Inflammation Lecture and CILS Lecturer: Julianne Qualtieri, MD 11/5/13 Lecture; 11/6/13 CILS Learning Objectives:

1. Define acute and chronic inflammations. 2. Contrast acute and chronic inflammations in regard to common causes and morphologic manifestations. Identify acute or chronic inflammation in a microscopic image. 3. Define granulomatous inflammation and list causative agents. Identify granulomatous inflammation in a microscopic image. 4. Describe the vascular events associated with acute inflammation. 5. Describe the sequence of cellular events involved in leukocyte extravasation and phagocytosis. 6. Identify the molecules that are primarily involved in leukocyte rolling, endothelial adhesion, transmigration and interstitial migration. 7. Describe basic pathophysiologic mechanisms involved in chronic inflammation. 8. List important chemical mediators of inflammation. 9. List important actions of chemical mediators. 10. Describe systemic manifestations of inflammation.

Key Words:
Inflammation Acute inflammation Chronic inflammation Granulomatous inflammation Extravasation Phagocytosis Rolling Adhesion Transmigration Chemical mediators

Blood and Lymphatics: Immunology Infrastructure Fundamentals of Cellular Medicine Course November 7th, 2013 10am Julianne Qualtieri, M.D. qualtije@ucmail.uc.edu Vocabulary or key words presented in session:

bone marrow -cytosis eosinophil lymph macrophage

hematopoietic stem cell (HSC) -penia basophil lymph node cortex dendritic cell

differentiation neutrophil lymphocyte lymphoid follicle innate immunity

thymus monocyte plasma cell lymph node medulla adaptive immunity

Objectives: At the end of this session the student will: 1. Diagram the basic life cycle of a hematopoietic stem cell and how this pertains to normal hematopoiesis and the development of stem cell disorders. 2. Describe anatomic structures needed for surveillance of the entire human body pathogens. 3. Describe the major differences and similarities between innate and adaptive immune responses and which types of leukocytes are involved in each. 4. Explain the events necessary to complete an adaptive immune response to a foreign antigen and where each event occurs in the body. 5. Become familiar with complete blood count (CBC) and peripheral blood smear abnormalities and how these can be used to guide patient diagnosis and treatment. against

Assigned Reading: R&C: PBD, 8th Ed., Chapter 2 (pp. 43-77). Reading Assignments: Guyton & Hall Medical Physiology Chapter 33: Macrophage and neutrophil responses during inflammation through Leukopenia pp. 428-431 Chapter 33: Beginning through Lifespan of white blood cells pp. 423-424 Chapter 34: Beginning through Millions of specific types of lymphocytes are stored in the lymphoid tissue pp. 433-435 Robbins and Cotran Pathologic Basis of Disease Chapter 6: Tissues of the immune system pp. 188-190 NOTES:

11/5/13 Lecture Acute/Chronic Inflammation

Types of inflammatory responses Acute inflammation Chronic inflammation Granulomatous inflammation

Why does inflammation occur? Inflammation is the bodys protective response to eliminate pathogens and remove injured tissues remove injured tissues. Injuries from chemical physical and micro trauma. Body has a set way to deal with insult -- given sequence of innate events that occur upon insult to the body (first line defense, reproducible) before a more specific response can be deployed. Coordinated dance between vascular, tissues and recruited cells (immune cells). Also adaptive response (with lymphocytes in the blood, spleen) this often requires the extravascular accumulation of blood/lymphocytes. We want a very protein rich environment in our area of injury want lots of immune cells/blood flow (Involves extravascular accumulation of fluid and leukocytes). Eloquent system. Injury can happen anywhere/anytime. Our body works to be ready at all times to acquire an injury anywhere. These coordinated events involve: Vascular Reactions Local Tissue Reactions Recruitment of Innate and Adaptive Immune cells Balance of inflammation is needed to prevent unwanted responses (below). Excessive response Sepsis body is trying to wall off/fight of bacterial infections, some cytokines cannot be neutralized and get into the circulation causing some of the inflammation events throughout the entire body -- which is bad. Inappropriate response Autoimmune diseases in response to our own antigens Allergic reactions arent noxious to us, but our body determines as noxious. Ineffective response Immunodeficiency states dont have an appropriate response to any trigger Inflammation is easily recognizable clinically. 4 Cardinal sign of inflammation --- Heat, Redness, Swelling & Pain Often come about quite rapidly ACUTE INFLAMMATION: Arises quickly Short duration provided it is an effective response Hyperemia increased blood (accounting for redness/ blood) Neutrophils WBC Causes of acute inflammation: Infection

Foreign bodies -- immune system is military based, looking for non-self (splinter, microorganism, etc) Tissue necrosis -- dead tissue Trauma Physical and chemical agents -- chemical exposure, caustic agents Immune reactions -- inappropriate antibody response (autoimmune, allergic, etc.)

**Chronic inflammation involves a lot of the same events just the timeline is different and a lot of the cellular components are different Many of the mediators between acute and chronic inflammation are similar Vascular Reactions Vasodilation histamine secreted by mast cells in the damaged tissues increased blood flow to the area, lots of proteins, nutrients, cells to fight infection accounts for redness and heat Increased permeability happens in the venules, occurs through a variety of mechanisms: endothelial cells retract creating gaps (endothelial gaps) provide flux of fluid, plasma proteins and cells into the tissue this gets the cells right to the site of the infection/trauma. transcytosis physical defect: Sometimes the trauma just damages the tissue releasing a lot of fluid, also releasing hemorrhage. edema (tumor): This is often the result of the influx of fluids, plasma proteins and cells. Vascular Stasis: Blood is typically whizzing by quickly. There needs to be vascular stasis, so the cell will dilate creating turbulent flow. This will get the leucocytes to jump around and literally bump into the sides of the vessel. This has a similar theory to a chemical reaction, the more they bump into it, the more likely they will be to bind to an endothelial cell. Exudate vs. Transudate Exudate Local cells release cytokines and they tell the endothelial cells to retract allowing cells, proteins and fluid to come out for good. In an inflammatory reaction we usually are dealing with an exudate (fluid that filter from the circulatory system into lesions or areas of inflammation). Transudate something wrong with pressure, protein synthesis oncotic pressure. Fluid is literally squeezing out. This is typically pathologic. Abnormal. Not related to an inflammatory response Not good. Normal situations -- balance. So that we dont swell up. Cellular Reactions Just getting a Leukocyte from the blood into the tissue is quite a project. Cells cant pass the cellular (capillary) epithelium (to keep so form hemorrhaging everywhere). They pass via a controlled process, where we have just the cells we want leaving the lumen while keeping the others (RBCs) within the blood vessels (lumen). Extravasation leaving of the cells from the blood into the tissues.

Stasis is going to increase bumping against the walls Rolling to a stop Selectins: express (on endothelial cells) just at the right time (only in areas of inflammation), because we dont want neutrophils sticking to the surface of every vessel in the body. This would not be good. These are expressed under the influence of cytokines. Neutrophils bind to selectins causing them slowing down and cascade in a process called rolling Adhesion -- tight adherence to the epithelium in preparation for transmigration Integrins: Binding of selectines and activation of neutrophils expressed integrin on the surface of both the neutrophil and the endothelium cell. This tight binding of integrin is what allows the neutrophil to completely stop and anchor in to get ready to squeeze through the endothelial cells. Transmigration -- literally squeezing through the junctions of the epithelium. PCAM-1 (CD31): Allows transmigration, or squeezing through. Margination leukocytes being pushed to the edge of the vessel Any defect in these molecules can cause clinical problems to fight off infections.

Chemotaxis tells the cells Exactly where to go. Helps to cells locate the injury Leukocytes (cells) from the blood are attracted to the site of injury by a chemical gradient of signaling. Chemical gradient triggers signaling which activates the cytoskeleton within the cells and they crawl towards the site of injury, propelled by their cytoskeleton network. Molecules that promote chemotaxis are often foreign molecules such as amino acids unique to bacteria (Formyl-Methionyl)The body immediately says this isnt a part of me and starts creating a chemogradients. Activation activation enzymes are closely coordinated, because we do not want them activated in the wrong place or at the wrong time. Signal the cell to adhere to endothelium, bind to tissues, create its own cytokines to help bring in more cells and to create toxic molecules to actually kill the cell Phagocytosis The whole point of acute inflammation is to bring the immune cells to the site of inflammation so that they can gobble up stuff phagocytosis. Macrophages/Neutrophils are the main gobblers Must recognize there is something to eat Enhance by recognition of strange chemical structures (bacterial cell wall), opsinization or covering of a material with something such as compliment. Phagocytic cells have receptors for compliment. If immunoglobins are bound to a particle they will likely be recognized, because macrophages have a Receptor for the FC portion of immunoglobins. Engulf: We have binding of foreign thing. Need to get inside cytoplasm. If antibody binds compliment, triggers membrane of cell to undergo invagination creating the phagosome. The purpose of this is to bring the micro organism/particle into its own compartment within the cell because we are going to be destroying this micro organism by releasing some really toxic chemicals which we do not want released to other tissues and other organisms.

Eat (Kill/Degrade): largely by oxygen-dependent mechanisms (Reactive oxidative species ~~ bleach, which produce H2O2 that reacts with NO species causing free radical damage) when a phagosome fuses with a lysosome. Most bacteria do not have a way to respond to free radical damage. Bodys have ability to neutralize free radicals. Chronic Inflammatory states like atherosclerosis, Cancers, etc. This happens when our inactivaters (neutralize free radicals) get overwhelmed and we have to much free radical production.

Acute Inflammation Timeline Edema -- rush of fluid (few hours) Neutrophils -- day Monocytes -- slower, but longer lived cell capable of phagocytosis and presenting antigens to activate other portions of the immune system if it is necessary... (couple days)

Typical time Course for Acute Inflammation 0-12 h: vasoactive changes edema 12-24 h: beginning infiltration of neutrophils 24-36 h: peak of neutrophil infiltration 36-96 h: influx of macrophages begin process of repair, granulation tissue, angiogenesis and, in some cases, collagen deposition (patch over holes literally) 96 h-3 wk: resolution or formation of a scar Morphology of Acute Inflammation: Appendicitis: o normal appendix: shiny outer surface and fine vasculature. o inflamed appendix (acute appendicitis): inflammatory stimulus such as -- fecal matter can back up and bacteria can clog it by bacteria proliferating. The local epithelium becomes injured, swollen, red, hemorrhage, white extradative material ( composed of neutraphils,bacteria, etc) etc (from all the blood flow). Acute Meningitis: o Causes inflammation along the meninges. Cerebral Disfunction. Lethal. Can see a lot of little neutrophils little cluster of grapes, ants crawling around multi-lobated nuclei. Lots of neutrophils dying off, thats why the nuclear material is becoming dark and pigmented, in addition to the anucleic debris in there. Acute inflammatory cells in bacteria. We like inflammation because it can help us combat microorganisms, the problem is that the reactive species and harmful enzymes can be released outside of the cell Neutrophils only survive for about a day and than they die off, which is when the toxic species can be released and cause damage to our tissues. Lot of collateral damage (local destruction) involved in acute inflammation. The body tries to avoid this, but it would rather have some local destruction than an infection that overtakes and overwhelms the organism. Option after acute inflammatory response happens:

Resolves Macrophages will eat up dead bacteria, cells will be cleared away through the lymphatics, completely normal tissue. Neutrophils are a little over activated, the enzymes can destroy the vascular in the tissues. Resulting in an abscess. Which is a collection of neutrophils, bacteria, cell debris, which cant really be cleaned up because the main supply route (to supply nutrients, macrophages for fibrious, etc) has been cut off. The Neutrophils Shot their own foot. This will eventually result in healing, but will be more of a scar. Abscess's can be difficult to treat. Often antibiotics are given to stimulate clearance of the micro organism, however antibiotics are transported through the vasculature which has been destroyed -this is difficult. No way to get the antibiotics to the abscess require surgery to clear the dead tissue. Another option is maybe we didnt do such a good job clearing the agent so now it is time to call out the special troops -- chronic inflammation which will increase angiogenesis, bringing more nutrients to the area, more cells to gobble things up, lymphocytes (provide specified killing power). Will typically result in chronic damage/fibrosis. The more Macrophages that come in, the more fibrotic responses are elicited.

CHRONIC INFLAMMATION Prolonged duration Concurrent tissue injury and repair Macrophages, lymphocytes and plasma cells

First line of defense has failed, need to bring in some more powerful weaponry, which is mediated by macrophages, lymphocytes, plasma cells (these more powerful mechanisms have their own kind of collateral damage). This is why we can get tissue injury. Macrophages play roles in both acute and chronic responses Lymphocytes & Plasma cells: are ONLY seen in chronic responses NOT seen in acute responses. Chronic inflammation can start while acute inflammation is still present. (Back-Up) Can see both at the same time not separate entities Common Causes of Chronic Inflammation: Basically didnt get rid of the agent Acute Inflammation Persistent Infections Prolonged Irritation foreign substances Autoimmunity Macrophages are similar to a garbage truck, but smart. Recruit other macrophages and secrete pieces of cells on their surface Macrophages are produced via a feedback loop Antibody B-Lymp

Direct Kill T-Lymph Activaetion of Macrophages results in Tissue injury and Fibrosis. However, to much fibrosis can be bad, harming organs. Tissue injury results by secreting reactive oxidative species. Morphology of Chronic Inflammation: Lots of lymphocytes, plasma cells (which make antibodies to neutralize) and macrophages laying down fibrosis, bands of collage, forming nodules (characteristic of hepatitis). When chronic inflammation occurs in the liver, like in Hep C, the biliary system can become chocked off and inhibit bile production. Chronic Inflammation is present in rheumatoid arthritis resulting in joint deformalities chronic inflammation of the synovial, lymphocytes and plasma cellsr eacting to the bodys own antibodies. Granulomatous Inflammation: Special Form of chronic inflammation Epithelioid Macropahges Macrophage can eat, but not digest organism. Becomes multi-nucliate in their own attempted to wall-off and neutralize the infection. Causes: o TB o Fungal o Asbestos (Non-infectious) Granulomas form walled of infection/foreign particle We probably have old granulomas because we have been exposed to Histoplasma capsulatum fungal in the Ohio River Valley. If we had a weakened Immune system and were unable to do this, this could become very dangerous. Foreign body Granuloma Common to see in pathologic specimens with previous surgeries dissolvable suture Foreign material that is not pathologic whatsoever. Granulona does not equal Granulation Tissue: Granuloma inflammation/Walling Off organisms or Insult. Generally not helpful. Granulation Tissue Healthy, regenerative tissue with a lot of fibroblast laying down scar tissue. This is how the body heals itself. Good. Chemical Mediators: Come from cells/Liver(constantly synthesizing proteins) Good that the liver is constantly creating these, so there is little lag time and control of the process. Want strong mediators to be carefully controlled while we want some of the other ones around all the

Chemical Mediators: A lot of them have checks and balances against one another (most of then can also have both pro- and anti- inflammatory responses) Vasoactive amines --- histamine, vasodilation Plasma proteases --- break down destructive enzymes Arachidonic acid metabolites --- control inflammatory response Platelet-activating --- factor activation of epithelium migration Cytokines --- recruiting cells Chemokines -- Nitric oxide --- vasodilation Lysosomal enzymes -- Reactive oxygen species --- Tissue Damage Fever: Triggered by cytokines (TNF-alpha, IL-1 and IL-6) triggering the hypothalamus to constrict the arterials and conserve heat, raising the body temperature. When the temperature is raised we are capable of synthesizing heat shock proteins this can be done only when we have a fever. Leukocytosis: Elevated WBC, accelerated release from marrow and increased immature neutrophils During acute inflammation we have more proteins around to provide/scavenge nutrients to eliminate tissue damage and enhance the immune response. Acute Phase Proteins: C-reactive protein opsonization 6 to 10 hours response time can be used as a laboratory test to evaluate the whole inflammatory burden in someones body. Fibrinogen coagulation Alpha(1)-antitrypsin protease inhibitor Haptoglobin binds hemoglobin C3 -- complement Ceruloplasmin bind copper

Alpha(2)-macroglobulin protease inhibitor

Systemic Effects of Inflammation:

Laboratory Evaluations of Inflammation: White Blood Cell Count (How many leukocytes have been recruited?) o Normal: 4,500 10,000 /microL o Leukocytosis: 10,000 20,000 /microL o Leukemoid reactions are often confused pith acute leukemia, but there are no bad effects from them o Cell Type in the blood can help us tell what is going on Clinically Neutrophilia Bacterial Infections Burns Tissue Necrosis Eosinophilia Parasitic Infections Allergic Disorders Colleen Vascular Disease Monocytosis (become macrophages) Chronic Infections TB Malaria IBD Lymphocytosis Viral infection Bordetella pertusisi w/ monocytosis in TB

Erythrocyte Sedimentation rate (ESR) o Lots of proteins will interfere with the net negative charge and the RBCs stick together.

o sediment more rapidly than monodispersed RBCs o An increased sed rate is considered non-specific evidence of an inflammatory process C- Reactive Protein (CRP) o Secreted rapidly by the liver in response to inflammation o Elevation persists while inflammation exists Serum Protein electrophoresis o Serum proteins are classified on the basis of their electrophoretic mobility: o albumin 1 2

Clinical Implications of the inflammatory response. FYI: Leuko = White (When WBCs pile up, they look white) 11-7-13 Blood and Lymphatics NOTES: Components of an Effective Immune System: Headquarters Bone marrow Endless source of recruits Bone marrow Bone Marrow: hematopoietic cells (Multipotent, self-renewal, differentiation, special microenvironment, Can multiple without external signal, sometimes allowing it to get out of control --- this can often result in a cancer (block in differentiation, and division goes on and on). Aplastic anemia: STEM cells for RBCs die off.) Erythroid Precursors where red cells come from hanging out on the iron islands Myeloid Precursors: the granulocyte precursor cell in bone marrow

Megakaryocytes: produce platelets sit in sinusoids of bone marrow and poop out platelets. Lymphocytes Lymphocyte Primary Maturation Occurs in the bone marrow Ig gene rearrangement occurs in the bone marrow Assembly of the b-cell receptor in the bone marrow At his point they leave the bone marrow called nave b-cells, they havent met their antigen (epitope) yet. If they dont find their match, they will die. Peripheral lymph node tissues are where they will meet up. -- Only respond to epitopes that are foreign to the body. Granulocytes lymphocytes, etc develop from this. Major components of cellularity of Bone Marrow. Because a neutrophils life span is 24h and we need a ton of them. Granulocyte maturation: Major leukocytes of our blood. Start out as blasts in which the nucleus is the major important organelle high nucleus/cytoplasmic ratio. As the cells mature, DNA condenses and nucleus shrinks. All this maturation occurs in the bone marrow. Only see mature form in the blood. Plasma cells: produce antibodies Monocyte/macrophages Stromal cells less clear that they are directly involved in disease processes, however they do provide support and lay down a nice supportive network often via fibrogen. Fibroblasts/reticulin Adipose -- bone marrow is replaced by fat eventually Osteoclasts/blasts Endothelial cells play important role in release of cells/signaling Transportation Blood and Lymphatics Blood: Less direct Lymphatics: Express Shuttle, Direct connection to lymph nodes, Coordinate Immune Response Have to be careful, because the more transportation you open up, the more chance there is for intruders (microbes tumors cells) to get in. So you have to be careful about this, so that they do not spread all over the place). Lymphatics also provide a drainage route. Lymph fluid will be dumped back into the circulation. Continuous surveillance Macrophages Ingests Digests Presents antigen to regional authorities Lymph nodes for tissues

Langerhans cells resident dendritic cells in the skin will pick up the antigen (if it enters through the skin) and start secreting cytokines. Acute inflammatory cascade will begin cytokine/chemokine release, local epithelial cells will start producing selectin (basically waving a flag to any passing neutrophil to come and help) there lymphocytes will slow down, adhere and enter the tissue helping to fight the infection. Macrophages will digest, display and take the antigen to the lymphatic's. This is already mobilizing that adaptive immune response. Macrophages are educating the lymphocytes in the lymph nodes about a new antigen, seeing if their antibody fits within it. B cell will go through hyper mutation, in hopes of getting an even better match for its antigen. Lot of deleterious mutations so a lot of less good b-cells. Germinal center is where lot of death and destruction occurs. A lot of macrophages in here eating up dead b-cells that didnt make it through the final selection At the end you get a b-cell with amazing affinity for the antigen differentiate into memory/plasma cells (plasma cells secrete antibodies) Spleen for foreign invaders of the blood stream known as the lymph node for the entire blood stream Dendritic cells: residents of the tissues, highly specialized antigen presenting cells. Gobbling up, showing antigens and traveling to lymph nodes. First responders Innate immunity Acute inflammatory response Recruited by cytokines Immediate/Acute Not antigen Specific Control infection while special forces are mobilized Neutrophils: Innate protection from bacteria and fungi Enzyme release Path of destruction Tissue destruction protective walling off Tissue destruction destroys circulation (could form abscess) Acute inflammation Predominant Leukocyte Eosinophil Innate protection from parasitic Infection Involved in allergic response (Eosinophil cause more harm than health in our bodies because we dont have parasites -- often involved in allergic response.) Enzyme release

Minor amount in blood (0-8%) Basophil Not sure why we have basophiles Release histamines Hypersensitivity reaction Very minor Leukocyte in blood (0-1%) Natural Killer Cells Innate response to viruses Antitumor activity Variable killer inhibitory receptors Cytotoxic granules release (like lymphocytes, but with granules) Special forces Adaptive immunity know exactly which cell needs to be target for degradation takes a little bit of time B and T cells Adaptive Immunity Self vs. Non-Self Recognition T cells: Targeting killing/cytokine release Intracellular Pathogens B cells Antibody production, specific to antigen Tag pathogens for phagocytosis (by macrophages) Activate complement Communication system Cytokines Weaponry/arms Radicals/NO/Enzymes