Inflammation and wound healing

Inflammation

Complex reaction in tissues that consists of responses in blood vessels and leukocytes

• Protective response, allows healing of injured tissues
• Inappropriate trigger/ poorly controlled inflammation -> problematic
• Acute – rapid onset (minutes), short duration (hours/days) -> exudation of fluid + plasma proteins + emigration of neutrophils -> elimination of offenders -> response subsides
• Chronic – may follow acute or be insidious onset, longer duration -> involves lymphocytes and macrophages + proliferation of blood vessels + fibrosis + tissue destruction
• Inflammation terminated when offending agent is eliminated = mediators broken down, leukocytes short life span in tissues + anti-inflammatory agents
• Injured tissue replaced with regeneration of native parenchymal cells or fibrosis (scarring)
• Four cardinal signs – redness, swelling, heat and pain

ACUTE INFLAMMATION

Rapid host reponse to deliver leukocytes + plasma proteins (Ab) to site of infection/ injury .

Stimuli = infection, tissue necrosis (ischaemia/ necrosis/ physical or chemical injury), foreign bodies, immune reactions (hypersensitivity)

3 components:

• Alterations in vascular calibre to increase blood flow
  • Vasodilation = increased blood flow (causes heat and redness) induced by histamine + NO
• Structural changes in microvasculature to permit leakage of plasma proteins and leukocytes
  • Increased permeability = loss of fluid + increased vessel diameter -> slower blood flow -> vascular congestion with cells -> leukocytes accumulate on vascular endothelium + adhere due to increased levels of adhesion molecules on vessel wall
  • Leakage =
    • Contraction of endothelial cells via histamine/ bradykinin/leukotrienes/neuropeptide substance P/others -> increased inter-endothelial spaces. Immediate transient response lasting 15-30 mins, or delayed prolonged leakage after 2-12 hours, lasing hours or days (eg. Late appearing sunburn)
    • Endothelial injury -> necrosis and detachment (eg. Burns)
    • Increased transcytosis via vesico-vascular organelles (increased in numbers via VEGF)
  • Increased lymph flow helps drain oedema fluid
  • Reaction of blood vessels:
    • Exudation = escape of fluid, proteins and blood cells into interstitial tissues/ body cavity
      • Exudate – high protein concentration, high cellular debris = high specific gravity
      • Presence indicates increased permeability of blood vessels
      • Pus is an exudate
    • Transudation = escape of fluid only from osmotic and hydrostatic pressure imbalance
      • Transudate – low protein concentration, little or no cellular debris = low specific gravity
    • Oedema = excess fluid in interstitial tissue, can be trans or exudate

• Emigration of leukocytes + accumulation at site + activation to eliminate offending agent
  • Neutrophils + macrophages = ingest/ kill bacteria, eliminate necrotic tissue, produce growth factors for repair. Can also induce tissue damage and prolong inflammation.
  • Three steps:
    • Recruitment
      • Leukocyte adhesion to endothelium: stasis = more leukocytes in peripheries of vessels (margination) -> adhere and detach repeatedly via selectins (rolling) -> adhere firmly via integrins (adhesion)
        • Selectins: P-selectin on endothelium and platelets, E-selectin on endothelium and L-seletin on leukocytes. Expression regulated by cytokines (TNF, IL-1) and P/E- selectin on endothelium bind to Sialyl-Lewis-X modified proteins on leukocyte. L-selectin on leukocyte binds to GlyCam-1/ CD34 on endothelium. Mediators stimulate redistribution of P-selectin from normal intracellular stores to endothelial cell granules (Weibel- Palade bodies).
        • Integrins: CD11/CD18 (beta 2/ LFA-1/Mac-1) bind to ICAM1 or VLA-4 (beta 1) bind to VCAM1. Chemokines increase binding avidity of integrins on leukocytes by changing them to high affinity state. TNF/IL-1 increase expression of ligands.
      • Migration across endothelium to vessel wall (diapedesis) = post capillary venules, chemokines act on adherent leukocytes -> stimulate migration via PECAM-1 (platelet endothelial cell adhesion molecule) or CD31 over endothelium -> traverse basement membrane via secreting collagenases -> migration to site. Bind to ECM via integrins and CD44 binding to matrix proteins = retained at site needed.
        • Leukocyte adhesion deficiency I: defect in beta2 chain shared by LFA-1 and Mac-1 integrins.
        • Leukocyte adhesion deficiency II: lack of Sialyl-Lewis-X
      • Migration to tissues toward chemotactic stimulus = locomotion oriented along chemical gradient. Chemoattractants – bacterial products (exogenous) + cytokines, complement, arachidonic acid metabolites (endogenous) -> bind to G protein-coupled receptors on leukocytes -> second messengers increase cytosolic calcium + activate guanosine triphosphates + kinases -> polymerization of actin at leading edge of cell + myosin filaments at back = filopodia pulls cell toward inflammatory stimulus.
        • Neutrophils predominate infiltrate 6-24 hours then replaced by monocytes 24-48 hours ß N are more numerous, respond rapidly to chemokines, attach more firmly to adhesion molecules.
        • Exception to above: Pseudomonas – N recruited for several days. Viral – lymphocytes first to arrive. Hypersensitivity – eosinophils.
    • Recognition of microbes/ necrotic tissue
      • Recognition via receptors = activation of leukocytes
        • Toll-like receptors (TLR) -> bacterial lipopolysaccharides/ proteoglycans/ lipids/ unmethylated CpG nucleotides + viral dsRNA. Present on cell surface and in endosomal vesicles – can detect extra cellular and ingested microbes.
        • G protein- couple receptors (neutrophils and macrophages) -> bacterial proteins containing N-formylmethionyl residues + chemokines/C5a/ platelet activating factor/ leukotrienes
        • Receptors for opsonin -> leukocytes express R for proteins as part of opsonization = coating microbe to target for phagocytosis. Opsonins = antibodies (IgG – Fcg receptor), complement proteins (fragment C3 – type I complement R), lectins.
        • Receptors for cytokines -> interferon g secreted by NK cells/ T lymphocytes – activates macrophages.
    • Removal of offending agent
      • Phagocytosis: recognition and attachment of particle (via mannose/ scavenger/ opsonin R) -> engulfment (phagocyte membrane zips up around microbe – fusion of phagosome with lysosome – phagocytic vacuole) -> killing/ degradation of material
        • Efficiency enhanced by opsonisation
      • Destruction via:
        • ROS + NO = phagocyte oxidase activated -> reactive oxygen and nitrogen species via respiratory burst + enzyme myeloperoxidase (MPO) from azurophilic granules in neutrophils -> ROS damage lipids/ proteins/ nucleic acid + MPO causes halogenation (binding halide to cellular constituents) -> microbial death
        • Action of substances in leukocyte granules: Lysosomal enzymes (elastase, lysozyme) + defensins + cathelicidins + lactoferrin -> killing within phagolysosome
    • Other functional responses
      • Macrophages – growth factors -> stimulate proliferation of endothelial cells, fibroblasts and synthesis of collagen -> repair

Leukocyte mediated tissue injury:

• Part of normal defense reaction, surrounding tissues suffer ‘collateral damage’ (septic shock)
• Inappropriate inflammatory response against host tissues (ARDS, autoimmune)
• Host reacts excessively to usually harmless environmental substances (asthma)

Defects in Leukocyte function

• Genetic = leukocyte adhesion deficiency, chronic granulomatous disease (X-linked or autosomal recessive -> decreased oxidative burst), MPO deficiency, Chediak- Higashi Syndrome (mutations in lysosomal membrane)
• Acquired = bone marrow suppression, radiation/chemotherapy, diabetes, sepsis

Termination of Acute Inflammation

• Neutrophils have short half lives and undergo apoptosis within few hours
• Mediators are short lived

Mediators of Inflammation: generated from cells/ plasma proteins, produced in response to various stimuli, various cellular targets:

Arachidonic Acid metabolite pathway

Coagulation and Kinin Systems:

• Activated Hageman factor (XIIa) -> 4 systems
  • Kinin = vasoactive kinins
  • Clotting = thombin (inflammatory properties)
  • Fibrinolytic = degrades fibrin  to produce fibrinopeptides (induce inflammation)
  • Complement = anaphylatoxins + mediators
• Kallikrein feeds back and stimulates Hageman factor = amplification of reaction.

Outcomes of acute inflammation:

• Complete resolution
• Healing by fibrosis
• Chronic inflammation

Morphologic patterns of acute inflammation:

= Dilation of small vessels, slowing of blood flow + accumulation of leukocytes and fluid in extravascular tissue.

• Serous = outpouring of thin fluid from plasma or secretions of mesothelial cells of body cavities. Accumulation of this fluid is called effusion. Eg. Skin blister.
• Fibrinous = deposition of fibrin in extracellular space -> scarring if not removed. Eg. Fibrinous pericarditis.
• Suppurative or Purulent; Abscess = large amounts of pus consisting of neutrophils, liquefactive necrosis and oedema fluid. Abscess is a localised collection of purulent inflammatory tissue. Eg. Acute appendicitis.
• Ulcers = local excavation produced by sloughing of inflamed necrotic tissue. Eg. Peptic ulcers

Chronic Inflammation

Prolonged duration (weeks or months) in which inflammation, tissue injury and attempts at repair co-exist.

Causes – persistent infections (mycobacteria, fungi), immune mediated (RA, MS) or prolonged exposure to toxic agents (silicosis).

Morphologic features – infiltration with mononuclear cells (macrophages, lymphocytes, plasma cells) + tissue destruction + healing by fibrosis/ angiogenesis.

Role of Macrophages:

• Mononuclear phagocyte system (reticuloendothelial system)
• Arise from bone marrow -> monoblast -> monocyte in blood -> macrophage in tissues
• Activated by microbial products, cytokines/chemical mediators = increased levels of lysosomal enzyme/ ROS/ NO + increased mediators
• Assist in tissue destruction
• Short lived inflammation – macrophages die or drained by lymph, chronic inflammation – macrophages persist due to ongoing recruitment and proliferation

Other cells:

• Lymphocytes: T cells produce cytokines and IFN-g -> activates macrophages
• Plasma cells: develop from activated B cells, produce Ab
• Eosinophils: abundant in immune reactions mediated by IgE/ parasitic infections -> eotaxin recruits -> granules contain major basic protein -> toxic to parasites, lysis of epithelial cells
• Mast cells: express R binds Fc portion of IgE Ab -> release histamine and PG (anaphylactic shock)

Granulomatous inflammation = distinctive pattern of chronic inflammation (TB, syphilis)

• Granuloma –aggregation of macrophages transformed into epithelium-like cells, surrounded by mononuclear leukocytes.

Systemic effects of inflammation

“Acute phase response” =

• Fever is a response to pyrogens (exogenous via bacteria, endogenous TNF/IL-1) -> stimulate PG synthesis in vascular/ peri-vascular cells of hypothalamus -> elevated temperature
• Acute phase proteins (CRP, fibrinogen, amyloid, hepcidin) synthesis upregulated by cytokines in liver
• Leukocytosis = leukemoid reaction/ left shift, accelerated release of cells from bone marrow
• Septic shock

Tissue Regeneration and Repair

Normal Tissues:

• Regeneration = proliferation of cells and tissues to replace lost structures
• Repair = fibroproliferative response, “patches up” tissue

Usually mixture of both in healing process – depends on proliferative capacity of cells, integrity of ECM and resolution/ chronicity of inflammation.

• Fibrosis = extensive collagen deposition in chronic inflammation

Regeneration:

• Size of cell population depends on rate of proliferation, differentiation and death
• Proliferative activity:
  • Labile = continuously dividing (derived from stem cells) such as surface epithelia, lining mucosa of salivary glands/ pancreas/ biliary tract, columnar epithelium of intestine/ uterus, bone marrow
  • Quiescent = low level of replication such as parenchymal cells of liver/kidneys/pancreas and mesenchymal cells such as fibroblasts/ smooth muscle, vascular endothelium, lymphocytes/ leukocytes (eg. Liver regeneration after partial resection)
  • Non dividing = cannot divide further, such as skeletal/cardiac muscle cells and neurons
• Stem cells = capacity to generate differentiated cell lineages, maintained via two mechanisms:
  • Obligatory asymmetric replication (daughter cell retains self renewing capacity while other enters differentiation pathway)
  • Stochastic differentiation (stem cell population maintained by balance in stem cell divisions) -> two self renewing stem cells OR two cells that will differentiate
  • Types:
    • Embryonic stem cells (ES) = inner cell mass of blastocyst are pluripotent stem cells, can differentiate into any cell lineage. Focus of research to repopulate damaged organs
    • Somatic (adult) stem cells = multipotent stem cells which continuously divide
      • Bone marrow – haemopoetic stem cells (HSC) -> all blood cell lineages, and multipotent stromal cells (MSC) -> chondrocytes, osteoblasts, adipocytes, myoblasts or endothelial cell precursors depending on which tissue they migrate to
      • Liver – progenitor cells in canals of Hering -> oval cells -> hepatocytes OR biliary cells
      • Brain – neural stem cells (NSC) -> neurons, astrocytes, oligodendrocytes in subventricular zone and dentate gyrus of hippocampus
      • Skin – stem cells at hair follicle bulge, interfollicular areas of surface epidermis and  sebaceous glands
      • Intestinal epithelium
      • Skeletal muscle – myocytes do not divide, growth occurs by replication of satellite cells beneath basal lamina
      • Cornea – limbal stem cells (LSC)
    • Induced pluripotent stem cells = differentiated adult tissues reprogrammed to become pluripotent by transferring nucleus into enucleated oocyte (reproductive cloning – Dolly the sheep 1997)

Repair by healing, scar formation and fibrosis

• Severe/ chronic tissue injury -> damaged parenchymal cells and stromal framework -> healing cannot be accomplished by regeneration alone
• Repair = deposition of collagen + ECM required -> scar formation
• Features: inflammation, angiogenesis, migration and proliferation of fibroblasts, scar formation and connective tissue remodelling
• Angiogenesis: blood vessel formation via branching of existing network or recruitment of endothelial progenitor cells (EPC) from marrow
  • Steps – Pre-existing vessel:
    • Vasodilation by NO + VEGF induced increased permeability of vessel
    • Proteolytic degradation of basement membrane of parent vessel by matrix metalloproteinases (MMPs) + disruption of endothelial cell-to-cell contact via plasminogen activator
    • Migration of endothelial cells towards angiogenic stimulus
    • Proliferation of endothelial cells
    • Maturation of endothelial cells + remodelling into capillary tubes
    • Recruitment of peri-endothelial cells to form mature vessel
  • Growth factors and receptors:
    • VEGF – secreted by mesenchymal and stromal cells, induced by hypoxia/ TGF b/ TGF a/ PDGF -> binds to VEGF receptors and promotes all steps of angiogenesis
    • ECM proteins (angiopoietins 1 and 2), PDGF and TGF b help stabilise new vessels

Cutaneous wound Healing = 3 phases

• Inflammation:
  • Injury -> platelet adhesion and aggregation -> clot at wound surface
  • Clot stops bleeding, scaffold for migrating cells which are attracted via release of growth factors, cytokines and chemokines
  • VEGF = increased vessel permeability and oedema underlying wound
  • Dehydration at external clot surface = scab
  • <24 hours, neutrophils release proteolytic enzymes to clear out debris and bacteria
• Proliferation:
  • Formation of granulation tissue -> proliferation and maturation of connective tissue cells -> re-epithelialisation of wound surface
  • 24-72 hrs, fibroblasts and vascular endothelial cells proliferate = granulation tissue – histologic features: presence of angiogenesis and proliferation of fibroblasts
  • By 5-7 days, granulation tissue fills area and neovascularisation is maximal
  • Neutrophils replaces by macrophages 48-96 hrs -> release chemokines (TGF/TNF/ PDGF/ IL-1/ EGF) which promote migration of fibroblasts, increased collagen synthesis, decreased degradation of ECM
    • TGF b is most important fibrinogenic agent
• Maturation
  • ECM deposition, tissue remodelling and wound contraction
  • Collagen fibers present at margins of excision -> abundant, bridge excision
  • Epithelial cells move from wound edge to margins of dermis, depositing basement membrane components -> continuous epithelial layer formed, fuse in midline (slower in secondary intention) -> resumes normal thickness and surface keratinization
  • Scar formation (week 2) = original granulation tissue converted into pale, avascular scar composed of spindle shaped fibroblasts + collagen + ECM components
  • Wound contraction = myofibroblast network, reduced surface area, brings dermal edge closer together -> important in secondary intention
  • Connective tissue remodelling = replacement of granulation tissue by changes in ECM, degradation via MMPs
  • Recovery of tensile strength = increased fiber size and cross linking of fibrillar collagen (type I), takes ~ 2 months as balance between synthesis and degradation is achieved

HEALING BY PRIMARY INTENTION = limited epithelial and connective tissue cell deaths/ minimal disruption of epithelial basement membrane -> re-epithelialisation to close wound occurs with relatively thin scar. Eg. Surgical incision closed with sutures.

HEALING BY SECONDARY INTENTION = extensive loss of cells and tissue -> intense inflammatory reaction -> abundant granulation tissue -> extensive collagen deposition -> substantial scar, generally contracts. Eg. Excisional wounds.

Factors that influence wound healing:

• SYSTEMIC = Nutrition (protein deficiency, lack of vitamin C -> inhibits collagen synthesis), metabolic and circulatory status (microangiopathy associated with delayed healing), hormones (glucocorticoids -> anti-inflammatory, reduced collagen synthesis)
• LOCAL = infection (persistent tissue injury and inflammation), mechanical factors (early motion, separating wound edges), foreign bodies, size/location/wound type

Pathologic aspects of repair:

• Deficient scar formation = wound dehiscence and ulceration
• Excessive scar formation = hypertrophic or keloid scarring
• Formation of contractures = deformities

Fibrosis: Excessive deposition of collagen and other ECM components

• Persistent stimulus, chronic inflammation -> activation of macrophages and lymphocytes à
  • growth factors (PDGF/FGF/ TGF b) = proliferation of fibroblasts, endothelial cells
  • cytokines (TNF/ IL-1,4,13() = increased collagen synthesis
  • decreased metalloproteinase activity = decreased collagen degradation

Last Updated on August 19, 2021 by Andrew Crofton