrations in the basal layer of the epidermis in normal
skin and co-localises with the CD44 receptor expressed by keratinocytes
migrating over the provisional matrix of wound granulation tissue. Suppression of
CD44 expression and consequential decreased HA binding results in defective
inflammatory responses, decreased skin elasticity and impaired healing.
In contrast to wounds in the adult, foetal wounding is characterised by a lack of
fibrotic scarring. The HA content of foetal wounds remains high for longer periods
than in adult wounds. Whilst it is attractive to confer a causal relationship
between these two observations many other differences exist between the foetal
and adult wounds. For instance the foetal wound is a sterile environment with a
relatively lower capacity for generation of the early inflammatory phase of
healing. However applied HA has been demonstrated to induce scarless healing
of adult tympanic membranes.
RE-MODELLING AND SCARRING
Collagen is constantly being degraded and re-synthesised even in normal intact
skin. Following injury its rate of synthesis increases dramatically along with an
increase in degradation. Collagen synthesis decreases to normal levels by day
21 after wounding. Remodelling of the collagen fibres by degradation and resynthesis allows the wound to gain strength by re-orientation of collagen fibres.
The resulting scar is less cellular than normal skin and never achieves the same
tensile strength as uninjured skin.
Collagen degradation requires specific enzymes known as collagenases. These
are members of the matrix metalloproteinase (MMP) family. MMPs are produced
by a number of cell types including, macrophages, fibroblasts and keratinocytes.
Their production is inducible and regulated by cytokines, growth factors,
hormones and contact with ECM components. Pro-inflammatory cytokines such
as TNF and IL-1 appear to be major inductive factors whilst TGF- inhibits
procollagenase production and induces synthesis of specific inhibitors of MMPs
known as TIMPs (Tissue inhibitors of metalloproteinases).
Remodelling can continue for up to two years after injury as the healed wound
becomes covered with mature tissue. The relative weakness of the scar
compared to normal skin is a consequence of the collagen fibre bundle
orientation and abnormal molecular cross linking. The fibres in normal skin are
relatively randomly ordered whilst in scar tissue more of the fibres run in parallel.
As well as poor cosmesis scar tissue tends to contract abnormally so that normal
function can be lost where there are large areas of scarring such as following
burn wounds.