Inflammation and Repair - 4

Dr.CSBR.Prasad, M.D.

v3-CSBRP-May-2012

Recognition of Microbes and Recognition of Microbes and Dead Tissues Dead Tissues

• Recruitment• Recognition• Removal

3Rs

v3-CSBRP-May-2012

Recognition of Microbes and Recognition of Microbes and Dead Tissues Dead Tissues

Leukocytes express several receptors that recognize external stimuli and deliver

activating signal

v3-CSBRP-May-2012

v3-CSBRP-May-2012

v3-CSBRP-May-2012

v3-CSBRP-May-2012

Recognition of Microbes and Recognition of Microbes and Dead Tissues Dead Tissues

Leukocyte – Receptors:1. Toll-like receptors (TLRs) 2. G protein – coupled receptors 3. Receptors for opsonins 4. Receptors for cytokines

v3-CSBRP-May-2012

Recognition of Microbes and Recognition of Microbes and Dead Tissues Dead Tissues

Leukocyte – Receptors:1. Toll-like receptors (TLRs) Bind microbial products 10 mammalian TLRs have been identified Mediate cellular responses to bacterial products

LPS / EndotoxinBacterial proteoglycansLipidsUnmethylated CpG nucleotides (abundant in bacteria /

viruses)Function through receptor-associated kinases to stimulate

the production of microbicidal substances and cytokines by the leukocytes

v3-CSBRP-May-2012

Recognition of Microbes and Dead Recognition of Microbes and Dead Tissues Tissues

Leukocyte – Receptors:1. Toll-like receptors (TLRs)

2. G protein–coupled receptors Found on neutrophils, macrophagesRecognize:

Bacterial peptides with N-formylmethionyl residuesChemokinesProducts of complement such as C5aLipid mediators [PAF, PGs, and LTs]

Ligand binding induces ExtravasationProduction of microbicidal substances (ROS).

v3-CSBRP-May-2012

Recognition of Microbes and Recognition of Microbes and Dead Tissues Dead Tissues

Leukocyte – Receptors:1. Toll-like receptors (TLRs)2. G protein–coupled receptors

3. Receptors for opsonins: Leukocytes express receptors for proteins that coat

microbesOpsonins include: Ig, C, and lectinsPhagocytes have:

• FcγRI (for Fc fragment)• CR1 (for C3b)• Receptor for plasma Lectins [mannan-binding lectin]

Receptors promotes phagocytosisv3-CSBRP-May-2012

Receptors for opsonins

v3-CSBRP-May-2012

Recognition of Microbes and Recognition of Microbes and Dead Tissues Dead Tissues

Leukocyte – Receptors:1. Toll-like receptors (TLRs)2. G protein–coupled receptors3. Receptors for opsonins

4. Receptors for cytokines: Leukocytes express receptors for cytokines

Interferon-γ (IFN-γ)IFN-γ is the major macrophage-activating

cytokine

v3-CSBRP-May-2012

v3-CSBRP-May-2012

Removal of the Offending Agents Removal of the Offending Agents

v3-CSBRP-May-2012

Removal of the Offending Agents Removal of the Offending Agents

1. Recognition of microbes by the receptors 2. Leukocyte activation:

o Increase in cytosolic Ca2+ & o Activation of enzymes:

Protein kinase C and Phospholipase A2

3. Destruction of microbes o Phagocytosis and o Intracellular killing

v3-CSBRP-May-2012

Killing & DegradationKilling & Degradation

• Antibacterial substances kill the bacteria • Killed bacteria are degraded by the

hydrolytic enzymes• These mechanisms may not degrade

some eg: Myc.TB

v3-CSBRP-May-2012

Killing & DegradationKilling & Degradation

Following mechanisms facilitate this process:

A. INTRACELLULAR Mechanisms:1. Oxidative – by free radicals of O2

i. MPO-dependedntii. MPO-independent

2. Oxidative – by lysosomal granules3. Non-oxidative mechanisms

B. EXTRA CELLULAR Mechanisms

v3-CSBRP-May-2012

Killing & DegradationKilling & Degradation

Following mechanisms facilitate this process:

A. INTRACELLULAR Mechanisms:1. Oxidative – by free radicals of O2

• This mechanism produces reactive O2 metabolites (O.

2, H2O2, OH., HOCl, HOI, HOBr)

• Respiratory burst by activated leucocytes requires presence of NADPH oxidase

• Liberation of superoxide anion O.2

v3-CSBRP-May-2012

v3-CSBRP-May-2012

Superoxide is subsequently converted into H2O2 which has

bactericidal properties

v3-CSBRP-May-2012

Superoxide radicals O.2 are

utilized in MPO-dependent and MPO-independent killing

v3-CSBRP-May-2012

MPO-dependent killingH2O2-MPO halide system

v3-CSBRP-May-2012

v3-CSBRP-May-2012

Killing & DegradationKilling & Degradation

Following mechanisms facilitate this process:

A. INTRACELLULAR Mechanisms:1. Oxidative – by free radicals of O2

i. MPO-dependedntii. MPO-independent

2. Oxidative – by lysosomal granules3. Non-oxidative mechanisms

B. EXTRA CELLULAR Mechanisms

v3-CSBRP-May-2012

Killing & DegradationKilling & Degradation

Oxidative – by Lysosomal granules

• The preformed lysosomal granules discharged into the phagosomes

• They inlcude proteases, trypsinase, phospholipase and ALP

• This induces proteolysis

v3-CSBRP-May-2012

Killing & DegradationKilling & Degradation

Following mechanisms facilitate this process:

A. INTRACELLULAR Mechanisms:1. Oxidative – by free radicals of O2

i. MPO-dependedntii. MPO-independent

2. Oxidative – by lysosomal granules3. Non-oxidative mechanisms

B. EXTRA CELLULAR Mechanisms

v3-CSBRP-May-2012

Killing & DegradationKilling & DegradationNon-oxidative mechanisms (do not require oxygen for bactericidal activity)

Include the following:1. Granules:

lysosomal hydrolases, cationic proteins, lipases, DNAses

These enzymes cause lysis with in phagosome 2. Nitric oxide:

Formed by nitric oxide synthase Similar to ROS in their action Potent microbial killers Produced by endothelial cells and by activated

macrophages v3-CSBRP-May-2012

ONOO. damages the lipids, proteins, and nucleic acids

Killing & DegradationKilling & Degradation

v3-CSBRP-May-2012

Killing & DegradationKilling & Degradation

Following mechanisms facilitate this process:

A. INTRACELLULAR Mechanisms:1. Oxidative – by free radicals of O2

i. MPO-dependedntii. MPO-independent

2. Oxidative – by lysosomal granules3. Non-oxidative mechanisms

B. EXTRA CELLULAR Mechanismsv3-CSBRP-May-2012

Killing & DegradationKilling & Degradation

EXTRA CELLULAR Mechanisms:

Immune mechanismso Ab mediated lysiso Cell mediated cytotoxicity

v3-CSBRP-May-2012

Is inflammation Is inflammation always protective?always protective?

v3-CSBRP-May-2012

Release of Leukocyte Products Release of Leukocyte Products and and

Leukocyte-Mediated Tissue Injury Leukocyte-Mediated Tissue Injury

v3-CSBRP-May-2012

Release of Leukocyte Products and Release of Leukocyte Products and Leukocyte-Mediated Tissue Injury Leukocyte-Mediated Tissue Injury

• Normal tissue is also damaged in some inflammatory processes

• These mechanisms are similar to antimicrobial defense

• Once the leukocytes are activated, their efector mechanisms do not distinguish between offender and host

v3-CSBRP-May-2012

Release of Leukocyte Products and Release of Leukocyte Products and Leukocyte-Mediated Tissue Injury Leukocyte-Mediated Tissue Injury

• Collateral damage is more common in TB, Leprosy and viral infections

• Inflammatory responses to self Ags – Autoimmunity

• Excessive reaction to harmless environmental substances may result in Allergic diseases eg: asthma

v3-CSBRP-May-2012

Clinical Examples of Leukocyte-Induced Injury

Disorder Cells & molecules involvedACUTEARDS Neutrophils Transplant Rejection Lymphocytes, Abs, CAsthma EøGlomerulonephritis PMNs, Mø, C, AbsSeptic shock CytokinesCHRONICArthritis Lymphocytes, macrophages; AbsAtherosclerosis Macrophages, LymphocytesPulmonary fibrosis Macrophages, Fibroblasts

v3-CSBRP-May-2012

Mechanisms of entry of lysosomal contents into the extracellular milieu

• Frustrated phagocytosis• Phagocytosis of membrane-damaging

substances• Premature release of lysozymes before

the formation of phagosome

v3-CSBRP-May-2012

Mechanisms of entry of lysosomal contents into the extracellular milieu

Premature release of lysozymes before the formation of phagosome

v3-CSBRP-May-2012

Defects in Leukocyte Function Defects in Leukocyte Function

v3-CSBRP-May-2012

• Inherited defects in leukocyte adhesion – LAD

• Inherited defects in phagolysosome function – Chédiak-Higashi syndrome

• Inherited defects in microbicidal activity – Chronic granulomatous disease

• Acquired deficiencies – Cancer chemotherapy

Defects in Leukocyte FunctionDefects in Leukocyte Function

v3-CSBRP-May-2012

Disease Defect GENETIC

LAD Defective adhesion molecules

CGD Decreased oxidative burst

MPO deficiency defective MPO—H2O2 system

Chédiak-Higashi syndrome Lysosomal membrane

ACQUIRED

Bone marrow suppression: tumors, radiation, and chemotherapy

Production of leukocytes

Diabetes, malignancy, sepsis, chronic dialysis

Adhesion and chemotaxis

Leukemia, anemia, sepsis, diabetes, malnutrition

Phagocytosis and microbicidal activity v3-CSBRP-May-2012

Chediak-Higashi Syndrome

v3-CSBRP-May-2012

E N DE N D

v3-CSBRP-May-2012

Laboratory Findings Laboratory Findings in Inflammationin Inflammation

v3-CSBRP-May-2012

Complete Blood Count (CBC)

Total RBCTotal WBC Hemoglobing(HB) Hematocrit (HCT) RBC Indices: MCV,MCH,MCHC WBC Differential

v3-CSBRP-May-2012

Laboratory Findings in Inflammation

Leukocytosis :15,000-20,000 cells/ul (4,000-10,000 cells/ul)

Leukemoid reaction:40,000-100,000 cells/ul

v3-CSBRP-May-2012

Laboratory Findings in Inflammation

“Left Shift”: an increase in the number of immature neutrophils

Immature neutrophils: Bands or stabs

Meta or Juvenile

Myleocytev3-CSBRP-May-2012

“Left Shift”

Baso Eos Meta Stabs Segs Lymph Mono

70 20 3321 1

Normal

75 8 11210 3

Left Shift

v3-CSBRP-May-2012

Laboratory Findings in Inflammation

Neutrophilia : bacterial

Lymphocytosis : viral

Leukopenia:many viruses

Eosinophilia: parasites & allergies

v3-CSBRP-May-2012

Laboratory Findings in Inflammation

Erythrocyte Sedimentation Rate (ESR) will be increased

v3-CSBRP-May-2012

Erythrocyte Sedimentation Rate (ESR)

0

10

20

30

40

50

60

70

80

90

100

mm

1hr

The distance, in mm, the RBC fall in 1 hr

is the Sed Rate

0

10

20

30

40

50

60

70

80

90

100

mm

v3-CSBRP-May-2012

Acute Phase Proteins

During an inflammatory response a number of interleukins(IL) are produced

IL-6 stimulates the hepatic production of a number of proteins ,called acute phase

proteins

v3-CSBRP-May-2012

Acute Phase Proteins

Fibrinogen

C-Reactive Protein (CRP)

C3

C4

Ceruloplasminv3-CSBRP-May-2012

Acute Phase ProteinsAcute Phase Proteins are normally found

in the blood at low concentrations, but following hepatic stimulation by IL-6

their concentration increases

Detection of elevated levels of acute phase proteins is an indication of an

inflammatory responsev3-CSBRP-May-2012

v3-CSBRP-May-2012

Ligands• In biochemistry and pharmacology, a ligand (Latin ligare = to bind) is a

substance that is able to bind to and form a complex with a biomolecule to serve a biological purpose.

• In a narrower sense, it is a signal triggering molecule, binding to a site on a target protein.

• The binding occurs by intermolecular forces, such as ionic bonds, hydrogen bonds and Van der Waals forces. The docking (association) is usually reversible (dissociation). Actual irreversible covalent binding between a ligand and its target molecule is rare in biological systems. In contrast to the meaning in metalorganic and inorganic chemistry, it is irrelevant whether the ligand actually binds at a metal site, as it is the case in hemoglobin.

• Ligand binding to a receptor alters the chemical conformation, that is the three dimensional shape of the receptor protein. The conformational state of a receptor protein determines the functional state of a receptor. Ligands include substrates, inhibitors, activators, and neurotransmitters. The tendency or strength of binding is called affinity.

• Radioligands are radioisotope labeled compounds and used in vivo as tracers in PET studies and for in vitro .

v3-CSBRP-May-2012

WOW!

The movement of leucocytes from out of the blood vessels into the tissues spaces is known as

DIAPEDESIS

v3-CSBRP-May-2012

O2.

Fe++

(Hydroxyl radical)

MPO-independent killingMPO-independent killing

v3-CSBRP-May-2012