Chronic inflammation and wound healing1 Cellular components

Chronic inflammation and wound healing1 Cellular components WWAMI lecture Nicole Meissner-Pearson Outcome of acute inflammation • Complete resolution – due to elimination of the offending agent and regeneration of injured tissue with normal function • Healing by connective tissue replacement (fibrosis/scar formation) – Occurs after large tissue destruction, – fibrinous exudation into serous cavities – tissue without regeneration capabilities • Progression to chronic inflammation – Resulting in granuloma formation to wall off injurious agent and tissue fibrosis (scar formation) Definition of chronic inflammation an inflammatory response of prolonged duration (weeks – months - years) provoked by the persistence of the causative stimulus simultaneous presence of acute inflammation, tissue destruction and repair Causes of chronic inflammation • Infectious organisms that resist clearance and form a persistent infection in tissue or undrained abscess cavities – e.g mycobacterium tuberculosis, actinomycetes, treponema palidum and Staph aureus (in bone and pleural cavities) • Exposure to irritant non-living foreign material that can not be removed – implanted materials into wounds (wood splinters), inhaled materials (silica, asbestos), deliberately introduced material (surgical suture material or prosthesis) • Potentially normal tissue components as seen in auto-immune diseases – Beta islet cell in diabetes mellitus type I, Acetyl cholin receptor in Myastenia gravis Characteristics of chronic inflammation • Infiltration of mononuclear cells • Tissue destruction • Healing with scar formation and fibrosis The dominant cellular player in chronic inflammation is the tissue macrophage Blood monocyte migrate into tissue within 48 hours after injury and differentiate Tissue macrophage (RES) Kupffer cell (liver) Microglia (CNS) Histiocytes (spleen) Alveolar macs (lung) It is joined by lymphocytes and plasma cells, however mast cells and eosinophils are as well involved in chronic allergic diseases Lymphocyte Plasma cell In chronic inflammation macrophage accumulation persists by different mechanisms • Continued recruitment of monocytes from the circulation • Local proliferation • Prolonged survival and immobilization During chronic inflammation macrophages serve to eliminate injurious agents and initiate repair- however, they are as well responsible for much of the tissue injury that occurs Tissue macrophage Activated T cell or NK cell Non Immune activation: Endotoxins, fibronectin, chemical mediators IFN-g Activated macrophage Fibrosis (Scaring) Tissue injury Toxic oxygen metabolites Metallo-proteases Coagulation factors AA metabolites and NO Growth factors involved in fibroblast proliferation (PDGF,TGFb,FGF) Angiogenesis factors (FGF,VEGF) Collagen deposition (IL-13 and TGFb) Outcome of chronic inflammation • Ulcers • Fistulas • Granulomatous diseases • Fibrotic diseases (Scaring) • and combinations of the above Examples of severe chronic inflammatory diseases • Tuberculosis • Sarcoidosis • Rheumatoid arthritis and other connective tissue diseases • Inflammatory bowl diseases (Crohns disease, ulcerative colitis) • Silicosis and other pneumoconioses • Peptic ulcer of the duodenum and stomach • Liver cirrhosis • Bronchial asthma Mechanisms of regeneration, wound healing and repair Repair of tissue damage can be broadly separated into two processes • Regeneration – Restitution of lost tissue • Tissue with high proliferative capacity = labile tissue (e.g hematopoietic cells, epithelial cells of skin and gastrointestinal tract regenerate from stem cells) • Quiescent tissues = stable tissue which normally have low levels of replication, however can undergo rapid cell division when stimulated (e.g parenchymal cells of liver, kidney, pancreas; mesenchymal cells as lymphocytes, fibroblasts, smooth muscle cells, endothelial cells…) • Healing – may restore original structures but results in collagen deposition and scar formation • in tissue where scaffold is disrupted • or damage occurs in non dividing = permanent tissue (e.g central nervous system, skeletal and cardiac muscle) Regeneration requires • Presence of stem cells for renewal • or tissue cells that are capable to divide in response to growth factors Intact tissue scaffold • Most of the processes that are referred to as “regeneration “ in mammalian organs are actually compensatory growth processes that involve cell hypertrophy and hyperplasia (e.g liver regeneration) Stem cells • They are undifferentiated cells that do not yet have a specific function. • They can replicate for a long period of time and give rise to differentiated cells. • In every cell division one cell retains its self renewing capacity while the other cell can undergo differentiation (“asymmetric replication”) Two types of stem cells • embryonic stem cells – derived from the inner cell mass of a blastocyst from in vitro fertilized eggs – are pluripotent and can generate all tissues • adult (somatic) stem cells – they are present in small numbers in various tissues of the adult body – are typically programmed to form different cell types of their own tissue and are therefore multipotent – in tissues with high turn over (hematopoietc system, epithelial lining of the gut and skin) they are instrumental in renewal – although present in a variety of permanent non-dividing tissues they are not very active Bone marrow contains two different types of adult stem cells: the hematopoietic stem cell and the bone marrow stromal cell Potential plasticity of hematopoietic stem cells Hematopoietic stem cell transplantation: an established treatment option for hematological disorders and cancers • Hematopoietic stem cells can be retrieved from the peripheral blood or the bone marrow and identified by the expression of the CD34 marker • 2X106 HSC/KG body weight of recipient are needed for a successful autologous HSC transplantation Under steady state conditions the number of CD34+ cells in peripheral blood is 1-5/mm3 Mobilization procedures of CD34+ stem cells into the peripheral blood can be accomplished by administration of G-CSF or GM-CSF to the donor and can increase the HSC count in the peripheral blood 50 fold • • Repair by Healing (Scarring) • Healing is a fibro-proliferative responses that “patches” rather than restores tissue and involves the following processes – Induction of an inflammatory response to remove dead and damaged tissue – Proliferation of parenchymal and connective tissue cells – Angiogenesis (blood vessel formation) and formation of granulation tissue – Synthesis of ECM proteins and collagen deposition – Tissue remodeling – Wound contraction – Acquisition of wound strength It usually leads to scar formation and does not lead to complete restitution of the injured tissue • Angiogenesis = growth of new blood vessels • Angiogenesis occurs in the healthy body for healing wounds and for restoring blood flow after tissue injury • Healthy angiogenesis is tightly controlled by a serious of “on” and “off switches (Angiogenic growth factors versus angiogenesis inhibitors) • In many serious diseases the body loses control over angiogenesis and angiogenesis-related diseases occur when new blood vessels grow excessively or insufficiently Rheumatoid arthritis Cancer AIDS complications Blindness Excessive Psoriasis Angiogenesis Stroke Insufficient Infertility Heart disease Scleroderma Ulcers Angiogenesis / Neovascularization is critical to chronic inflammation and fibrosis, tumor growth and vascularization of ischemic tissue Sprouting VEGF and Angiopoietins are the most important angiogenic factors Anti-VEGF Fab-fragment treatment is used in tumor therapy as well as wet macular degeneration Normal macular Wet macular degeneration Macular degeneration is a group of diseases characterized by the breakdown of the macular leading to the loss of central vision. It is the leading cause of vision loss in patients over the age of 55 years Wet macular degeneration is rapidly progressive and accounts for 10% of cases with age related AMD. Is the result of abnormal vessel growth beneath the macula resulting in bleeding. The first currently approved anti-VEGF drug for vitrial injection is MACUGEN R (pegaptanib) Role of extracellular matrix in wound healing and scar formation • Extracellular matrix (ECM) is formed by specific secreted macromolecules that form a network on which cells grow and migrate along ECM is secreted locally and forms a significant proportion of the tissue volume ECM sequesters – water that provides turgor to soft tissues – and minerals that provides rigidity to skeletal muscles – Forms a reservoir for growth factors • • • ECM proteins assemble into two general organizations – Interstitial matrix (present between cells) – Basement membrane [BM] (produced by epithelial and mesenchymal cells and is closely associated with the cell surface) Three groups of macromolecules constitute the ECM • Fibrous structural proteins – Collagen – Fibrillins • Adhesive glycoproteins – – – – Cadherin Integrins Immunoglobulin family Selectins • Proteoglycans and Hyaluronic Acid • Fibrous structural proteins – Collagens • Collagens are the most abundant proteins • 27 different types • Type I,II, III, V and XI are the most abundant (interstitial or fibrillar collagens) • Provide tensile strength of tissue • Fibrillar collagen requires hydroxylation of proline and lysine in procollagen which is dependent on Vitamin C • Type IV is the main component of Basemant membrane and forms sheets) – Elastins and Fibrillins • • • • Provide tissue with the ability to recoil Elastins are found in large vessels, uterus, skin and ligaments Fibrillins form a scaffolding for the deposition of elastins Marfan syndrome is an inherited autosomal dominant defect in fibrillin synthesis. Without the structural support provided by fibrillin, many tissues are weakened, which can have severe consequences, for example, ruptures in the walls of major arteries. • Proteoglycans and hyaluronic acid – Proteoglycans (mucoproteins) are formed of glucosaminoglycans (GAGs) covalently attached to core proteins and are highly negatively charged • Biophysical functions due to ability to fill space, bind and organize water molecules and repel negatively charges molecules – They are ideal lubricating fluids in the joint due to high viscosity and low compressibility • Biochemical functions are mediated by specific binding of GAGs to other macromolecules – e.g Antithrombin III (AT III) binds tightly to heparin and heparan sulfates and inactivates factor II, IXa and XIa thus controlling blood coagulation – Proteoglycans (such as Syndecan) act as reservoirs for growth factors secreted into the ECM by binding the latter. Chronic inflammation 2 Cutaneous wound healing Granulomatous inflammation Nicole Meissner-Pearson Cutaneous wound healing is generally divided into three overlapping phases • Inflammation • Granulation tissue formation and reepithelialization • Wound contraction, extracellular matrix deposition and remodeling The phases of cutaneous wound healing Injury leads to accumulation of platelets and coagulation factors. Coagulation results in fibrin formation and release of PDGF and TGF-b and other inflammatory mediators by activated platelets. This leads to more Neutrophil recruitment which signals the beginning of inflammation (24 h). After 48 h macrophages replace neutrophils. Neutrophils and macrophages are responsible for removal of cellular debris and release growth factors to reorganize the cellular matrix. At 72 hours the proliferation phase begins as recruited fibroblasts stimulated by FGF and TFG-b begin to synthesize collagen. Previously formed fibrin forms initial matrix for fibroblasts Collagen cross-linking and reorganization occurs following months after injury in the remodeling phase of repair. Wound contraction follows in large surface wounds and is facilitated by actin-containing fibroblasts (myofibroblasts) TGF-b functions as a central regulator of tissue repair and negatively regulates both acquired and adaptive immunity Lack of the TGFb1 gene in mice results in excessive tissue inflammation and autoimmunity resulting in death of the animals, however increased activity leads to excessive scar formation and loss of organ function Skin wounds are classically described to heal by either primary or secondary intention and the distinction is made by the nature and extent of the wound Healing by first intention: • wounds with clean opposing edges (surgical incision, should form a narrow scar due to small amount of granulation tissue required to fill the gap) Healing by second intention: • wounds with separated edges (trauma that requires abundance of granulation tissue for wound closure) – Granulation tissue consists of newly formed blood vessels, macrophages, fibroblasts and loose ECM framework – As collagen accumulation increases, the granulation tissue scaffolding is converted into a mature scar composed of mature spindle-shaped fibroblasts, dense collagen and elastic fibers. – The mature scar does not contain vessels Complications of wound healing • Deficient scar formation – Wound dehiscence – Ulceration • Excessive formation of scar tissue – Keloid (excessive collagen deposition) – Exuberant granulation (proliferation of fibroblasts that inhibits re-epithelialization) – Desmoid (aggressive fibromatosis, semi-malignant) • Contraction Wound dehiscence Wound ulceration Keloid Contracture Factors that influence wound healing Systemic factors • Malnutrition – Protein deficiency – Vitamin C deficiency (inhibition of collagen synthesis) • Metabolic status – e.g Diabetes mellitus • Consequence of microangiopathy – Cortison treatment • inhibits inflammation and collagen synthesis • Circulatory status – Inadequate blood supply due to ateriosclerosis – Varicose veins (retarded venous drainage) Factors that influence wound healing (continue) Local Factors • Infection (single most important reason for delayed wound healing) • Foreign bodies – suture material, bone and wood splinters …. • Mechanical factors – Early movement – Pressure Granulomatous inflammation Granulomas are millimeter size nodules of chronic inflammatory cells that can be isolated or confluent. Granuloma formation is the result of dealing with indigestible substances or pathogens and walls them off The essential component are modified macrophages named epithelioid cell (because of shape). Epithelioid cells can form multinucleated giant cells. Epithelioid cells are surrounded by a collar of lymphocytes and occasionally plasma cells. Fibrous connective tissue often surrounds granulomas (remodeling of tissue) Areas within the granuloma can undergo necrosis (prototype: caseous necrosis in tuberculosis). Necrosis can lead to calcification or liquefaction and formation of a cavern if drained. Examples of granulomatous inflammation Tuberculosis • Specific infections (immune granuloma): Mycobacteria (tuberculosis, lepprosy…) syphilis, brucellosis, … Foreign bodies: – endogenous ( keratin, necrotic bone or adipose tissue uric acid crystals) – Exogenous (wood, silica, asbestos, silicone…) Specific chemicals: – Beryllium Drugs – Allupurinol, phenylbutazone, sulphonamides (in liver) Unknown origin – Sarcoidosis (although granuloma typically form to defend the host against known injurious agents, they can develop for unknown reasons and become injurious themselves) – Hypersensitivity pneumonitis • Foreign body aspiration • • Berrylliosis • Granuloma: a hallmark of tuberulosis • • • Infectious bacilli are inhaled by droplets Infectious dose is estimated by a single bacterium Bacteria are phagocytosed by alveolar macrophages A localized inflammatory response recruits more mononuclear cells The granuloma consists of a kernel of infected macrophages surrounded by foamy macrophages and a ring of lymphocytes and a fibrous cuff (containment phase) Containment usually fails when the immune status of the patient changes; the granuloma caseates, ruptures and spills into the airway • • • Microscopic and macroscopic appearance of tuberculosis Characteristic tubercle with caseating necrosis in center Cavity formation due to liquefaction and drainage of TBC lesion Macroscopic lesion in TBC Pulmonary Granulomatous inflammation: Tuberculosis, Sarcoidosis, hypersensitivity Pneumonitits Normal lung Characteristic tubercle with caseating necrosis in center Characteristic sarcoid noncaseating granuloma of the lung with many giant cells Hypersensitivity Pneumonitits with loosely formed interstitial granuloma Sarcoidosis versus Hypersensitivity Pneumonitits • Sarcoidosis – is a systemic disease of unknown origin characterized by non-caesating granulomas in many tissues – 90 % of cases have primarily pulmonary manifestations – Histological diagnosis is made by exclusion – Prevalence is higher in women – Is 10 X higher in American blacks versus whites – Histologically the lesions are distributed primarily along lymphatics, around blood vessels and – The process is driven by CD4-T cells – Intra-aveolar and interstitial accumulation of CD4-T cells results in CD4:CD8 ratios of 5:1 and 15:1 • Hypersensitivity Pneumonitis (HP) – also called extrinsic allergic alveolits – HP is caused by sensitization to repeated inhalation of dusts containing organic antigens – The dust can be derived from a variety of sources such as dairy and grain products, animal dander and others – The most common antigen are thermophilic actinomycetes (farmer’s lung) and avian proteins (bird fancier’s disease) – The disease is immunologically mediated – The immune pathogenesis involves macrophages, CD4 T-cells and particularly CD8 T-cells Pathogenesis of immune Granuloma formation: a result of delayed type hypersensitivity reaction Alveolar macrophages appear first to come in contact with the offending agent (e.g are infected by mycobacteria) Activated macrophages produce IL-1, TNF-a, IL-12, IL-6 TGF-b and IL-10 and process the antigen Dendritic cells migrate to the regional lymphnode and activate antigen specific T-cells, which under the influence of IL12 produce IFN-g and further activate recruited macrophages Release of TNF-a and IL-8 leads to recruitment of neutrophils and monocytes from the circulation, IL-15 and RANTES leads to recruitment of T cells. This is followed by the organization of cells into a tight granuloma and development of epithelioid cells. Chronic granulomatous inflammation can result into connective tissue deposition and fibrosis (tissue remodeling) Epithelioid cell Bi-hiliar lymphadenopathy and pulmonary infiltrate in sarcoidosis Severe pulmonary changes with honey comb like structures suggesting fibrosis Mechanisms of fibrosis Nicole Meissner-Pearson Mechanisms of Fibrosis: a result of chronic inflammation and repair • Fibrosis: excessive accumulation of extracellular-matrix components such as collagen that is produced by local fibroblasts leading to a permanent fibrotic scar Macrophages and fibroblasts are the main effector cells involved in the pathogenesis of fibrosis • • • Pro-fibrotic mediators such as TGFb and IL-13 amplify this process The degradation of collagen is controlled by Matrix-Metalloproteinases (MMPs) and are activated by IFN-g Therefore the net increase of collagen within a wound is controlled by the balance of these opposing mechanisms Although severe acute injuries can cause marked tissue remodeling. Fibrosis that is associated with chronic injury (repetitive) is unique in that the adaptive immune response is thought to have an important role • • TH-2 cytokines IL-4 and IL-13 lead to “alternative” activation of macrophages • Macrophages differentiate into at least two functionally distinct populations depending on whether they are exposed to TH-1 or TH-2 cytokines TH-1 cytokine activate NOS2 in classically activated macrophages whereas TH-2 cytokines IL-4 and IL-13 preferentially stimulate Arginase-1 (ARG1) leading to an alternative activation pathway ARG1 promotes the generation of polyamines and L-proline via metabolism of L-arginine to L-ornithine and activation of ODC and OAT Polyamines are crucial for cell growth and L-proline is a substrate for collagen synthesis • • • A balance between TH-2 and TH-1 cytokines is necessary to promote healing but inhibit excessive fibrotic tissue remodeling Therapeutics that modulate this balance may be beneficial in patients suffering from fibrotic diseases. Drugs that directly inhibit TGF-b1 and IL13 might prove the safest and most effective approach Fibrotic tissue remodeling can result in loss of organ function • Fibrotic changes can occur in various vascular diseases including – Cardiac diseases – Peripheral vascular diseases • They affect as well main organ systems like – – – – Skin Lung Liver Kidney Tight skin and skin necrosis due to Sclerodermia Lung fibrosis due to sarcoidosis Liver cirrhosis Liver cirrhosis macroscopic and microscopic normal liver histology Liver cirrhosis • Cirrhosis represents the common histological pathway for a wide variety of liver diseases • It is defined histologically as a diffuse hepatic process characterized by the presence nodular proliferation of hepatocytes surrounded by bands of fibrosis Most common causes of liver cirrhosis in the USA (updated Nov. 2005; Davis C. Wolf, New York Medical College) • Hepatitis C (26%) • Alcoholic liver disease (21%) • Hepatitis C plus alcoholic liver disease (15%) • Cryptogenic causes (18%) • Hepatitis B (with or without Hepatitis D, 15%) • Miscellaneous(5%) Clinical consequences and signs of liver disease • Characteristic signs – – – – – – – – – Jaundice and cholestasis Hypoalbuminemia Hyperammonemia Hypoglycemia Palmar erythema Spider angiomas Hypogonadism Gynecomastia Muscle wasting • Portal hypertension due to fibrosis – – – – Ascites Splenomegaly Hemorrhoids Caput medusa Jaundice (note yellow sclera and skin) Spider angioma Palmar erythema Laboratory evaluation of liver diseases • Disruption of Hepatocyte integrity » Elevated serum aspartate amino transferase (AST = sGOT) » Elevated serum alanine amino transferase (ALT= sGPT) • Biliary excretory function » Elevated total bilirubin (unconjugated and conjugated) direct bilirubin = conjugated indirect bilirubin = unconjugate Plasma membrane enzymes » Elevated serum alkaline phosphatase (ALP) » Elevated serum g-glutamyl transpetidase (gGT) • Hepatocyte function » Decreased serum albumin » Hypoglycemia » Decreased clotting time (early decrease of PT due to short half live of factor VII, cholestasis results in decreased uptake of vitamin K) » Elevated serum ammonia Bilirubin metabolism •Bilirubin = end product of heme degradation • Majority is derived from the break down of senescent erythrocytes •Heme is oxygenized to biliverdin and reduced to bilirubin by phagocytes •Bilirubin is virtually water insoluble and needs to be bound to albumin to be transported to the liver •Upon uptake into the liver bilirubin will be conjugated with one or two molecules of glucuronic acid by bilirubin UDP-glucuronyl transferase in the endoplasmatic reticulum •Conjugated bilirubin is non toxic, watersoluble and is excreted into bile •Most bilirubin glucuronides are deconjugated in the intestines to urobilinogen and 20% is reabsorbed and promptly excreted via bile Cholestasis and jaundice • Hepatic bile formation serves two major functions: – Emulsification of dietary fat (bile acids are strong detergents) – Elimination of systemic waste products (bilirubin, excess cholesterol and other water insoluble products) Jaundice is the result of tissue accumulation of unconjugated or conjugated bilirubin in tissues. It becomes evident when plasma levels exceed 2mg/dl. Clearance of bilirubin can be disturbed by – Excessive production (e.g. hemolysis) – Reduced hepatocyte uptake (e.g. drugs, hepatocellular disease) – Impaired conjugation (enzyme defect or overwhelmed system) – Cholestasis Clinically, Cholestasis is any condition in which substances normally excreted into bile are retained : – Increased serum concentrations of conjugated bilirubin is the principal sign of cholestasis – Conjugated bilirubin is water soluble and is secreted in urin (dark urine) – Complete cholestasis leads to decoloration of feces Cholestasis may present with jaundice and pruritus due to deposition of bilirubin and bile acids in peripheral tissues • • • Histopathologically Cholestasis is defined by the appearance of bile within the elements of the liver • Mechanisms of Cholestasis are broadly classified into: – Hepatocellular (impaired bile formation, histological appearance of bile within hepatocytes = feathery degeneration, and canalicular spaces leading to hepatocyte injury) – Obstructive (impairment of bile flow usually due to physical obstruction of bile duct [extraheaptic stone/tumor]; bile plugging of the interlobular bile ducts, portal expansion and bile duct proliferation) Unrelieved obstruction leads to portal tract fibrosis and ultimately end-stage bile stained cirrhotic liver disease Role of Kupffer cells and Stellate cells in induction of cirrhosis Portal hypertension: increased resistance to portal vein blood flow • Causes – Pre-Hepatic • Obstructive thrombosis of portal vein – Intra-Hepatic • Fibrosis • Schistosomiasis • Granulomatous diseases – Post-Hepatic • Sever right heart failure • Constrictive pericarditis • Hepatic vein out flow obstruction Portal hypertension in cirrhosis results from increased flow resistance at the level of sinusoids and compression of terminal hepatic veins by perivenular scaring The four clinical consequences: Ascites Formation of porto-systemic shunts Congestive splenomegaly Hepatic Encephalopathy Life threatening complications of chronic liver disease and fibrosis • Hepatic failure leading to – Hepatic Encephalopathy (due to ammonemia) – Coagulopathy – Multiorgan failure • Portal hypertension from cirrhosis – Esophageal varices with bleeding – Ascites with spontaneous peritonitis – Hepatocellular carcinoma (very long term)