Phagocytosis

1. Phagocytosis Dr Alok Tripathi Department of Biotechnology aquaimmuno@yahoo.co.in 09795894495

9. 1. Endothelial activation and Rolling: Neutrophils constitutively express ligands and receptors (e.g. sialylatedcarbohyrates and L-selectin respectively) which interact with reciprocal receptors and ligands (e.g. P- and E-selectin and GlyCAM-1 respectively) on endothelial cells. Blood vessels near sites of inflammation dilate and blood flow reduces, this allows these interactions to take place and the neutrophilsmarginate and roll along endothelium in these areas. Inflammatory mediators (esp. IL-1 and TNF) increase expression of E and P-selectin on endothelial cells which can then bind mucins (sialyl -Lewisx, PGSL) on the neutrophil and this allows tethering of marginated cells. 2. Activation: activation of the neutrophil is essential for extravasation. Neutrophils are activated by complement components (iC3b, C5a), inflammatorylipid mediators (eg. leukotrienes (LTB4), PAF), cytokines (IL-8; MIP1b) and bacterial products (e.g. N-formylated peptides such as N-formyl-methionyl-leucylphenylalanine (fMLP)). Once activated neutrophils must gain access to the infected tissue. They do this using adhesion molecules (selectins, integrins, intercellular adhesion molecules (ICAMs)). 3. Arrest: activation of neutrophil causes conformational change in integrin (LFA-1), which allows it to bind to ICAM. Local inflammatory mediators also cause upregulation of integrins (e.g. LFA-1, Mac-1) on neutrophils which interact with receptors (e.g. ICAM-1, ICAM-2) upregulated on endothelium. These stronger interactions allow the neutrophil to stop rolling and cross the endothelium into tissue in a process known as diapedesis 4.  Migration: Next, the Neutrophil responds to a group of molecules called chemoattractants to make its way between the endothelial cells of the blood vessel by a process called diapedesis.Chemoattractants include complement protein C5a, bacterial products (e.g. fMLP), lipid mediators (LTB4, PAF) and  cytokines (in particular IL-8, also MIP1b). These chemoattractants also form a chemical gradient in the tissue, and the neutrophilmigrates up this gradient (a process calledchemotaxis) in order to find the site of infection. During this process the neutrophil is activated and ready to kill on its arrival.

10. How are Macrophages activated? Normal tissues harbour low numbers of resident macrophages that are long lived cells (months compared with neutrophils (6h in circulation, 2-3 days in tissue). They come under a number of tissue specific names Kupffer cells (liver), Microglia (brain), Histiocytes (skin), Mesangial cells (kidney). These cells are not activated and would not automatically be able to kill a pathogen – these cells require to undergo activation in order to get their pathogen killing processes going. This is important, as most of the time these macrophages are digesting the normal cell debris or dead cells that result from normal wear and tear in tissues. Classically activated macrophages can be found in 2 activation states: i. Primed – this happens in response to IFNg, which is produced mainly by T cells. This causes macrophages to increase the expression of Class II molecules on their surface, and upregulate phagocytosis and some killing mechanisms. Natural Killer (NK) cells can act as an innate source of IFNg, allowing macrophage activation to occur in advance of adaptive immune responses (see Figure). ii. Hyperactivated – this requires the presence of pathogen derived molecules, the best studied is lipopolysaccharide (LPS), which is abundant on the surface of gram negative bacteria. LPS causes macrophages to secrete inflammatory mediators such as IL-1, IL-6 and TNF, and also fully upregulate their killing machinery However, macrophages can also be alternatively activated through engagement of different cellular receptors, or mediators of the innate and adaptive immune responses (see Figure 1 from Gordon (2003) Nat. Rev. Immunol.) Macrophage numbers in tissues are topped up by migrating monocytes from the blood and approximately 24 hours after infection, large numbers of monocytes are actively recruited from the blood to the site of infection. IL-1 induces expression of ICAM on endothelial cells which interacts with beta integrins (e.g. Mac1(CD11b:CD18)) to induce rolling and adhesion. Like Neutrophils, Monocytes follow gradients of chemokines to the site of infection of which, IL-8 and MCP (monocytechemoattractant protein) are particularly active.

11. How do phagocytes kill pathogens? Once formed, the pathogen-containing phagosome can then fuse with other cellular compartments containing microbiocidal products. Neutrophils have 3 main microbiocidal compartments: Primary granules - contain serine proteases, lysozyme  and phospholipase A2, highly acidic. Secondary granules - similar to primary, but also include lactoferrin, elastase and collagenase Tertiary granules - at leading edge of migrating neurophil and contains gelatinases capable of degrading basement membranes Macrophages kill pathogens by fusing phagosomes with lysosomes which have similar constituents to the neutrophil primary granule.In addition to these mediators, phagocytes also posess a variety of oxygen dependent killing mechanisms. Both phagocytes produce a respiratory burst which produces superoxides and hydrogen peroxide. Neutrophils contain an enzyme, myeloperoxidase, which can convert superoxide into hypochlorite (very effective bleach). Macrophages contain an enzyme called nitric oxide synthase which is activated by IFNg and TNF (or LPS) treatment which produced nitric oxide, which is very effective at killing.