Immunodiagnostics Cytokines

What are cytokines?

Small soluble proteins that serve as chemical messengers

-Enables communication and coordination between widely dispersed immune cells

Cytokines regulate?

Both innate immunity and adaptive immune response

Cytokines are often released to in response of?

Infectious stimuli

-LPS

-Flagellin

Define Autocrine

Cytokine that acts locally

-Impacts only the producing cell

Define Paracrine

Impacts a few nearby cells

Define Endocrine

Cytokines that transmit their messages throughout the entire body

What are the properties of Cytokines?

A network of cytokines work best

-Pleiotropy

=One cytokine has many different actions

-Redundancy

=Different cytokines have the Same effect

-Synergy

=Some cytokines enhance each others effects

-Antagonism

=Some cytokines Counteract each other

What is meant by a Cytokine Cascade?

One Cytokine induces the production of additional cytokines

Describe the regulation and dysregulation of cytokines:

1. Balanced signaling

-Proinflammatory cytokines activate immune defense

-Antinflammatory cytokines shut them down once threat is controlled

2. Controlled production

-Cytokine synthesis is tightly switched on and off

3. Limited action

-Mechanisms like feedback inhibitors and soluble receptors prevent prolonged signaling

Name the Proinflammatory cytokines

1. Interleukin-1

2. Tumor necrosis factor (TNF-a)

3. Interleukin-6

4. Chemokines

5. Interferons gamma

Name the anti-inflammatory cytokines

1. Transforming growth factor-B (TGF B)

2. IL-1RA

3. IL-10

4. IL-13

5. IL-35

What is the main role of cytokines in Innate Immune response

1. Reduce inflammation

2. Recruit effector cells (neutrophils/monocytes) into inflammed tissues

3. Produce systemic effect like fever or increased APRs

What are the five classes of cytokines in innate immunity?

1. Interleukins

2. Tumor necrosis factors

3. Chemokines

4. Growth factors

5. Interferons

What triggers Interleukins?

Microbial products

Describe IL-1a

1. Produced by phagocytes and epithelial cells

2. Stays in cytoplasm and only released during cell death

3. Attracts immune cells to damaged tissues

Describe IL-1B

1. Secreted from macrophages, monocytes, and DCs

2. Major driver of systemic inflammation, production of acute phase proteins and FEVER

Describe IL-1RA

1. IL-1 receptor ANTAGONIST

2. produced by macrophages/monocytes/DCs

3. Blocks IL-1a activity (anti-inflammatory)

Describe IL-6

1. Pleiotropic and plays a role in acute phase reactions

2. Stimulates fever with IL-1

3. Activates B and T Cells

-Stimulates B cells to proliferate and differentiate into plasma cells

What is the clinical relevance of Elevetaed IL-6?

Autoimmune diseases, cytokine storm, and severe covid-19

Describe Tumor Necrosis Factor Alpha

1. Most prominent cytokine of TNF

2. Major trigger = LPS from GN bacteria

3. Comes from activated monocytes and macrophages

Describe the Local effects of TNFa

1. Vasodialation and increase in vascular permeability

-Immune cells and proteins can enter tissue

2. Increased MHC class II, adhesion molecules and chemokines

3. Activates macrophages and other innate cells

Describe the Systemic effects of TNFa

1. Works with IL-1 to produce fever and APRs

2. Increase Hematopoiesis

3. EXCESS TNFA = SEPTIC SHOCK

Describe Chemokines

1. Enhance motility and promote migration of WBCs towards chemokines (Source)

2. Modulate adhesion of WBCs to endothelial cells lining the blood vessels facilitating diapedesis

-Involved in inflammatory response of many diseases

What four main families are chemokines based on the position of N-terminal cysteine residues?

CXC

CC

C

CX3C

Describe Transforming Growth Factor Beta

1. Induces antiproliferative activity in many cells

2. Regulates cell growth, differentiation, apoptosis, migration and the inflammatory response

3. Helps down-regulate the inflammatory response when no longer needed

What if TGF-B really important for?

Tissue remodeling and wound healing

-Stimulates fibroblasts and extracellular maxtrix deposition

-Can lead to scarring

Describe Hematopoietic Growth Factors

1. Stimulate proliferation, differentiation and maturation of progenitor cells in the bone marrow

2. Includes SCF, IL-3 and colony-stimulating factors

List the types of hematopoietic growth factors:

1. Erythropoietin

2. Thrombopoietin

3. Granulocyte colony-stimulating factor

4. Macrophage colony-stimulating factor

5. Granulocyte-macrophage colony-stimultaing factor

Describe Interferons

Part of our innate defense against both DNA and RNA viruses

-Interfere with viral replication

-Activate NK cells

-Enhance expression of MHC class I proteins

-Used as treatments for some autoimmune diseases

Differentiate between IFNa and IFNB

1. IFNa

-Produced by dendritic cells and macrophages

2. IFNB

-Made by a variety of virus-infected cells

Describe Interferon-γ

1. Primary cytokine of Th1 cells in adaptive immune

2. Stimulates antigen presentation by class I and II MHC molecules

3. Potent activator of macrophages

4. Regulate CD4+ Th cells, CD8+ cytotoxic cells and NK cells

Describe Interferon-λ

1. Mainly produced by DCs

2. Primary role in defense against viral infections at epithelial barriers of the GI and respiratory tract

3. Modulate innate and adaptive immune responses, autoimmunity and tumor growth

Cytokines in Adaptive immunity are secreted mainly by?

T helper cells

What are the 4 subclasses of T helper cells?

1. Th1

-Induced by DC production of IL-12 and IFNy

2. Th2

-Induced by IL-4, IL-25, IL-33

3. Th17

-IL-1, IL-6, IL-23, TGFB

4. Follicular helper T cells

-IL-6, 12, 21, 23 and TGFB

Describe Th1 Cells

1. Induced by IL-12 and IFNy

2. Secrete IFNy and TGF-B

3. Function: to activates CD8+ cytotoxic T cells and macrophages

4. Boosts cell-mediated immunity against intracellular pathogens

Describe Th2 cells

1. Induced by IL-4

2. Secrete IL-4 and IL-5

3. Activates mast cells, eosinophils and B cells

4. Boosts humoral immunity against parasites

-B cells produces IgE (Involved in allergic response)

Describe Th17 cells

1. Induced by IL-6 and IL-23

2. Secretes IL-17 and IL-22

3. Recruits and activates neutrophils, Strengthens mucosal barriers and antimicrobial protein production

4. Enhances barrier immunity at mucosal to defend against extracellular bacteria and fungi

Describe T regulatory cells

1. Induced by TGF-B

2. Secretes IL-10 and TGF-B

3. Inhibits activation of T cells, B cells and APCs

4. Suppresses excessive immune responses, promotes resolution of inflamation, maintains self-tolerance

Massive overproduction of cytokines can cause a condition called?

Hypercytokinemia aka Cytokine storm

-Dysregulation of pro-inflammatory cytokines

-Leads to hypotension, multi-organ failure and death

What is an example of cytokine therapy?

1. Administration of hematopietic growth factors to improve RBC count in severe anemia

2. IFNa used to boost anti-viral immunity in HepB and C infections

3. Many cytokines stimulate anti-tumor responses in cancer

What is an Anti-cytokine therapy?

Targets chronic inflammation

1. Disrupts interaction between cytokines and their receptors

2. Monoclonal antibodies function as cytokine agonists

3. Hybrid proteins contain cytokine receptor binding site to Ig constant regions block cytokine activity

Whats the difference between immunoassays and Bioassays?

Immunoassays

-Measure presence and quantity of cytokines

Bioassays

-Measure biological activity of cytokines

What is a sandwich ELISA?

antigen is measured between a layer of capture antibodies and layer of detection antibodies. Helps to measure cytokine because second reporter antibody shows color change

What are the three clinical assays for cytokines?

1. ELISPOT assay

-Similar to ELISA performed on in-vitro activated WBCS

2. Multiplexed ELISAs

-Can detect pro and anti inflammatory cytokines in one reaction

3. Microbead assays

-Allow for simultaneous detection of multiple cytokines in a single tube

List the ELISpot assay principle steps:

1. Plate is coated with an antibody that capture cytokine of interest

2. Cells of interest are added and secrete cytokine

3. Secreted cytokines are trapped by the anitbodies

4. Detection antibodies or conjugated to enzyme reporter

5. Substrate is added that creates an insoluble colored spot= cytokine secreting cell