TARGETING ADHESION MOLECULES
for ANTI-INFLAMMATORY INTERVENTION

   Adhesion molecules, key elements in the regulation of inflammatory cell migration, are emerging as promising new targets for interrupting the inflammatory process. Identifying these endogenous chemicals and defining their actions and regulation is a science all its own, but the products of current research will be the pharmaceutical agents of the near future. Interfering with the action of these compounds by chemical or immune means, or enhancing their action by genetic manipulation have been shown to effectively treat a number of inflammatory and immunodeficiency disorders.
    The processes involved in inflammation are overwhelmingly complex, especially in light of the fact that we are still only learning about them. The initial stage of the inflammatory response involves increased blood supply to locally affected tissues and increased capillary permeability to facilitate two essential aspects of the inflammatory process: 1) Migration of various types of immune cells across local vascular endothelium to sites of injury or infection and 2) Increased vascular permeability to large serum molecules like complement, antibodies, and kininogens. In order to understand the intricacies of adhesion molecules and their involvement in certain disease processes, it is necessary to discuss the larger process of immune cell migration.
    Migration provides the specific functions of each unique inflammatory cell type to local tissues. It is also a means of ensuring that the small number of cells specifically sensitized to a particular antigen are actually exposed to that antigen for processing and mounting an appropriate immune response. Recirculation of antigen-presenting cells (APC’s) into the lymphatic drainage pathways affords lymphocytes access to those antigens in the lymph nodes (or spleen in the case of blood-borne antigen). The secondary lymphoid tissues are the site of initial clonal expansion of antigen-specific lymphocytes in preparation for release into the efferent lymphatics, general circulation, and eventually target tissues.
    The timing of migratory activities as well as the relative numbers of cells involved depend largely on the nature of the antigenic challenge and the site of that challenge. In the case of infection, neutrophils are first on the scene and remain the dominant inflammatory migratory cell type for several days. Lymphocytes and mononuclear phagocytes begin appearing after the first day, followed by CD8+ T-lymphocytes and B-lymphocytes. If the offending antigen is eradicated in this acute process, resolution follows rather quickly; but when the antigen cannot be cleared, chronic inflammation ensues, characterized by accumulation of large numbers of mononuclear phagocytes and CD4+ T-lymphocytes, with relatively few neutrophils. Following asthmatic exacerbations, eosinophils, basophils, and macrophages prevail in the bronchial tissues; and eosinophils accumulate in the case of parasitic infection.

    Migratory leucocytes can be found throughout all tissues under normal circumstances; and those found circulating in the blood or lymph are in transit from one tissue site to another. Various migratory patterns are dictated not only by the particular cell type, but its state of activation or differentiation.
    Neutrophils and monocytes -- the phagocytes -- migrate to tissue sites of infection or trauma from the bone marrow. The monocytes further differentiate into macrophages that may return to secondary lymphoid tissues to act as antigen-presenting cells, while neutrophils remain in peripheral tissues to perform their local functions in mediation of the inflammatory response.
    Virgin lymphocytes migrate to the secondary lymphoid tissues from the thymus and bone marrow for activation and further differentiation. B-lymphocytes and memory T-lymphocytes seed other lymphoid tissues, while activated T-lymphocytes migrate to sites of inflammation.
    Dendritic cells (skin Langerhans cells, for instance), originating as stem cells from the bone marrow and colonizing other organs, may migrate to local lymph nodes to act as APC’s.
    Migratory patterns are also dictated by the location of antigenic challenge. Subpopulations of migratory cells tend to be selective for specific areas of the body, demonstrated by the fact that migratory cells isolated from the gut or the spleen return to those organs upon reinfusion. They seem to know where they belong. Preferential migration can also be a function of the nature of vascular endothelium, which varies greatly in different tissues and areas of the body, even among non-lymphoid vascular beds. But lymphoid endothelium of high endothelial venules (HEV’s) in secondary lymphoid tissues is particularly different from non-lymphoid endothelium, being particularly suited to facilitate large migratory volumes in the lymphoid tissues. These high-volume characteristics tend to be induced at sites of chronic inflammation, which of course facilitates migration into those local tissues.
    The first of the two phases of cell migration requires attachment of a circulating cell to the vascular endothelium. The process is controlled not only by the presence of appropriate adhesion molecules on the surface of the migratory cell in the proper state of activation, but by adhesion molecules expressed on the cell surfaces of vascular endothelium. Determination of which cells migrate across different endothelial beds is made by 1) the surface charge of the interacting cells, 2) the hemodynamic shear force in the vascular bed, and 3) the expression of complementary sets of adhesions molecules on both the migratory cell and the endothelium.
    Migrations occur preferentially when surface charge and hemodynamic shear are low in the presence of selective adhesion molecules. Adhesion molecules expressed by endothelial cells of lymphoid HEV’s tend to be sulfated and heavily glycosylated to bind circulating T-lymphocytes and direct them into the lymphoid tissues. Different sets of adhesion molecules are expressed in different lymphoid HEV’s as well as on the endothelial cells of vascular beds in inflammatory sites. These unique sets of adhesion molecules (previously called vascular addressins) are the basis for the homing tendencies of the different leukocyte populations, inducing different groups of cells to return to mucosal lymph nodes, Peyers’ patches, etc. from which they originated.
    Adhesion molecules provide an effective means for cells to interact with each other. There must be mutual attraction of migratory cells to the appropriate sites, as well as between endothelial tissue cells, which must separate from each other and/or their extracellular matrix to facilitate the passage of migratory cells. Adhesion molecules are proteins expressed and membrane-bound on cell surfaces, but they may penetrate the cell membrane and attach to the cytoskeleton to facilitate movement and provide traction against surrounding cells or extracellular matrix.
    The localization of adhesion molecules on specific cell surfaces in specific numbers or concentrations can be closely manipulated in order to regulate relative attractions between cells that must necessarily vary according to function and phase. They may be stored within each cell in vesicles for immediate release and application on the cell surface; or new adhesion molecules can be produced and transported to the cell surface, a process that can require several hours. The specific affinity or avidity of individual adhesion molecules can also be manipulated, as can the specificity for particular ligands (molecules to which the adhesion molecules adhere). A given cell logically utilizes a combination of these means of adjusting its attraction to other cell types in response to changing needs.

Four Means of Adhesion Manipulation

* Release of stored adhesion molecules from intracellular vesicles
* Synthesis of new adhesion molecules
* Changing the affinity of expressed adhesion molecules
* Reorganization of expressed adhesion molecules

     Cell migration involves a staggering number and variety of adhesion molecules that can be conveniently categorized into four distinct structurally-related families:
1. The immunoglobulin supergene family, expressed or inducible on vascular endothelium include
    A. Cellular adhesion molecules (CAM’s)
      (1) Intracellular adhesion molecules (ICAM-1 and ICAM-2)
      (2) Vascular cellular adhesion molecule (VCAM-1)     
      (3) Mucosal addressin CAM-1 (MAdCAM-1) 2.

2. Integrins are membrane glycoproteins composed of both alpha and beta chains (heterodimeric – one alpha and one beta chain), non-covalently bound polypeptide chains that both penetrate the cell membrane to bind different ligands via divalent cations (Mg2+ or Ca2+). Each of eight beta chains combines with one of sixteen alpha chains to form identifiable groups with different specificities. Integrins are the main source of a cell’s response to its extracellular matrix and other cells, present in greater numbers but exerting far less individual affinity for receptors than other adhesion molecules. These types have been characterized.
    A. Beta1 integrins assist in the adhesion of cells to extracellular matrices.
    B. Beta2 integrins regulate adhesion of leucocytes to other immune cells or vascular endothelium.
    C. Beta3 integrins involve neutrophils and platelets and their interaction in inflammation or tissue damage.

3. Cadherins form molecular links between adjacent cells with zipper-like bundles of actin filaments

4. Selectins also penetrate the cell membrane with extracellular components (domains) similar to those of proteins involved in control of the complement system as well as the epidermal growth factor receptor. They bind to ligands with carbohydrate components (as opposed to the other types of adhesion molecules that bind other proteins) via a calcium-dependent carbohydrate recognition domain (CRD).
    A. E-selectin is selectively expressed by endothelium in response to exposure to specific cytokines and act on carbohydrate ligands on leucocytes, particularly neutrophils. Along with ICAM-1, E-selectin is involved in recruiting leucocytes and macrophages into sites of inflammation.
    B. P-selectin is expressed by platelets and endothelium and acts on carbohydrate ligands platelets, endothelium, and neutrophils.
    C. L-selectin is expressed on leucocytes and acts on carbohydrate ligands of endothelium and HEV.

     Defective interactions between adhesion molecules play significant roles in a number of identified disease processes. In cancer, the adhesive properties of tumor cells change as a tumor evolves, allowing cells to detach from the tumor mass and migrate, thus facilitating all three essential characteristics of neoplastic cells: Uncontrolled growth, local invasiveness, and ability to metastasize. Loss of E-cadherin as well as overexpression of certain integrins are specifically associated with tumor invasiveness. Clinical correction of such abnormal adherence activities could keep malignancies from spreading.
    Autoantibodies attacking various adhesion molecules are integral in a number of skin disorders. Beta1 integrins are known only in the basal layer of healthy epidermis, but they are observed in the suprabasal differentiating skin in psoriasis and during wound healing. The abnormal expression produces epidermal hyperproliferation, perturbed differentiation of keratinocytes, and inflammation characteristic of psoriasis, indicating a possible genetic cause and a potential mechanism of eventual pharmacological intervention in the disease process.
    Two inherited diseases involve abnormalities of the leukocyte adhesion molecules. Leukocyte adhesion deficiencies Type I and Type II both present with leukocytosis and recurrent infections; and they involve adherence abnormalities in chemotaxis, phagocytosis, and synthesis of fucosylated carbohydrate ligands of the selectins.
    Inhibition of selectins by administration of antibodies against P-selectin or L-selectin as well as well as treatment with soluble carbohydrate selectin ligands protects against cardiac ischemia. This is accomplished by preventing neutrophil migration and the ensuing vascular damage characteristic of overexpression of intravascular adhesion molecules in ischemia and reperfusion. Similar tactics are proposed for preventing graft rejection, where expression of adhesion molecules is typically increased with the resultant enhanced infiltration of inflammatory cells into the grafted tissue. Infusion of antibodies against ICAM-1 and integrin (alpha)L(beta)2 definitively improves survival in laboratory allografts.
    Evidence implicates overexpression of vascular adhesion receptors in the pathophysiology of rheumatoid arthritis. Therapeutic agents in current use (notably the corticosteroids and colchicine) are shown to decrease the expression of ICAM-1 and E-selectin by endothelium, so immunotherapy via administration of antibodies against these adhesion molecules is being evaluated in clinical trials.
    Even the common cold involves adhesion molecules, the rhinovirus infecting nasal epithelial cells by appropriating ICAM-1 as its receptor. Involvement of adhesion molecules is suspected and being investigated in a number of other infectious diseases as well.
    With such overwhelming evidence as the mysteries begin to unfold in this first of two phases of the migratory process, it is no wonder that a major focus of pharmaceutical development will implement agents that target some aspect of the function of adhesion molecules. As function and regulation of adhesion molecules become more succinctly defined, manipulation of those parameters is destined to become a boon to pharmaceutical manufacturers in treating various diseases. Such pharmacological or biological agents promise mechanisms of action tightly focused on processes more closely associated with the causes of these diseases rather than the symptoms. The tremendous investments now being made in research on these processes will pay off by facilitating more effective treatment with fewer side effects.