Complement System, Inflammation, and Acute Phase Reaction - Immunology PDF

The complement system, inflammation and acute phase reaction Dalma Papp 1  The natural or innate immune system Recognition, Mobilization, Destruction Cells: In the inte...

The complement system, inflammation and acute phase reaction Dalma Papp 1  The natural or innate immune system Recognition, Mobilization, Destruction Cells: In the interstitial space: phagocytes/monocytes - macrophages, dendritic cells In circulation: monocytes, neutrophil granulocytes, NK (natural killer) cells Humoral components: In circulation: antimicrobial enzymes, proteins, and peptides (e.g., defensins) – proteins of the complement system! Response capability and time – limited capabilities, but fast! No antigen specificity. Recognition: The duty of the natural immune system contains the detection of pathogens, the danger signals, and the self-deficiencies. The first line of defense, which must always be active. A sufficient number of cells must constantly be present, as it provides protection immediately without the need for prior activation or cell division. Humoral: Humoral factors – soluble participants: Antimicrobial peptides capable of directly destroying pathogens in body fluids, enzyme cascade systems activated by invaders, and various small molecules, called cytokines, that influence the functioning of different cells and ensure "communication" between them. The response capability does not improve with repeated infection; unfortunately, there is no memory. It recognizes a limited number of structures with the same receptors, and the activity and protection cannot be transferred to another individual. However, the response time is very short (within hours) and constant participants are always present. Upon detecting danger, it immediately activates. The quantity of certain components increases in response to infection, such as the production of acute- phase proteins during acute inflammatory processes, and the bone marrow boosts immune cell production. Immune cells gather at the site of infection, increasing the 2 local cell count. Activating signals (e.g., cytokines—produced by macrophages, these initiate inflammatory processes at the infection site) enhance its functioning. Opsonization also enhances the response, which will be explained shortly. Macrophages phagocytize pathogens, recognize them with their receptor cells, and destroy them upon first encounter. Granulocytes destroy pathogens during the complement process and in the acute phase. NK cells (natural killer cells) are a type of white blood cell, and their important role is to eliminate tumor and virus-infected cells. Dendritic cells play a crucial role in recognition, functioning alongside macrophages as tissue sensors. This is a non-antigen-specific system, where its cells recognize the molecular patterns of pathogens (PAMP – Pathogen Associated Molecular Patterns, which are absent in higher organisms but essential for pathogen survival). In contrast, the cells of the adaptive immune system primarily recognize the fine structure of proteins. https://dea.lib.unideb.hu/server/api/core/bitstreams/d8dd311b-4994-4503-8074- 05a7b9b1016d/content https://www.immbio.hu/phocadownload/basic_immunology_dentist/Immunologia_ala pjai_Fogasz_2019_6.pdf http://zsuzsanna.emri.uni-eger.hu/public/uploads/immunvalasz_5e75fa39b0a31.pdf Szemán-Nagy Gábor György jegyzetek 2  Structure of the complement system Components: Approximately 30 types of proteins (glycoproteins) that can complement the function of antibodies. Their inactive proenzymes are present in the blood plasma (e.g., pro-C3) along with receptors that bind the activation fragments of the components. Activation: Inactive protein -> proteolytic cleavage -> active protein Enzyme cascade There are 3 different activation pathways: Alternative pathway: (MAC - Membrane Attack Complex): Continuously active part, triggered by the presence of foreign surfaces. Classical pathway: Activated by the adaptive immune system – Opsonization – Creates opsonized surfaces that trigger activation. MB-Lectin pathway: Activated by mannose-binding lectin. Inhibition of pathogen-independent activation So, the complement system is essentially an enzyme cascade system in an inactive state within blood plasma and sometimes other bodily fluids. It's composed of factors that activate each other in a chain reaction. The proenzymes are crucial protein molecules involved in catalytic processes, and once activated, they transform into enzymes. Through the three pathways, these inactive proteins become true proteases, capable of causing cell damage and fulfilling their protective functions. In the classical pathway, activation is triggered by antibodies, which play a key role in recognizing pathogens and initiating this defense mechanism. 3  Functions of the complement system Opsonization (e.g., biological activity of C3b, C4b components): C3b and C4b components bind to the surface of pathogens, acting as "tags" that are recognized by phagocytic cells like macrophages and neutrophils, facilitating their engulfment and destruction. MAC – Formation of the Membrane Attack Complex: forms a pore in the membrane of pathogens, leading to their lysis. It is the terminal step of the complement activation cascade, with components like C5b, C6, C7, C8, and C9 assembling to puncture the pathogen's cell membrane, causing its contents to leak out and the cell to die. Chemical Messengers: Complement components also act as chemical messengers that regulate and amplify immune responses. These molecules signal immune cells, guiding them to the site of infection or inflammation and facilitating their activation and coordination. Anaphylatoxins: Anaphylatoxins, such as C3a and C5a, are small peptide fragments produced during complement activation. They act as potent inflammatory mediators by recruiting immune cells to the infection site, promoting vasodilation, and increasing vascular permeability, contributing to inflammation and anaphylaxis if not controlled. Opsonin Molecules: Opsonins are extracellularly binding protein molecules that facilitate the recognition of pathogens by the immune system. They enhance the ability of innate immune cells to recognize and respond to pathogens more quickly. While a pathogen itself may not always be readily recognized by a phagocyte, an opsonized pathogen is much easier to identify. This is because phagocytes recognize complement components or antibodies bound to the pathogen through opsonin receptors, such as complement receptors (CR) or Fc receptors. Opsonins, which attach to the pathogen's surface, act as a bridge between the pathogen and the phagocytes, marking them for destruction. This process enhances the efficiency of the immune response by promoting the engulfment (phagocytosis) of pathogens by phagocytic cells like macrophages and neutrophils. Moreover, opsonins also tag antibody-bound antigens (immune complexes), further improving their clearance from the body. Membrane Attack Complex (MAC): The final steps of the complement activation cascade result in the assembly of the MAC, a pore-forming structure made of proteins. This pore is inserted into the target cell's or bacterium's membrane, making it permeable to various substances. The disturbance of the cell's osmotic balance eventually leads to cell lysis and death. This mechanism is crucial in destroying pathogenic cells directly. 4 Chemical Messengers and Anaphylatoxins: Following activation, certain complement components function as chemotactic factors, acting as chemical signals that attract immune cells to the site of infection or complement activation. They guide immune cells to the area where they are needed most, coordinating a more robust immune response. Additionally, some components, known as anaphylatoxins (such as C3a and C5a), play a key role in triggering local inflammation. These molecules increase blood vessel dilation and the permeability of capillary walls, allowing immune cells to reach the infected or inflamed tissue more effectively. However, if this process is uncontrolled, it can lead to excessive inflammation or anaphylaxis, which can be harmful. https://dea.lib.unideb.hu/server/api/core/bitstreams/d8dd311b-4994-4503-8074- 05a7b9b1016d/content 4  Components of the complement system Plasma Proteins: C (C1 - C9 + subunits, e.g., q, r, s), produced in zymogen form in the liver. Activation: proteolytic cleavage Triggering factor: pathogen Recognition molecules: PRM (Pattern Recognition Molecules) Example: Pro-C3 -> C3 Activating Molecules Complement (cell surface) receptors (CR) (FC) Regulatory Factors: Factor B, D, H, I, P (properdin) Regulatory Proteins: decay accelerating factor (DAF); CR1, protein-S (vitronectin); C1 inhibitor (C1-INH, serpin); C4 binding protein (C4-BP), C5 (starting step of MAC) Membrane proteins that prevent damage to self-cells Other important elements: Mannose-binding lectin (MBL); MBL-associated proteases MASP-1 and MASP-2 The complement system recognizes various microorganisms and pathogen- associated molecular patterns (PAMPs) with the help of soluble pattern recognition molecules (PRMs). Protease: Protein breakdown, so when it is activated, it will cleave another component, resulting in the breakdown into smaller and larger units: (a) The smaller subunit will enter the microenvironment. (b) The larger subunit will bind to the pathogen and facilitate its phagocytosis or lysis. EXCEPTION!!!: In the case of the C2 protein, it is reversed (a: large, b: small). C3 is the most important protein of the complement system (glycoprotein). Main synthesis site: The liver, in the pro-C3 form. Activated by all three pathways. Abundantly present in the plasma of vertebrates. Single-chain - Transforms into the mature two-chain C3 protein, consisting of α- and β-chains connected by a single disulfide bond. Plays a key role in mediating all biological functions of the complement 5  system. Figure: Schematic representation of the C3 chain structure and its physiological degradation products. The single-chain pro-C3 is converted into the mature C3 protein when a furin protease removes four arginine residues, resulting in the formation of C3 α- and β-chains. https://www.sciencedirect.com/topics/pharmacology-toxicology-and- pharmaceutical-science/complement-component-c3 https://www.studocu.com/hu/document/szegedi-tudomanyegyetem/immunologia/5- eloadas-a-komplementrendszer-es-mukodese/21249564 5  Activation of the complement system Antibody-dependent: Classical Pathway: Activated by the adaptive immune system - Opsonization - Creation of opsonized surfaces that trigger activation. Antibody-independent: Alternative Pathway (MAC): Continuously active, activated by the presence of foreign cell surface structures. BM-Lectin Pathway: Activated by mannose-binding lectin. The essence of all three pathways is that the C3 component is cleaved, which activates C5 (also cleaved), leading to the formation of the lytic complex (MAC). Alternative Pathway: C3 activation occurs spontaneously if there is something to bind to. It can bind to anything recognized as foreign, then cleave it, leading to cell lysis—MAC (Membrane Attack Complex)—which forms a pore in the lipid bilayer. Interestingly, some pathogens can create structures similar to human cells, increasing their survival chances. BM-Lectin Pathway: Activation of mannose-binding lectin, also known as the mannose-binding lectin pathway. It is most effective against bacteria with cell walls because its activation requires the presence of accessible mannose on the antigen. Mannose on the pathogen is targeted and destroyed. Classical Pathway: Activated by antibodies and antigens, meaning the adaptive immune system triggers it. This leads to phagocyte recruitment and the appearance of inflammatory peptide mediators. Could someone explain what an antibody and an antigen are? 6  Antigen: A substance, typically protein in nature (e.g., bacteria or virus), that the immune system identifies as foreign and thus triggers a defensive reaction or immune response against it. Antibody: A protein produced by the immune system whose role is to recognize and bind to foreign substances identified as antigens (e.g., bacteria and viruses), then destroy them either directly or indirectly. 6  Activation of the complement system Antibody-independent: Lectin Pathway Steps Activators: Mannose side chains on bacterial cell walls and membranes. C1 Component Replacements: MBL (C1q): Mannose-binding lectin. MASP-1 (C1r) and MASP-2 (C1s): Mannose-associated serine proteases 1 and 2. Recognition Molecule: MBL (including ficolins, collectins). Pattern: Carbohydrate structures - PAMPs (Pathogen-Associated Molecular Patterns), DAMPs (Damage-Associated Molecular Patterns). Lectin Pathway: In the bloodstream, it searches for mannose side chains. When it finds them, MBL (mannose-binding lectin) binds to them. MASP-1 and MASP-2 also bind from the blood, with MASP-1 cleaving MASP-2. C4 then binds and is cleaved by the activated protein, with its larger subunit binding to the bacterial surface. C2 also binds and is cleaved by the activated protein, with its larger subunit binding alongside C4b to the bacterial surface, forming the C4bC2a complex, which then acts as a C3 convertase. From this point, the process is the same as in the classical pathway. https://www.immbio.hu/phocadownload/basic_immunology_dentist/Immunologia_al apjai_Fogasz_2019_6.pdf 7  Activation of the complement system Antibody dependent Initial steps of the classic route Activators: Antigen-antibody complex (IgM, IgG1, IgG2, IgG3) C1, C2, C3, C4 components Recognition molecule: C1q Pattern: IgG, IgM, but C-reactive protein, pentraxin and RNA, DNA, other polyanionic structures may also be present, PAMP C1q-> C1r -> C1s-> C4 -> C2-> C3-> C5-> C7-> C8-> C9 Classical Pathway: Antibodies and antigens activate the adaptive immune system, leading to phagocyte recruitment and the appearance of inflammatory peptide mediators. Antibodies produced by the body (IgG or IgM immunoglobulins) bind to antigenic determinants on the surface of a bacterium. In the presence of Ca++, the inactive C1 complex (inhibitor) binds to the antibodies, causing a conformational change in C1q (which binds). This activates the r and s subunits, with r cleaving s. C4 then binds (a binding protein, cleaved by r), forming the b and a subunits. The b subunit binds to the bacterial surface. The still-active complex then cleaves C2, producing the b (a precursor to kinin, a catalyst for edema) and a subunits. The a subunit binds to the bacterial surface alongside C4b, forming the C4bC2a complex (C3 convertase, capable of cleaving C3). C3 is then cleaved and binds to the existing two-subunit complex, forming the C4bC2aC3b complex, which acts as C5 convertase and initiates the formation of the lytic complex. https://www.immbio.hu/phocadownload/basic_immunology_dentist/Immunologia_al apjai_Fogasz_2019_6.pdf 8  Activation of the complement system Antibody independent Alternative route steps Activators: Pathogen surface B, D, P factors Recognition molecule: no specific molecule (presumably properdin, for example, has such a function) Pattern: PAMP Alternative Pathway: The C3 protein spontaneously hydrolyzes in the aqueous environment of the human body (where it is abundant), forming C3i (which is continuously present in the blood because it is constantly being produced). C3i can bind to Factor B, allowing Factor D to cleave Factor B into two subunits. The larger b subunit remains, forming the C3iBb complex, which is short-lived but capable of binding to C3 if available. Amplification Loop Process: The C3iBb complex can bind and cleave C3, functioning as a C3 convertase. This cleavage produces C3b, which can again bind to Factor B. This binding allows Factor B to be cleaved into Bb, which also acts as a C3 convertase. The key difference is that this C3 convertase is generated due to the convertase effect, not spontaneously. The process continues as follows: the released C3b binds to Factor B, which is then cleaved, and another C3 binds and is cleaved, and so on. Control of Spontaneous C3 Activity: The amplification loop only occurs if an activating membrane (bacterial or foreign origin) is present. In the absence of a bacterial membrane, C3b can only bind to our 9 own membrane, which contains DAF (Decay-Accelerating Factor). DAF prevents C3b from binding to Factor B, thereby blocking the C3b-B interaction and the amplification loop. The already formed C3b-B convertase is then dissociated by Factor H (a protein), and the bound C3 is degraded by Factor I (a protease). DAF designates Factor I to perform this task (it acts as a marker protein). The same process occurs with Bb, ensuring that the amplification loop is constantly interrupted. Upon Encountering an Activating Membrane: Stabilization and Activation: The C3bBb convertase binds Factor P (properdin), which stabilizes the complex and enables it to bind and cleave additional C3. From this point, it acts as C5 convertase. This process acts as an additional safeguard for the other two pathways, providing a robust response against previously unknown pathogens. The recognition of the activator surface is not a directed process (unlike the directed DAF recognition); rather, the absence of DAF induces activation, leading to rapid and effective bacterial destruction through the amplification loop. https://www.immbio.hu/phocadownload/basic_immunology_dentist/Immunologia_ala pjai_Fogasz_2019_6.pdf https://www.studocu.com/hu/document/szegedi-tudomanyegyetem/immunologia/5- eloadas-a-komplementrendszer-es-mukodese/21249564 9  Main components and effector effects https://www.immbio.hu/phocadownload/basic_immunology_dentist/Immunologia_al apjai_Fogasz_2019_6.pdf This is an overview for us to helt understand all of the processes and coponents. 10  Late phase of complement activation - MAC Convertase C5 in classical and lectin pathways Convertase C5 in alternative pathway Lytic Pathway: The three pathways can operate in parallel if conditions permit, converging at the C5 convertase stage. This leads to the activation of the lytic pathway and the formation of the Membrane Attack Complex (MAC). Components: C5, C6, C7, C8, C9. Process: The C5 convertase formed earlier cleaves only C5; the other components only increase the size of the complex and the pore. The C5 complex binds to C5 and cleaves it, producing two subunits, with the larger b subunit remaining and accumulating. Once enough of these subunits accumulate, they bind to C6, allowing C7 to bind. C7 has a hydrophobic region that anchors the complex to the bacterial membrane. At this point, C8 binds, which by itself forms a small pore. However, this pore is still small, so C9 monomers come in large numbers to expand the pore. The lytic complex is not only effective against bacterial infections but also assists in the elimination of virus-infected cells via the alternative pathway, as viral infections can also lead to the loss of DAF proteins. https://www.immbio.hu/phocadownload/basic_immunology_dentist/Immunologia_al 11 apjai_Fogasz_2019_6.pdf 11  Inhibition of the complement system Pathogen-independent Activation Inhibition Inhibitory Molecules - e.g., FHR (Factor H-Related proteins). Complement Regulatory Proteins (in serum, on cell surfaces) - e.g., Factor H (FH), C1 inhibitor. Lack of Signaling In the body, the complement system's pathogen-independent activation is kept in check by various inhibitory molecules. The regulation of complement activation by various complement regulatory proteins, which are constantly present in the serum and on cell surfaces, prevents damage to the body's own cells. The absence of these proteins on pathogen surfaces can lead to activation even without specific recognition of the microbe. Antigen presentation: T cells recognize peptides presented by antigen-presenting cells when these peptides are associated with MHC molecules. The peptide and MHC bind simultaneously to the T cell receptor as a complex, triggering complement system activation. Similar to NK cells, the activation of the complement system can be triggered by the absence of "self" signaling, not just by the recognition of pathogen- specific patterns. Factor H (FH) is an important regulatory protein of the alternative pathway, inhibiting the formation of C3 and C5 convertases. Factor H-related proteins (FHR) can also inhibit C3 convertase. The C1 inhibitor, when present in high concentrations in the blood serum, acts as an inhibitor of C1r and C1s, which are involved in the classical pathway. 12 https://dea.lib.unideb.hu/server/api/core/bitstreams/d8dd311b-4994-4503-8074- 05a7b9b1016d/content https://edit.elte.hu/xmlui/bitstream/handle/10831/43441/Doktori%20%C3%A9rtekez% C3%A9s_Cserhalmi%20Marcell.pdf https://core.ac.uk/download/pdf/51314945.pdf 12  Insufficient operation of the system Diseases Caused by Inadequate Function Classical Pathway: Hereditary angioedema (C2b overproduction). Predisposition to systemic lupus erythematosus (SLE) (opsonized immune complexes precipitate in tissues instead of remaining in solution, causing inflammation). Lectin Pathway: Increased susceptibility to bacterial infections in infants and immunosuppressed patients (due to the lack of activation of the lectin pathway). Alternative Pathway: More frequent infections caused by pyogenic bacteria (due to ineffective opsonization). Increased incidence of bacterial infections (due to lack of opsonization and absence of the lytic complex). Sensitivity to Gram-negative bacteria (due to failure to attack the bacterial cell outer membrane). https://dea.lib.unideb.hu/server/api/core/bitstreams/d8dd311b-4994-4503-8074- 05a7b9b1016d/content 13  Inflammatory process Process of Removal and Restoration Triggers: Exogenous: Infection, chemical effects, radioactive effects, active radicals; necrosis, foreign substances. Endogenous: Tumor; intense local immune reaction; severe metabolic disturbance. Inflammation is responsible for eliminating harmful effects that appear in the body and thereby restoring the original state and function. It is a crucial element of the body's defensive mechanisms. Falus András - Inflammation and Acute Phase Response Exogenous: Bacteria and viruses causing infection; acids, bases, burns; necrosis (tissue death). Active radicals, for example, are produced by macrophages, which are necessary for destroying foreign and tumor cells. Unfortunately, these can also have tissue-damaging effects. The nature and timing of the inflammation process are also important. Today’s lecture will focus in detail on acute inflammation. http://zsuzsanna.emri.uni-eger.hu/public/uploads/2011-0001-524- immunologia_59f039dfa2d5c.pdf 14  Acute inflammation Acute: immediate response, acute, short-lived - It is induced by the natural immune system against a harmful substance. Leukocyte, plasma protein and fluid flow to the site of injury. Local nature, in the area of affected ssues - It takes place at the site of pathogens, damage, in the affected area Vascular changes: The diameter of blood vessels increases, the permeability of the vessel wall/capillaries increases, activation of the blood coagulation system Triggers the acute reaction - pathogen, any foreign substance, danger signal Symptoms: Swelling (oedema) Skin redness (blush) Local feeling of warmth (calor) Pain (dolor) Loss of function (functo laesia) The innate immune response creates an inflammatory state at the site of infection. The localized inflammation in the tissues, where the pathogen has entered, is essential for defending against microbes. Processes occur that facilitate the control of infections and promote the development of an effective immune response. In inflamed tissue, blood vessels dilate, causing more blood to flow through the affected area. At the same time, the permeability of capillaries increases, allowing antimicrobial substances in the blood plasma, such as complement system proteins, as well as various cells, to leave the circulation and enter the peripheral tissues in large quantities. Hyperemia develops, and the diameter of the blood vessels increases so that the larger volume of blood flows more slowly. The increased permeability of the blood vessel walls allows immune response cells, including leukocytes, to migrate into the tissues. The blood coagulation system also activates, isolating severely infected areas and preventing pathogens from entering the bloodstream. A danger signal might be the release of cytokines that trigger inflammation. We experience this as uncomfortable, painful swelling or edema. This process also 15 increases the amount of lymph entering the lymphatic vessels, which transports some of the antigens to the surrounding lymph nodes, where antigen-specific lymphocytes can be activated. https://dea.lib.unideb.hu/server/api/core/bitstreams/d8dd311b-4994-4503-8074- 05a7b9b1016d/content Szemán-Nagy Gábor György jegyzetek 15  Acute inflammation Cellular Changes Participants: Macrophages: Immediate destruction of pathogens and production of cytokines. Dendritic Cells: Transport and presentation of antigens. Epithelial Cells: Play a primary role in activating neutrophil granulocytes (through IL-12) and recruiting monocytes. Types of Cytokine Production: Distant: Autocrine: Affects the cell that produces the cytokine. Endocrine: Affects distant organs, such as the brain/hypothalamus, leading to fever. Proximate: Paracrine: Affects nearby cells such as those in the liver, bone marrow, blood vessels, and smooth muscle tissue. It acts through opsonization and signals neutrophil granulocytes. Initiating the Immune Response: The activation of the immune response can be triggered by the involvement of just a few cell types. Macrophages and dendritic cells, which are dispersed near potential pathogen entry sites beneath the body's external and internal surfaces, play a crucial role in this process. Additionally, epithelial cells that form the boundary surfaces may also participate in the alerting process. These few types of cells possess nearly every type of pattern recognition receptor. The primary role of dendritic cells is not to destroy pathogens but to transport antigens to the lymph nodes and present them to T cells, thus activating the adaptive immune system. They exit from the blood into the tissues. In contrast, macrophages immediately perform effector functions, including the destruction of pathogens. The presence of pathogens activates macrophages through their pattern recognition receptors. In cases of minor infections, the body may not require further immune response. For larger infections, an acute inflammatory response is initiated, and inflammatory cytokines are produced. These act as cytotoxins. Macrophages also migrate from the blood into the tissues. More on cytokines will be discussed later. 16  Liver: Proteins reaching the acute phase are continuously produced. Bone Marrow: Increased production of leukocytes, or white blood cells. Blood Vessel Cells: Endothelial cells separate, disrupting the connections to allow neutrophil granulocytes to reach the site of inflammation; the surrounding membrane of the vessels also becomes fragmented. Smooth Muscle Tissue: Relaxes to allow the cross-sectional area of the vessels to increase. Neutrophil Granulocytes: Activated within the tissues. They move rapidly through the blood and are stopped by adhesion molecules produced in response to cytokines. Cytokines anchor the granulocytes. The cytokine Interleukin-12 (IL-12) will activate them. They are stored in large numbers in the bone marrow. In the later stages of inflammation, cytokines that act as growth factors are produced to aid in tissue regeneration, such as stimulating connective tissue cell division or promoting the growth of new blood vessels at the damaged sites. https://dea.lib.unideb.hu/server/api/core/bitstreams/d8dd311b-4994-4503-8074- 05a7b9b1016d/content Szemán-Nagy Gábor György jegyzetek 16  Acute inflammation Maturation of mononuclear phagocytes and dendritic cells involved in the inflammatory process Members of the Monocyte-Macrophage System: Bone Marrow Precursors: (monoblast, promonocyte) Mature monocyte Free monocyte Immobilized macrophages Let's see how this process occurs and where it begins. Somehow, these cells need to be produced, mature, and move. Monoblasts constitute an extremely small fraction of the bone marrow precursors. Promonocytes, which arise from monoblasts, are capable of phagocytosis and contain non-specific esterases, acid phosphatases, and arylsulfatases. After the final maturation division, the resulting monocytes leave the bone marrow within approximately 24 hours. Unlike granulocytes, monocytes do not form a bone marrow reserve. Consequently, monocytes make up only 0-4% of the bone marrow cells. They often have a kidney-shaped nucleus, characterized by chromatin aggregates arranged near the nuclear membrane, connected by thin strands. Upon exiting the circulation, monocytes continue to mature, increasing in size, and the number of lysosomes and mitochondria in the cells also grows. Monocytes then transform into tissue macrophages. The elements of the so-called Mononuclear Phagocyte System (MPS) include blood, pleural, and peritoneal cavity monocytes, tissue-resident macrophages (known as histiocytes), osteoclasts, Kupffer cells (liver), alveolar macrophages (lungs), microglia (brain), and Langerhans cells (skin). Members of the Monocyte-Macrophage System: 17  Bone Marrow Precursors: Monoblast: The earliest stage in the monocyte lineage, found in the bone marrow. Promonocyte: An intermediate stage that develops from monoblasts. Mature Monocyte: These are released from the bone marrow into the bloodstream, where they circulate until they migrate into tissues. Tissue Macrophages: Once monocytes enter tissues, they differentiate into macrophages. These are specialized for tissue-specific functions and can be further classified based on the tissue they inhabit (e.g., Kupffer cells in the liver, microglia in the brain). Free Macrophages: Macrophages that are not fixed to a specific tissue but can move freely within the tissues to perform their functions. Immobilized Macrophages: Macrophages that are anchored in specific tissues or structures, often playing a role in maintaining tissue homeostasis and responding to local stimuli. These components work together to carry out various immune functions, including pathogen phagocytosis, antigen presentation, and tissue repair. https://slideplayer.hu/slide/2202425/ https://dea.lib.unideb.hu/server/api/core/bitstreams/d8dd311b-4994-4503-8074- 05a7b9b1016d/content https://kortan.semmelweis.hu/targyak/kortan/gyakorlat/hemat/regi_jegyzet/mo- ly/modiffn.html 17  Acute inflammation Maturation of mononuclear phagocytes and dendritic cells involved in the inflammatory process Members of the Monocyte-Dendritic Cell System: Bone Marrow Precursors (monoblast, promonocyte) Mature Monocyte Pre-conventional DC (Dendritic Cells) Conventional DC Plasmacytoid DC Dendritic cells are the most important antigen-presenting cells, serving as a bridge between the innate and adaptive immune responses. They are phagocytic cells with extensions, found in lymphoid organs, mucosal surfaces, and the parenchyma of organs. They originate from the myeloid lineage, sharing a common ancestor with monocytes. Their precursor is the monocyte, which differentiates into a conventional dendritic cell. Most dendritic cells are conventional DCs. Conventional DCs are mobilized from the skin and mucosa in response to microbial stimuli and migrate to lymphoid organs to present antigens to T cells. Another group of DCs, the plasmacytoid DCs, act during the early stages of viral infection. They recognize viral nucleic acids and produce interferon, which has antiviral effects. These cells are of lymphoid origin. https://slideplayer.hu/slide/2202425/ https://dea.lib.unideb.hu/server/api/core/bitstreams/d8dd311b-4994-4503-8074- 05a7b9b1016d/content https://kortan.semmelweis.hu/targyak/kortan/gyakorlat/hemat/regi_jegyzet/mo- ly/modiffn.html http://web.med.u-szeged.hu/medmicro/dok/fresh/20180508_2/eloadas.pdf 18  Acute inflammation - cell activation https://slideplayer.hu/slide/2202425/ https://dea.lib.unideb.hu/server/api/core/bitstreams/d8dd311b-4994-4503-8074- 05a7b9b1016d/content Macrophages respond to pathogens through receptor activation with phagocytosis and cytokine secretion. Like macrophages, DCs recognize the molecular patterns of microbes and secrete cytokines in response to them. 19  Acute inflammation - activation of macrophages Pattern Recognition Receptors (PRRs) Macrophages play a significant role in defending against infections by phagocytosing bacteria, damaged cell debris, and apoptotic cells, as well as in tissue repair and regeneration following tissue damage and inflammation. In addition, they present antigens, produce cytokines and other biologically active molecules, regulate T-cell activation, and thus influence the maintenance of tissue homeostasis and the immune response. They are also crucial in the regeneration following inflammation and wound healing. Macrophages respond to pathogens through receptor activation, leading to phagocytosis and cytokine secretion. 1.Figure: When a bacterial cell binds to a macrophage's recognition and phagocytic receptor, the process of engulfment and degradation inside the macrophage begins. 2.Figure: When a bacterial component binds to a signaling receptor, it induces cytokine production. Transcription occurs in the cell nucleus because macrophage function and appearance are significantly affected by microenvironmental signals, which trigger different gene activation programs and influence their transformation, 20 known as macrophage polarization. Two extremes of macrophage polarization are characterized by completely different molecular patterns and functional properties. However, recent studies show that the complex molecular microenvironment can induce a wide spectrum of macrophage polarization states. Danger signals, such as cytokines and interleukins, can also lead to additional macrophages arriving at the site of inflammation. https://slideplayer.hu/slide/2202425/ https://dea.lib.unideb.hu/server/api/core/bitstreams/d8dd311b-4994-4503-8074- 05a7b9b1016d/content https://dea.lib.unideb.hu/server/api/core/bitstreams/b632c625-4d04-41d7-8f47- 093d9a0a8f83/content 20  Acute inflammation - activation of dendritic cells Similar to macrophages, dendritic cells recognize microbial molecular patterns and secrete cytokines in response. Immature dendritic cells (imDC) migrate to the nearest lymph node with the engulfed pathogen. During this migration, they mature and present peptides derived from the pathogen on their MHC molecules to Th cells. The same type of dendritic cell can elicit different immune responses depending on the environmental stimuli or the pathogen it encounters. Immature dendritic cells are found in the body's surface layers (skin, pharynx, upper esophagus, rectum) as well as in the mucous membranes of the respiratory and digestive systems. At these sites, immature dendritic cells can uptake antigens through various mechanisms, which may originate from pathogens or dying cells. The uptake of antigens and other signals (e.g., cytokines) leads to the maturation/activation of dendritic cells. After maturation, they lose their antigen uptake function and migrate to the lymph nodes, where they present the processed antigen to T cells. https://slideplayer.hu/slide/2202425/ https://dea.lib.unideb.hu/server/api/core/bitstreams/d8dd311b-4994-4503-8074- 05a7b9b1016d/content https://dea.lib.unideb.hu/server/api/core/bitstreams/b632c625-4d04-41d7-8f47- 093d9a0a8f83/content https://dea.lib.unideb.hu/server/api/core/bitstreams/e3651fdd-201a-4d43-90c6- 21 b52c7a32edb7/content https://www.nature.com/articles/s41565-020-00810-2 21  Acute inflammation - differentiation and activation of neutrophil granulocytes Neutrophil granulocytes, similar to macrophages, play a crucial role in clearing dead or tumor cells and opsonized microbes. They are often referred to as professional phagocytes. Based on their staining characteristics, granulocytes are classified into three types: neutrophils, eosinophils, and basophils. Neutrophil granulocytes (polymorphonuclear cells) have a lobulated, multi-lobed nucleus and characteristic cytoplasmic granules. These cells represent the first line of immune defense against pathogens that have breached physical barriers. Their main function is the recognition and engulfment of infectious microbes, which can occur through interactions between PAMPs and the cells' PRR receptors, as well as through FcRs and CR1 and CR3 complement receptors if the pathogen is opsonized with IgG or complement proteins. Neutrophil cells exhibit active phagocytosis and a more intense oxidative burst compared to macrophages, though they can also destroy harmful microbes through oxygen-independent mechanisms. Granulocytes are effectively activated by pathogen-derived substances and various cytokines, responding quickly to chemotactic stimuli, making them highly effective in resolving acute inflammation. Activated neutrophils, along with substances released from damaged tissue cells, stimulate additional cells—macrophages, mast cells, and endothelial cells. The factors released during this process alter the metabolism and cell surface structures of circulating granulocytes, leading to their exit from the 22 bloodstream and migration to inflamed tissues. Neutrophil granulocytes are stored in the bone marrow and differentiate from myeloblasts into neutrophil granulocytes. Their lifespan is extremely short; they spend 10-12 hours in circulation before undergoing spontaneous apoptosis. During inflammation, they arrive in large numbers at the site of infection, perform their effector functions (engulfing and degrading), and then die. In the fight against pathogens, three main antimicrobial processes can be distinguished: (a) phagocytosis—engulfing and digesting microbes, (b) degranulation—release of reactive oxygen species and cytotoxic enzymes, (c) NETosis—formation of neutrophil extracellular traps. Initially, neutrophil granulocytes flood the site of infection. These cells are highly effective at destroying engulfed pathogens using their extensive enzyme repertoire. Monocytes that enter the tissues, differentiating into macrophages under the influence of environmental factors (cytokines, chemokines, other inflammatory mediators), also anchor additional neutrophils by producing cytokines. It has also been recognized that activated neutrophils release various pro-inflammatory cytokines and surface molecules (MHCII) in a manner that allows antigen presentation to T cells and T cell activation (see later). https://slideplayer.hu/slide/8126246/ https://upload.wikimedia.org/wikipedia/commons/4/4a/Hematopoiesis_simple.png https://mob.aeek.hu/details.jsp?ITEMID=1939098 http://zsuzsanna.emri.uni-eger.hu/public/uploads/2011-0001-524- immunologia_59f039dfa2d5c.pdf https://www.semanticscholar.org/paper/Insights-into-the-Mechanism-of-Drug- induced-A-Study-Lobach/66072271c4dd6abc326cf08766a976ab610644f2 22  Cytotoxins Production Following Pathogen Detection - Alert! Cytokine Detection Receptors Cytokines + Receptors + Cell-Cell Communication -> Cellular Function Lymphokines, Monokines, Interleukins (IL) Chemokines Cytokines are hormone-like substances that can exert their effects even at very low concentrations. They can act in an autocrine manner on the cells that produce them, in a paracrine manner on nearby cells, or in an endocrine manner on distant cells and organs. The ability of a cytokine to affect a cell depends on the presence of the specific cytokine receptor on that cell. A single cell may have multiple types of receptors, allowing it to be influenced by several different cytokines simultaneously. These cytokines can inhibit, enhance, or modify each other's effects. Additionally, a single cytokine can affect different types of cells in various ways. Lymphokines are produced by lymphocytes, which is why cytokines are sometimes referred to as such. Monokines are cytokines produced by monocytes. Interleukins received their name by they facilitate communication between white blood cells/leukocytes. More than 30 different interleukins have been described, and they are typically distinguished by their numbers. https://dea.lib.unideb.hu/server/api/core/bitstreams/d8dd311b-4994-4503-8074- 05a7b9b1016d/content 23  Effects of cytotoxins Vasodilation and increased vascular permeability - IL-1, IL-2, TNF T-cell proliferation and differentiation - IL-2 Regulators of leukocyte activation and differentiation - IL-4, IL-12, IL-10 Involvement in inflammatory processes - IL-6 (proinflammatory cytokine) Growth factor - GM-CSF Inhibition of viral infections - interferons Induction of chemotaxis - chemokines (e.g., CXCL8) TNF – tumor necrosis factor IL – interleukin There are cytokines that act as growth factors, influencing the maturation and differentiation of various immune cells. For example, granulocyte-macrophage colony- stimulating factor (GM-CSF) enhances the formation of granulocytes and monocytes in the bone marrow. Chemokines are cytokines that attract cells with the appropriate chemokine receptors to their production site, inducing chemotaxis. These substances help lymphocytes in the bloodstream and dendritic cells migrating through tissues find the lymph nodes. In infected or inflamed tissues, large amounts of chemokines (e.g., CXCL8) and chemotactic factors (e.g., complement protein cleavage products) are produced, which recruit neutrophil granulocytes, monocytes, and other cells to the site of inflammation. The diagram shows systemic effects. https://dea.lib.unideb.hu/server/api/core/bitstreams/d8dd311b-4994-4503-8074- 05a7b9b1016d/content 24  Inflammatory cytotoxins ytokines: Interferons, TNF, interleukins, chemokines For example, cytokines that act at the site of inflammation + have endocrine effects include IL-6, IL-1, and TNFα. Endocrine Effects: Brain/hypothalamus -> Fever Liver -> Increased production of antimicrobial and acute-phase proteins (opsonins, complement system-activating factors) Bone marrow -> Enhanced white blood cell production Cytokines: Interferons, TNF, Interleukins, Chemokines In innate immunity and inflammatory processes, cytokines are those that are immediately released from non-specific immune cells following the pathogen's entry into the body. These include primarily mononuclear phagocytes, T cells, granulocytes, mast cells, fibroblasts, and endothelial cells. This group includes interferons, tumor necrosis factors (TNFα and -β), IL-1α and -β, IL-6, IL-10, IL-12, and inflammatory chemokines. These cytokines play a crucial role both in the development of inflammatory reactions following infection and in regulating the activation and differentiation of antigen-specific lymphocytes through their effects on various antigen-presenting cells. Endocrine – Systemic Effects https://dea.lib.unideb.hu/server/api/core/bitstreams/d8dd311b-4994-4503-8074- 05a7b9b1016d/content 25  Acute phase reaction Acute Phase Response: Infection or Trauma → Liver Cells → Production of Acute Phase Proteins → Sudden Increase in the Amount of Acute Phase Proteins in the Plasma Macrophages → Cytokines: IL-6, IL-1, TNFα → Systemic Inflammation, Septic Shock, Synthesis of Acute Phase Proteins (e.g., C3) IL-1 and IL-6 → Pyrogenic Effect TNFα → Cell-Damaging Effect → Liver → Acute Phase Response (APR) If the body is unable to spatially and temporally limit the inflammatory processes, these processes can affect organs that are distant from the site of the reaction. The acute phase response is the body's rapid defensive reaction to infection or trauma (e.g., burns), characterized by a sudden increase in the levels of certain proteins produced by liver cells in the plasma. In the acute phase response, as well as in systemic inflammation and septic shock, TNFα, IL-1, and IL-6 play key roles. The main source of all three cytokines is macrophages. Their induced biological effects significantly overlap, although they participate to varying extents in the synthesis of acute phase proteins or the manifestation of inflammatory symptoms. The levels of so-called acute phase proteins produced by the liver (see Chapter 8), such as C3, increase significantly in response to IL-1, IL-6, and TNF in the circulation. The pyrogenic (fever-inducing) effects of IL-1 and IL-6 are well known, and it is also recognized that elevated temperatures are unfavorable for pathogens but help protect host cells from the damaging effects of TNFα. Cytokine production associated with inflammation also affects protein synthesis in the liver, which is referred to as the acute phase response (APR), and the substances with altered serum concentrations as a result are called acute phase proteins or reactants. In the acute phase response, as well as in the development of systemic inflammatory processes and septic shock, TNFα, IL-1, and IL-6 play major roles. The main source of all three cytokines is 26 macrophages. IL-6, in particular, increases the secretion of certain proteins (positive acute phase proteins) in hepatocytes, while decreasing others (negative acute phase proteins). https://dea.lib.unideb.hu/server/api/core/bitstreams/d8dd311b-4994-4503-8074- 05a7b9b1016d/content https://slideplayer.hu/slide/8126246/ http://zsuzsanna.emri.uni-eger.hu/public/uploads/2011-0001-524- immunologia_59f039dfa2d5c.pdf 26  Acute phase proteins Protein Effect Positive CRP, MBL, C3, C4, B factor complement activation, opsonization Ceruloplasmin superoxide anion scavenger Haptoglobin hemoglobin binding, transport Fibrinogen blood clotting - coagulation Fibronectin cell adhesion Angiotensin Blood pressure control Negative Albumin, transferrin, transthyretin transport proteins In the acute phase response (APR), the proteins involved play diverse roles in various processes, ranging from blood clotting and complement activation to transport, underscoring the "interdisciplinary" nature of inflammation. As a result of this response, the liver alters the composition of blood plasma, effectively preparing the body for an efficient response. This change—primarily the increase in the concentration of the positive acute phase protein, fibrinogen—is responsible for the elevated erythrocyte sedimentation rate, which serves as a simple laboratory technique for detecting inflammation in the body. https://dea.lib.unideb.hu/server/api/core/bitstreams/d8dd311b-4994-4503-8074- 05a7b9b1016d/content https://slideplayer.hu/slide/8126246/ http://zsuzsanna.emri.uni-eger.hu/public/uploads/2011-0001-524- immunologia_59f039dfa2d5c.pdf 27  Acute phase proteins C-Reactive Protein (CRP): Binds to phosphocholine (PC) on the surface of bacterial cells (opsonization) -> Activates the complement system. Mannose-Binding Lectin (MBL): Binds to mannose, which is detected on the surface of pathogenic cells (opsonization) -> Activates the complement system. Ceruloplasmin: A copper-binding protein -> Enhances iron metabolism, improves protein functions, and scavenges superoxide anions/reactive radicals. Increased CRP levels can be an indicator of various issues. https://slideplayer.hu/slide/2129996/ 28  Outcome of acute inflammation Outcomes: Resolution/regeneration; secondary infection; sepsis; chronic inflammation; fibrosis/scarring Types of Exudate: o Serous: Clear, pale yellow fluid o Fibrinous: Thick, sticky fluid containing fibrin o Purulent: Pus-filled Pus: A whitish-yellow, yellow, or yellowish-brown discharge Produced during infection with pus-forming bacteria in vertebrates Consists of protein-rich fluid (liquor puris) + Dead cells (neutrophil granulocytes + dead tissue debris, necrotic cells) Brain abscess Exudate: Secretion, inflammatory fluid accumulation. Extracellular bacteria can proliferate in the intercellular space and on epithelial surfaces, such as in the bloodstream, connective tissue, and on the epithelium lining the respiratory and gastrointestinal tracts. These include pus-forming Gram-positive cocci (e.g., Staphylococcus, Streptococcus), Gram-negative cocci (e.g., Meningococcus, Gonococcus, two Neisseria species), Gram-negative rods (e.g., Escherichia coli), Gram-positive bacilli (e.g., anaerobic Clostridium species), and the spirochete Borrelia burgdorferi, which causes Lyme disease. These pathogens can cause disease in two ways. Firstly, they can initiate an inflammatory process that leads to tissue damage at the site of infection. Secondly, they can produce toxic substances, toxins, which may be present in the pathogen’s cell wall or released into the environment as soluble proteins. The former are known as endotoxins, and the latter as exotoxins. The endotoxin LPS (lipopolysaccharide) in the cell wall of Gram- negative bacteria stimulates cytokine production in macrophages, leading to inflammation. https://slideplayer.hu/slide/8126246/ https://semmelweis.hu/patologia2/files/2017/10/5_gyak_Gyulladas.pdf 29 Thank you for your attention! 30