Monocytes must also leave the circulation in response to inflammatory signals, and neutrophils and other granulocytes must be able to do so as well.

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1 Immunology Dr. John J. Haddad Chapter 15 Leukocyte Migration and inflammation Lymphocytes must recirculate in the body in search of antigens. Since antigens are brought to lymph nodes and the spleen, the lymphocytes must be equipped with mechanisms to enter these areas. In the case of lymph nodes, lymphoctes must leave the circulation, traverse the secondary lymphoid tissue, and then be returned to the blood if they do not encounter antigen. Sensitized cells must also be able to enter tissues like the skin, mucosa of the gastrointestinal, pulmonary and genitourinary tracts (tertiary extra-lymphoid sites) in response to inflammatory signals. Monocytes must also leave the circulation in response to inflammatory signals, and neutrophils and other granulocytes must be able to do so as well. This chapter deals with how these cells extravasate, or leave the circulation, and in response to what signals. Cell adhesion molecules (or CAMs) play a major role in these processes. There are four basic classes of CAMs (Figure 15-2): Selectins bind to negatively-charged sialic acid-containing carbohydrate moieties on the membranes of other cells in a Ca +2 ion-dependent manner. L-selectin is expressed on most leukocytes, and P-selectin and E-selectin are present on vascular endothelial cells Mucins are proteins rich in the hydroxyl-containing amino acids, serine and threonine, and these residues are highly glycosylated. L-selectin on naïve T cells binds to the mucins CD34 and GlyCAM-1 on high endothelial venules (HEVs) prior to extravasation (see below). The mucin PSGL-1 on neutrophils binds to E-selectin and P-selectin on inflamed vascular endothelial cells. Integrins are heterodomeric cell surface molecules consisting of a variety of α and β chains (See Table 15-1) expressed on leukocytes and also on many other cell types. They adhere to CAMs belonging to the immunoglobulin superfamily. Ig-superfamily adhesion molecules include ICAMs-1, -2 and 3, and VCAM. The adhesion molecule, MAdCAM-1, is expressed on mucosal endothelium, and it is unusual in that it has properties of both the Ig superfamily and mucin classes of adhesion molecules. A number of important adhesion molecules involved in leukocyte extravasation are summarized in Table Neutrophil extravasation In response to signals generated by injury or infection of tissue (see later), CAMs are expressed on blood vessels near the site of injury/infection. These endothelial cells that form these blood vessels now constitute inflamed vascular endothelium. In addition, chemokines are secreted in the vicinity of the injured/infected tissue which diffuse to the vascular endothelium and are suspended there on proteoglycans on the luminal surface. The chemokine IL-8 is among those to which neutrophils respond. Four steps can be distinguished in the process leading to neutrophil extravasation (Figure 15-3): Rolling Activation Arrest/adhesion Extravasation 71

2 Both rolling and arrest/adhesion can be studied in two experimental systems: Intravital microscopy, in which a small blood vessel in a living animal is manipulated so that cells flowing through it can be visualized by a microscope. Inflammation can be stimulated in the vessel, and neutrophils and/or lymphocyte rolling along the vessel wall or adhering to it can be observed. Experimental flow chambers can be constructed in which adhesion molecules such as ICAMs can be coated on the floor of the chamber and neutrophils or lymphocytes can be made to flow across the chamber in a stream of medium. Here, too, both rolling and arrest/adhesion can be studied. Characteristics of the four steps are as follows: 1. Rolling involves the neutrophil making its first contact with E-selectin on the vascular endothelial cell by means of a mucin-like CAM on the neutrophil membrane or sialyl Lewis x, a sialyated lactoseaminoglycan. This causes a temporary tethering, and the force of the blood streaming past breaks the contact, the cell rolls a bit and then becomes tethered again. Some break free and never reattach. Others progress to the next step. 2. In the activation step, local chemoattractants that include chemokines displayed on the proteoglycans on the luminal surface interact with a chemokine receptor on the neutrophil surface. As discussed in Chapter 12, this type of receptor spans the neutrophil s plasma membrane and signals by way of a G protein associated with the cytoplasmic domains of the receptor. This signal produces a conformational change in the integrin(s) on the neutrophil surface. The affinity of the integrin for its receptor on the endothelial cells goes up, and the neutrophil is activated. 3. The activated integrin adheres to the Ig-superfamily CAM on the endothelial cell and the rolling neutrophil is arrested and adheres to the vascular endothelium. 4. The steps by which the adherent neutrophil now begins to crawl and extravasate between the vascular endothelial cells is not well understood, but this is what happens next. Lymphocyte extravasation The extravasation of lymphocytes follows a similar set of events, but the process is controlled so that appropriate subsets of lymphocytes leave the circulation and enter the correct tissues. In general, this process is called lymphocyte homing or trafficking. Migration of naïve T cells into lymph nodes and other secondary lymphoid tissues except spleen is summarized in Figure They enter these tissues through specialized high endothelial venules (HEVs) that consist of cuboidal endothelial cells. These HEVs appear to require an antigenic environment in which to differentiate, since mice raised under germ-free conditions do not develop HEVs. HEVs express tissue-specific cell adhesion molecules called vascular addressins or homing receptors on their luminal surface. These are recognized by cell adhesion molecules on particular populations of lymphocytes that are meant to exit the circulation and enter the secondary lymphoid tissue at this point. The CAMs on the HEVs include mucins and members of the Ig-supergene family. Naïve T cells do not show a preference for a particular secondary lymphoid tissue. Rolling is initiated by L-selectin on the lymphocyte interacting with the vascular addressins CD34 and GlyCAM-1 (both mucins) on the vascular endothelium. Chemokines activate the integrin, LFA-1, on the T lymphocyte, and its adherence to ICAM-1 on the vascular endothelium results in arrest/adhesion. The T lymphocyte then extravasates. Effector T cells and memory T cells follow a similar series of events, except that their exit from the circulation is not at HEVs but rather at sites containing inflamed vascular endothelium. Such sites are recognized by the presence of chemokines and also tissue-specific CAMs. 72

3 For example, mucosal-specific effector T cells contain the integrins LFA-1 and LPAM-1 which bind to ICAM-1 and MAdCAM-1, respectively, on venule endothelia in the lamina propria of the gut. Also, L-selectin on the effector T cell binds to the mucin portion of MAdCAM-1 (Figure 15-5). In contrast, skin-specific effector T cells have a mucin-containing CAM (CLA, for cutaneous lymphocyte antigen) that binds to E-selectin on the dermal venule endothelium, and LFA-1 that binds to ICAM-1 (Figure 15-5). Effector T cells activated in the peritoneal cavity by the superantigen, Staphylococcal enterotoxin B, return to the peritoneal cavity in part by binding of an activated CAM called CD44 to a hyaluronic acid-containing CAM on venules that service the peritoneal cavity (Science 278:541 (1997)). Thus effector T lymphocytes and memory cells tend to home to the region of the body in which they were originally activated by virtue of their surface cell adhesion molecules that recognize vascular addressins on venule endothelium in those tissues. Mediators of Inflammation What are the identities of the mediator that attract neutrophils, monocytes and other cells to sites of tissue injury and infection? Chemokines Small polypeptides of amino acids that attract various leukocytes and cause expression and/or activation of integrins C-C subgroup Contains cysteines that are contiguous. Generally attract monocytes but not neutrophils. Examples are MIP-1, MIP-5 and RANTES. This subgroup is produced by monocytes, macrophages, endothelium and neutrophils, and attracts monocytes, macrophages, B cells and naïve T cells. C-X-C subgroup Contains cysteines separated by another amino acid. Generally attracts neutrophils but not monocytes. Example is IL-8. Secreted by monocytes, macrophages, endothelium, neutrophils and fibroblasts. Attracts neutrophils, basophils and T cells. Chemokines act through cell surface receptors that pass through the plasma membrane 7 times. They are coupled to large heterotrimeric G proteins, and activate a number of signaling pathways that affect the level of camp in the cell (reduce it), and also affect cell adhesion (expression or activation of integrins), cytoskeletal rearrangement, differentiation and proliferation, and actin polymerization. The latter process is due to activation of PLCγ2, an enzyme that cleaves membrane PIP 2 to generate DAG and IP 3 (Figure 15-8). Plasma enzyme mediators These are part of the innate non-specific immunity systems discussed in Chapter 1. They are activated in injured or infected tissue before there is any extravasation of monocytes or neutrophils, and they also continue to work after these processes begin. They all follow endothelial damage and are initiated through the action of the blood clotting protein, Hageman factor. The Kinin system prekallakrein activated Hageman factor kallekrein kininogen 73 bradykinin

4 Bradykinin is a vasoactive amine that causes vascular permeability, contraction of smooth muscle, vasodilation and pain. Kallakrein also cleaves C5 to C5a (analphylatoxin) and C5b. The C5a causes release of granules containing vasoactive amines (e.g. histamine) from mast cells. The blood clotting system activated Hageman factor blood clotting cascade thrombin fibrinogen fibrin + fibrinopeptides Fibrinopeptides induce vascular permeability and neutrophil chemotaxis. The proteoplytic enzyme, plasmin, degrades the blood clot, and the resulting peptides are chemotactic for neutrophils. The complement system The split products of complement (C3a, C4a and C5a) are anaphyatoxins, so activation of complement meads to smooth muscle contraction and vascular permeability. Also, C3q, C5a and C5b67 act together to induce monocytes and neutrophils to exit the vasculature, enter tissues and migrate toward the site of complement activation in the tissues. The result is accumulation of antibody-containing fluid and phagocytic cells at the site of inflammation. The lipid inflammatory mediators Membrane phospholipids are degraded by a phospholipase to arachidonic acid and lyso-platelet activating factor (Lyso-PAF). As shown in Figure 15-11, Lyso-PAF leads to platelet activation and aggregation, which subsequently lead to chemotaxis of eosinophils and degranulation of both eosinophils and neutrophils. The arachidonic acid enters two pathways the cyclooxygenase pathway leading to the synthesis of prostoglandins and thromboxanes, and the lipozygenase pathway that leads to the synthesis of leukotrienes. All of these final products are extremely potent. Thromboxanes lead to vasoconstriction and platelet aggregation. Prostoglandins lead to vascular dilation, an increase in vascular permeability and neutrophil chemotaxis. Leukotriene B4 leads to neutrophil chemotaxis. Leukotrienes C4, D4 and E4 lead to bronchial smooth muscle contraction. The cytokine inflammatory mediators The cytokines IL-1, IL-6 and TNF-α that are secreted during acute and chronic inflammatory responses lead to a variety of redundant and pleiotropic effects such as fever, synthesis of acute phase proteins by the liver and increased vascular permeability (Table 15-3). Also, IFN-γ is important in chronic inflammation by inducing T H 1 cell differentiation. The inflammatory process Neutrophils are the predominant cell type mediating inflammation. They are drawn to the tissues by signals of acute inflammation that act at the vascular endothelium. For example, thrombin and histamine stimulate production of more P-selectin on the vascular endothelium. IL-1 or TNF-α stimulate endothelial cells to express more E-selectin. These selectins adhere to mucins such as PSGL-1 on the neutrophils and initiate rolling. IL-8 and other chemokines activate neutrophil integrins and cause adhesion followed by extravasation. Neutrophils leave the circulation and 74

5 migrate up gradients of chemoattractants (e.g., IL-8, complement split products, fibrinopeptides, prostoglandins, leukotrienes) to the site of inflammation. In response to some of these signals, neutrophils increase expression of Fc receptors and complement receptors which facilitate phagocytosis of antibody- and complement-coated pathogens. Neutrophils also undergo a respiratory burst, generating reactive oxygen and nitrogen species. Release of some of these and of primary and secondary granules containing proteases, elastases, phospholipases and collagenases leads to pathogen destruction and also some tissue injury, the products of which accumulate in the area as pus. Acute inflammatory response Localized response (Figure 15-12) Swelling (tumor) Heat (calor) Redness (rubor) Pain (dolor) Loss of function Generally shows rapid onset and short duration. 1. vascular changes follow tissue injury 2. fluid leaves blood vessels 3. non-specific systems (kinin, clotting, fibrinolytic) are activated, and complement activation, histamine and prostaglandinr release occur 4. within a few hours, neutrophils roll, adhere to endothelial cells, extravasate, migrate to inflammatory site, destroy pathogens and secrete chemokines that attract macrophages hours after inflammatory response began, macrophages arrive, become activated and secrete IL-1, IL-6 and TNF-α. These can cause increased entry and activation of neutrophils and monocytes as well as lymphocytes. 6. The lymphocytes can contribute to the destruction, though the inflammation can proceed and conclude without the involvement of antigen-specific cells. 7. TGF-β can limit response and promote healing through accumulation and proliferation of fibroblasts and laying down of extracellular matrix. Systemic acute phase response Accompanies localized response, and includes: Fever Increased synthesis of ACTH and hydrocortisone Increased production of white blood cells Production of acute-phase proteins by hepatocyte IL-1, IL-6 and TNF-α play roles in induction of fever through the hypothalamus, and also in the synthesis of the acute phase proteins. C-reactive protein, one of the acute phase proteins, binds to the surface of many microorganisms, stimulating complement activation and C3b binding followed by phagocytosis. TNF-α stimulates vascular endothelium and macrophages to secrete M-CSF, G-CSF and GM-CSF, all of which stimulate hematopoiesis and lead to a transient increase in leukocyte production. Chronic inflammatory responses If the antigen persists, inflammation can become chronic. Occurs with difficult-to-clear pathogens. Also, selfantigens can cause continual autoimmune stimulation in chronic inflammatory disease. 75

6 Accumulation and activation of macrophages are hallmarks of chronic inflammation. Cytokines released stimulate fibroblast proliferation and collagen (extracellular matrix) production. Fibrosis occurs at the site. Granuloma forms a central mass of activated macrophages surrounded by activated lymphocytes IFN-γ secreted by T H 1, T C and NK cells activates macrophages. This perpetuates the inflammation. Microbicidal activity leads to tissue destruction. TNF-α produced by also causes tissue damage. A. Cerami and coworkers showed that a macrophage-derived factor was responsible for the severe wasting (loss of weight) seen in rabbits infected with trypanosomes. They purified and named the substance cachectin (the same wasting is seen in cancer patients and is called cachexia). This same wasting syndrome was seen in animals made transgenic for constitutively-expressed TNF-α. Cloning of the gene for cachectin showed that it is, in fact, TNF-α. Chronic inflammatory diseases These include Crohn s disease, Hashimoto s thyroiditis, Diabeted mellitus, Grave s Disease and Ulcerative colitis. HEV-like endothelial cells are observed in tertiary lymphoid areas of tissues experiencing chronic inflammation. These are sites of lymphocyte extravasation and express mucins recognized by the lymphocyte selectins. Trying to inhibit the development of these HEV-like regions may be a sensible approach to reducing the inflammation in such tissues. Other approaches to controlling inflammation include: Reduction of leukocyte extravasation using inhibitors of adhesion (e.g., antibodies to LFA-1 and ICAM-1). Corticosteroids (e.g., prednisone) These cause a decrease in the number of circulating lymphocytes due to induction of apoptosis. These are lipophilic molecules that can enter lymphocytes by diffusing through the lipids of the plasma membranes. They bind to receptors soluble in the cytoplasm and enable the receptor/steroid complex to enter the nucleus and affect gene expression. They also reduce the phagocytic and killing ability of macrophages. Chemotaxis, production of IL-1 and expression of class II MHC molecules are all reduced. Non-steroidal anti-inflammatory drugs (NSAIDs) are often used to treat inflammation and the accompanying pain. These include salicylates and other compounds that inhibit the cyclooxygenase pathway leading to prostaglandins and thromboxanes. The new Cox-2 inhibitors supposedly inhibit a form the cyclooxygenase enzyme specific to the immune system, thereby avoiding side effects caused by inhibition of a form of cyclooxygenase that is more widely distributed in the body and that does not contribute to inflammation. 76