Inflammatory Process. Mediators of Inflammation. Lymphocyte Recirculation.

What is inflammation?• In Latin: inflammare (to set on fire).

• It is body defence reaction in order to eliminate or limit the spread of injurious agent as well as to remove necrotic cells and tissues. Initiates the proces of repair

• Also a potentially harmful process. Components of inflammation that are capable of destroying microbes can also injury normal tissue

Excess inflammatory reactions

• Inappropriate inflammatory response when there are no foreign substances to fight off leads to autoimmunity

• Inflammatory process must be tightly regulated by immune system to avoid excessive damage normal tissue

• Components of the inflammatory proces include white blood cells and plasma proteins

Normally present in the blood The inflammatory reaction’s goal is to bring these to the site of

infection and/or tissue damage

• Inflammation is induces by chemical mediators produced by damaged host cells (Cytokines and other mediators)

Inflammation is normally

controlled and self-limited

Types of InflammationThe acute inflammatory response involves both localized (redness, swelling, heat, pain) and systemic responses.

Heat Redness Swelling Pain Loss of Function

Signs of acute inflammation

The local reactions are described as the cardinal signs and symptoms of inflammation

Signs of acute inflammation (Local manifestations of acute inflammation)

Redness (Rubor) & Heat (Calor) :• This is because the capillaries are filled up with more blood than usual (blood vessel dilation). (The increased volume heats the tissue and causes it to redden)

• Mediators promote the vasodilationoHistamineoSerotoninoBradykininoProstaglandin

•1x increase in arteriole diameter yields a 4x increase in blood flow

Swelling (Tumor) / Edema - caused by an accumulation of fluid

Edema fluid varies with the stage of inflammation• Initially vessel permeability is only slightly altered and no cells or proteins escape and the fluid is mainly water and dissolved electrolytes.

•As capillary permeability increases and plasma proteins escape the extravascular fluid becomes cloudy and more viscous. This is called exudate (contains a large amount of leukocytes) – extravasation of leukocytes

Signs of acute inflammation (Local manifestations of acute inflammation) cont.

Pain (Dolor)- The pain associated with inflammation, results in part from the distortion of tissues caused by edema.

Chemicals mediators (bradykinin, serotonin, and the prostaglandins) that stimulate nerve endings are released, making the area much more sensitive. Physical trauma may irritate pain receptors.

Loss of function (Functio laesa ) – may occur due to pain causing reflex guarding or muscle spasm.Loss of function may result from pain that inhibits mobility or from severe swelling that prevents movement in the area.

Signs of acute inflammation (Local manifestations of acute inflammation) cont.

The Inflammatory response consist of a vascular and cellular reaction

Vascular changeso vasoconstriction (momentary constriction of small blood vessels in the

area) – to decrease blood flow (very brief - first seconds after tissue injury).

o vasodilation - within minutes after tissue injury, there is an increase in vascular diameter resulting in an increase in the volume of blood in the area and a reduction in the flow of blood.

/The increased blood volume heats the tissue and causes it to redden/.

o Vascular permeability also increases, leading to leakage of fluid from the blood vessels, particularly at postcapillary venules. This results in an accumulation of fluid (swelling/edema)

o emigration of leukocytes from microcirculation

The Inflammatory Process Cellular Reaction

• Two types of leukocytes participate in the acute inflammatory response - the neutrophils and monocytes.

1st line of defense: neutrophils

o Within a few hours of the onset of vascular changes neutrofils adhere to the endothelial cells, and migrate out of the blood into the tissue spaces.

o Neutrophils phagocytose invading pathogens and release mediators that contribute to the inflammatory process

o Release chemokines that help to recruit macrophages - macrophage inflammatory proteins (MIP-1α and MIP-1β)

The Inflammatory Process Cellular Reaction

2nd line of defense: monocytes /macrophageso Macrophages arrive about 5-6 hours after an inflammatory

response beginso Macrophages exhibit increased phagocytosis and increased release

of mediators and cytokineso Activated tissue macrophages secrete IL-1, TNF-α and IL-6.

The Systemic Acute-phase Response

Acute inflammatory responses that are not restricted to the initiation site.

This response is marked by

• Hormone production (ACTH, hydrocortisone)

• Increased production of white blood cells (leukocytosis)

• Synthesis of acute-phase proteins in the liver

• The increase in body temperature

The acute inflammatory response involves both localized (redness, swelling, heat, pain) and systemic responses.

Acute Inflammatory Process

Acute Inflammation Process

Leukocyte extravasation

• Leukocytes must be able to get to the site of injury from their usual location in the blood

• The process of leukocyte movement from the blood to the tissues through the blood vessels is known as extravasation.

• As an inflammatory response develops, various cytokines and other inflammatory mediators act upon the local blood vessels, inducing increased expression of endothelial Cell Adhesion Molecules (CAM).

• The vascular endothelium is then said to be activated, or inflamed.

The leukocyte-extravasation cascadeLeukocytes leave the vasculature routinely through the following sequence of events:

1. Margination and rolling

2. Activation and adhesion

3. Transmigration via diapedesis

4. Movement of leukocytes within the tissue via chemotaxis

1. Margination and Rolling With increased vascular permeability, fluid leaves the vessel causing

leukocytes to settle-out of the central flow column and “marginate” along the endothelial surface.

• Endothelial cells and leukocytes have complementary surface adhesion molecules which briefly stick and release causing the leukocyte to roll along the endothelium until it eventually comes to a stop as mutual adhesion reaches a peak

E-selectin is also responsible for slow rolling interactions below 5mm/s and possibly the initiation of adhesion.

A chemokine such as IL-8 binds to a G-protein-linked receptor on the neutrophil, triggering an activating signal.

P-selectin is the most important selectin involved in rolling. Upon stimulation by trauma, P-selectin is rapidly surface-

expressed on the venular endothelium, and it makes the endothelium "sticky" to leukocytes.

2. Activation and adhesion• Rolling comes to a stop and adhesion results

• Interactions between integrins (leukocyte: LFA-1, Mac-1, VLA-4) and their immunoglobulin ligands (ICAM-1, VCAM-1) expressed on the endothelium mediate the adhesion of leukocytes. (ICAM-1 binds LFA-1/Mac-1, VCAM-1 binds VLA-4)

• Migration across the endothelium

• Occurs after adhesion within system of venules and capillaries via PECAM-1 (CD31) (platelet endothelial cell adhesion molecule) on endothelial cells, neutrophils, monocytes/macrophages, lymphocytes

3. Transmigration (diapedesis)

Mediators of Inflammation

Derive from cells or plasma proteins Cell-derived stored in granules and can be rapidly

secreted by exocytosis or synthesized de novo Plasma-derived produced in liver and present in

blood as inactive precursors Active mediators can be produced in response to

stimuli One mediator can stimulate release of other mediator Vary in range of cellular targets Short-lived

Cell-derived MediatorsMediator Sources Actions

Histamine Mast cells, basophils, platelets

Vasodilation, ↑ vascular permeability, endothelial activation

Serotonin platelets Vasodilation, ↑ vascular permeability

Prostaglandins Mast cells, leukocytes Vasodilation, pain, fever

Leukotriens Mast cells, leukocytes ↑ vascular permeability, chemotaxis, leukocyte adhesion and activation

Platelet-activating factor (PAF)

neutrophils, basophils, macrophages, mast cells

Vasodilation, ↑ vascular permeability, leukocyte adhesion, chemotaxis, degranulation, oxidative burst

Inflammatory Mediators

• Platelet-activating factor (PAF)

– It is generated from a lipid complex stored in cell membranes;

– It affects a variety of cell types and induces platelet aggregation;

– It activates neutrophils and is a potent eosinophil chemoattractant;

– It contributes to extravascularization of plasma proteins and so, to edema.

Cell-derived MediatorsMediator Sources Actions

Reactive oxygen species leukocytes Killing of microbes, tissue damage

Nitric oxide Endothelium, macrophages

Vascular smooth muscle relaxation, killing of microbes

Cytokines (TNF, IL-1) Macrophages, endothelium, mast cells

Endothelial activation, fever, pain, anorexia, hypotension, ↓ vascular resistance (shock)

Chemokines Leukocytes, macrophages

Chemotaxis, leukocyte activation

Plasma protein-derived mediatorsMediator Source Actions

Complement products (C5a, C3a, C4a)

Plasma (produced in liver) Leukocyte chemotaxis and activation, vasodilation

Kinins (bradykinin) Plasma (produced in liver) ↑ vascular permeability, smooth muscle contraction, vasodilation, pain

Proteases activated during coagulation

Plasma (produced in liver) Endothelial activation, leukocyte recruitment

Mediators of Inflammation

Role of mediators in inflammationRole in Inflammation Mediators

Vasodilation Prostaglandins, NO, Histamine

↑ vascular permeability Histamine, serotonin, C3a, C5a, bradykinin, leukotrienes C4, D4, E4, PAF, Substance P

Chemotaxis, leukocyte recruitment and activation

TNF, IL-1, chemokines, C3a, C5a, Leukotriene B4

Fever IL-1, TNF, Prostaglandins

Pain Prostaglandins, bradykinin

Tissue damage Lysosomal enzymes of leukocytes, reactive oxygen species, NO

The cells and mediators involved in an acute inflammatory response.

The cytokines TNF and interleukin 1 (IL–1) are released first and initiate

several cascades.

Short term friend, Long term foe

Types of Inflammation

1. Acute Inflammation

2. Chronic Inflammation

It is rapid response to injury or microbes and other foreign substances that is designed to deliver leukocytes and plasma

proteins to sites of injury

It is inflammation of prolonged duration (week to months to years) in which active inflammation, tissue injury and healing

proceed simultaneously

Differences between acute and chronic inflammation

Features Acute inflammation Chronic inflammation

Duration of corse Short [days] Long [ weeks to months]

Causative agents Physical and chemical damages; pathogen invasion

Presistent infection; presence of forein bodies; autoimmunity

Cardinal signs Pain; heat, redness, swelling; loss of function

Absent in any of cardinal signs

Fundamental cells Neutrophilsmacrophages

Lymphocytes [T and B]; macrophages; fibroblasts

Fluid Exudation and Edema

Present Absent

Fibrosis Absent Present

Angiogenesis (new vessel formation)

Absent Present

Primary Mediators Serotonin; Histamine; prostaglandins; tromboxane

IFN, TNF, Hydrolyzing enzymes

Differences between acute and chronic inflammation - Pathogenesis

Acute inflammation Chronic inflammation

1. Increased blood flow a. Transient vasocontriction upon

endothelial injuryb. Followed by released of cytokines

that promotes vasodilation leads to warmness and redness of injured area

2. Increased capillary permeability3. Migration of neutrophilsa. Rollingb. Adhesionc. Diapedesis4. Chemotaxis /movements of

neutrophils to the injurous agents/5. Leukocytes recruitment and

activation /leukocytosis/

1. Infiltration of mononuclear phagocyting cells

a. Macrophages /circulate as monocytes and reach site of injury within 24-48 hours and transform. Activated by numerous cytokines from the injured site/

b. T and B cells /Recruited and activated by APC like macrophages and DC/

c. B cells will be become plasma cells and produce antibodies

d. T cells will produce cytokines to activated the B cells and also macrophages

2. Tissue destruction (due to massive production of ROS, hydrolytic enzymes)

3. Tissue repair (Angiogenesis at the injured sites. Formation of granulomas. Fibrosis)

Inflammation declines spontaneously Mediators of inflammation are produced in rapid bursts only while the stimulus persists Mediators have short half-lives and are degraded after their release. Neutrophils also have short half-lives in tissues and die by apoptosis within a few hours after leaving the blood

Active termination mechanisms include Switch from pro-inflammatory leukotrienes to anti-inflammatory lipoxins Release of anti-inflammatory cytokines, including transforming growth factor-β (TGF-β) and IL-10, from macrophages Production of anti-inflammatory lipid mediators, called resolvins and protectins

Termination of acute inflammation

Lymphocyte recirculation• Lymphocyte extravasation is similar to neutrophil extravasation,

• Lymphocytes continuously travel from the blood to secondary lymphoid tissues and then return to the blood.

• Lymphocytes leave blood via high endothelial venules (HEVs) (HEVs - need to allow egress of naive cells from the circulation)

• Lymphocyte trafficking exposes antigen to a large number of lymphocytes

Extravasation of lymphocytes includes the same basic steps as neutrophil extravasation but some

of the cell-adhesion molecules differ.

• Activation of the integrin LFA-1, induced by chemokine binding to the lymphocyte, leads to firm adhesion followed by migration between the endothelial cells into the tissue

Naive Lymphocytes Recirculateto Secondary Lymphoid Tissue

Naive T cells tend to home to secondary lymphoid tissues through their HEV regions.

Naive lymphocytes express L-selectin

The initial interaction involves the homing receptor L-selectin and mucin-like cell-adhesion molecules such as CD34 or GlyCAM-1 expressed on HEV cells.

• A naive lymphocyte is not able to mount an immune response until it has been activated to become an effector cell.

• Activation of a naive cell occurs in specialized microenvironments within secondary lymphoid tissue (e.g., peripheral lymph nodes, Peyer’s patches, tonsils, and spleen)

Naive Lymphocytes Recirculateto Secondary Lymphoid Tissue

A second subset of memory/effector cells displays preferential homing to the skin.

This subset also expresses low levels of L-selectin but displays high levels of cutaneous lymphocyte antigen (CLA) and LFA-1, which bind to E-selectin and ICAMs on dermal venules of the skin.

Effector and Memory Lymphocytes AdoptDifferent Trafficking Patterns

Chemokine are important in controlling cell traffic to lymphoid tissues

Naive T cells:

• Express chemokine receptors CCR7 and CXCR4 which allow them to respond to chemokines (CCL21) expressed in lymphoid tissues;

• Respond to chemokines CCL18 and CCL19 produced by dendritic cells, which is thought to direct them to the appropriate T cell areas of lymph node where dendritic cells can present antigen to them

B cells:

• Express CCR7 and use similar mechanisms to migrate into the lymphoid tissues.

• B cells also express CXCR5, which allows them to respond to CXCL13, a chemokine produced in lymphoid follicles – B cells are therefore directed to the B cell areas of the node.

Thank you for your attentionThank you for your attention

• The duration and intensity of the local acute inflammatory response must be carefully regulated to control tissue damage and facilitate the tissue-repair mechanisms that are necessary for healing.

• TGF-β has been shown to play an important role in limiting the inflammatory response. It also promotes accumulation and proliferation of fibroblasts and the deposition of an extracellular matrix that is required for proper tissue repair.

Process of inflammation

The leukocyte-extravasation cascade• Leukocytes leave the vasculature routinely through the

following sequence of events:

– Margination and rolling

– Activation, Adhesion and transmigration

– Chemotaxis

They are then free to participate in: phagocytosis and degranulationLeukocyte-induced tissue injury

• Complement fragments and cytokines– It stimulates chemotaxis of neutrophils, eosinophils and

monocytes;– C3a, C5a increase vascular permeability;

• Cytokines – Interleukins (IL-1, IL-6, IL-8)• Stimulates the chemotaxis, degranulation of neutrophils and

their phagocytic activity• Induce extravascularization of granulocytes• Fever

– Tumor necrosis factor (TNF) and IL-8• Leukocytosis• Fever• Stimulates prostaglandins production

Inflammatory Mediators

• As an inflammatory response develops, various cytokines and other inflammatory mediators act upon the local blood vessels, inducing increased expression of endothelial Cell Adhesion Molecules (CAM).

• The vascular endothelium is then said to be activated, or inflamed.

The process by which leukocytes migrate in response to a chemical signal is called chemotaxis.

• Exogenous agents:Bacterial products

• Endogenous productsComponents of complement C5sLeukotriens LTB4Cytokines / Chemokines IL-8

• Prostaglandins

– Prostaglandins contribute to vasodilation, capillary permeability, and the pain and fever that accompany inflammation.

– The stable prostaglandins (PGE1 and PGE2) induce inflammation and potentiate the effects of histamine and other inflammatory mediators:

– They cause the dilation of precapillary arterioles (edema), lower the blood pressure, affect the phagocytic activity of leukocytes.

– The prostaglandin thromboxane A2 promotes platelet aggregation and vasoconstriction.

Inflammatory Mediators

• Leukotrienes

– The leukotrienes have been reported to affect the permeability of the postcapillary venules, the adhesion properties of endothelial cells, and stimulates the chemotaxis and extravascularization of neutrophils, eosinophils, and monocytes.

Inflammatory Mediators

Inflammatory Mediators

• Histamine

– It is found in high concentration in platelets, basophils, and mast cells.

– Causes dilation and increased permeability of capillaries (it causes dilatation of precapillary arterioles, contraction of endothelial cells and dilation of postcapillary venules).

Inflammatory Mediators

• Platelet-activating factor (PAF)

– It is generated from a lipid complex stored in cell membranes;

– It affects a variety of cell types and induces platelet aggregation;

– It activates neutrophils and is a potent eosinophil chemoattractant;

– It contributes to extravascularization of plasma proteins and so, to edema.

Inflammatory Mediators• Plasma Proteases

– The plasma proteases consist of:

• Kinins

– Bradykinin - causes increased capillary permeability (implicated in hyperthermia and redness) and pain;

• Clotting factors

– The clotting system contributes to the vascular phase of inflammation, mainly through fibrin peptides that are formed during the final steps of the clotting process.

Many of the integrins can bind to more than one ligand.e.g.:

• LFA-1 present on most lymphocytes binds to both intercellular CAM-1 (ICAM-1) and ICAM-2, which are expressed on endothelium;

• VLA-4 binds to vascular CAM-1 (VCAM-1) expressed on endothelium, or to fibronectin (an extracellular matrix component)

Chronic inflammation = long duration

• Duration - from several months to years

• Causative agent - non-degradable pathogens that cause persistent inflammation, persistent foreign bodies, overactive immune system reactions, autoimmune disease - self-perpetuating immune reaction that results in tissue damage and inflammation

• Major cells involved - Macrophages, lymphocytes, plasma cells and fibroblasts

• Outcomes - the destruction of tissue by inflammatory cells, thickening and scarring of connective tissue (fibrosis), angiogenesis (new vessel formation) death of cells or tissues (necrosis)

• High endothelial venules (HEV)

– Constitutively present in secondary lymphoid tissue

– Need to allow egress of naive cells from the circulation

• Post-capillary venules (PCV)

– Present in non-lymphoid tissues

– Injury and inflammation alters morphology to resemble HEV

– Need to allow egress of effector/memory cells to sites of infection

• Unlike naive lymphocytes, subsets of the memory and effector populations exhibit tissue-selective homing behavior.

A mucosal homing subset of memory/effector cells has high levels of the integrins LPAM-1and LFA-1, which bind to MAdCAM and various ICAMs on intestinal lamina propria venules

Effector and Memory Lymphocytes AdoptDifferent Trafficking Patterns

Inflammation-induced lymphocyte recirculation

• During the initiation of an adaptive immune response, a large number of naive lymphocytes are recruited specifically to the LNs draining the site of immunization or infection.

• This process facilitates the search of rare antigen specific lymphocytes by increasing the efficiency of screening cognate lymphocytes within the secondary lymphoid organs.

• Increased input of naive lymphocytes to the LN is in part mediated by a rapid increase in the number of HEVs.