Immunology #2 - Innate Immunity

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Predominant phagocytes

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Neutrophils & Macrophages

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Neutrophils

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- Neutrophils = 60% of the circulating leukocytes in peripheral blood.
- Neutrophils mature from precursor cells in the bone marrow
- 1/2 life ~ 8 hours

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Macrophages

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- Derived from monocytes
- 4% of circulating leukocytes
- When recruited into tissues - macrophages.
- More effective at phagocytosis than monocytes
- Have a much longer half-life than neutrophils
- Following their activation can serve as antigen presenting cells for CD4+ T cells

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Macrophages in different tissues

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- Blood - Monocytes
- Bone marrow - Monoblasts
- CNS - Microglial cells
- Liver - Kupffer
- Synovium - Synoviocytes
- Lungs - Alveolar macrophages

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Recognition of Pathogens

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- 2 Methods: Direct or Opsonin-mediated/Indirect
- Direct = without an intermediary protein
- Indirect = intermediary protein bound to an antigen, with a receptor on the phagocyte

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Direct Recognition

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- Uses pattern recognition receptors, (PRRs)
- PRRs = membranous or cytosolic receptors
- Ligands = PAMPS (pattern activation molecular patterns)

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NLRs

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- Group of cytosolic PRRs “nucleotide-binding oligomerization domain–like receptors"
- Nod1 and Nod2 proteins
- Mutations in Nod2 associated with Crohn’s

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TLRs

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- Group of membranous receptors that exist both on cytosolic vesicles and external cell membranes are the toll-like receptors (TLRs).
- TLRs are so named for their sequence similarity to the Drosphilia protein, Toll
- TLRs are expressed on a various cell types
- e.g. signaling via TLR2 or TLR4 leads to the activation of NF  B - then activates pro-IL-1 an inflammatory cytokine secreted by activated macrophages

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Opsonin-mediated Phagocytosis (Indirect Recognition)

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- Opsonins are the products of:
(i) complement activation (e.g., C3b)
(ii) B cell activation (e.g.,IgG )
(iii) cytokine mediated activation of hepatocytes (e.g., C-reactive protein, CRP).
- Interaction of any of these pathogen-bound opsonins triggers the process of phagocytosis.

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Receptor-Opsonin pairing

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1. Fcγ :FcγR
2. CRP: CRP-BS
3. C3b:CR1

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Phagosome

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- Vesicles that contain the engulfed pathogen
- Phagocytic vacuole serves as the “battlefield”
- Weapons include:
(i) lysosomal enzymes
(ii) reactive oxygen intermediates
(iii) reactive nitrogen intermediates

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Lysosomes

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- Fuse with the phagosome to form a fusion product - lysosomal granules are discharged
- Lysosomal granules contain many enzymes (including lactoferrin, lysozyme, and defensins) that are cytostatic/cytotoxic to microorganisms

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Lactoferrin

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Binds iron, thereby removing an essential ingredient for microbial growth

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Lysozyme

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Destroys muramic acid in bacterial cell walls

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Defensins

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Permeabilize bacterial and fungal membranes

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Myeloperoxidase

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Generates hypochlorite, a potent antimicrobial agent that mediates its function by halogenating bacterial cell walls

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NADPH oxidase

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Phagocytosis is accompanied by a respiratory burst via NADPH oxidase that uses oxygen, in the presence of cytosolic NADPH

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Reactions of NADPH Oxidase

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Nitric oxide

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- Lipid and water soluble gas that is cytotoxic/cytostatic to invading microorganisms.
- Many parasites and other intracellular organisms including viruses, intracellular bacteria, parasites, and fungi are susceptible to NO
- Even MORE powerful when NO reacts with reactive oxygen intermediates and generates reactive nitrogen intermediates (RNIs)

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Synthesis of NO

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L-Arginine to L-Citrulline and NO in the presence of oxygen.

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Roles of Cytokines in Regulation of NO Synthase

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- Activation occurs by 2 signals: TNF and IFNγ
- Down-regulation occurs: IL-10, IL-1 and TFG β
- TFG β = the MOST effective cytokine

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Activation of Macrophages

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- MCP-1 (CCL2) = chemoattractant for monocytes and macrophages
- IL-8 (CXCL8) = chemoattractant for neutrophils

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Cytokines secreted by activates macrophages

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IL-1, IL-6, IL-12, and tumor necrosis factor (TNF)

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TLR Family

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- PRR subtype is the TLR family ( TLR = Toll-like receptor)
- Extracellular with transmembrane domains

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NLR Family

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- Counterpart to the TLR family
- Largest of these families is the NALP/NLRP family of proteins that play a role in inflammation
- NALP3 protein, which has a leucine rich (LRR) domain that serves as the recognition portion for various ligands

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NALP3

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- NALP3 protein is dormant until it binds a ligand (directly or indirectly) to initiate assembly of the NALP3 inflammasome
- Important for processing of the pro-Caspase-1 zymogen to its active form (Caspase-1/IL-1 converting enzyme) induces proteolytic cleavage of
prointerleukin-1b and prointerleukin-18 (pro-IL-18) to their active forms

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The Inflamasome

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Macrophage Receptors

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PRRs, FcγR, CRP-R, CR1

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CD200Rs

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- Expressed primarily on cells of the myeloid lineage and T-cells
- CD4+ T cells express higher amounts than CD8+ T cells
- Memory cells express higher amounts of CD200R than naïve or effector cells

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Eosinophils

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- Bone marrow derived cells that exist both in the circulation and in tissues
- Only a small percentage of eosinophils released from the bone marrow remain in circulation
- 1/2 life ~ 8-10 hours.
- Major role: parasites - helminths
- FcεR that bind to IgE antibodies themselves bound to epitopes on helminths
- Major basic protein (MBP), and eosinophil cationic protein (ECP) = both toxic to helminths other parasites

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NK Cells

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- Arise from bone marrow precursors (lymphoid progenitor cell), and are found predominantly in the blood, spleen, and peritoneal exudate
- NK cells kill infected cells (e.g., viral infections) and some tumors
- Express both NK inhibitory receptors and NK activating receptors that interact with specific self-Class I MHC, MHC class I-like molecules and molecules unrelated to MHC
- IL-12 and IFN-y

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Dendritic Cells

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- Derived from bone marrow progenitor cells
- Most efficient of all antigen-presenting cells, particularly in a primary response to antigen.
- Present in all tissues; they express receptors for chemokines that direct them to the appropriate secondary lymphoid tissues following their encounter with antigen
- Under the influence of GM-CSF, bone marrow derived myeloid precursors mobilize to the circulation where they differentiate into immature dendritic cells - undergo further differentiation and maturation when they endocyose antigen