CHEMOTACTIC FACTORS:
FUTURE DISEASE INTERVENTION

    Last month I discussed the possibilities of interfering with the inflammatory response by inhibiting the action of adhesion molecules as a first phase of inflammatory cell migration. This first phase is characterized by activation of adhesion molecules on the surfaces of microvascular endothelium to arrest circulating lymphocytes by attaching to ligands on those mobile cells. Once stopped and attached to endothelial cells lining the lumen of a blood vessel, the lymphocyte migrates to the site of infection or trauma by passing between adjacent tissue cells or between tissue cells and their extracellular matrices. All of these intricacies depend upon the adhesion molecules, their micro-regulation, and their interactions.

Table 1
Steps In Inflammatory Cell Migration
* Tethering of circulating lymphocytes to endothelial cells is accomplished by activation of endothelial selectins interacting with their carbohydrate ligands on the lymphocytes. Circulating cells begin to roll when first contacted, which then eventually slows them to a stop, firmly adhered to the endothelial surface.
* Triggering of the arrested lymphocyte by endothelial surface molecules, chemotactic agents, and cytokines is possible once the cell is arrested, activating the inflammatory cell and initiating its migratory mission.
* Latching and activation escalate leukocyte integrin affinity to engage adhesion molecules on endothelial cells and begin movement.
* Migration between endothelial cells to the basement membrane and beneath the endothelium occurs with the aid of other adhesion molecules.
* Digestion of basement membrane components by enzymes released from the inflammatory cell facilitates migration into target tissues.

Chemotaxis: Phase 2 Of Cell Migration
    This month’s discussion focuses on phase 2 of the migratory process and the means by which inflammatory cells are recruited or guided to the site of inflammation. The chemotactic molecules involved in this phase provide another unique opportunity to disrupt disease processes and are currently being investigated for therapeutic applications. A variety of disease states has been associated with prevalence of various chemokines (see table below), so research on their roles in such diseases may inevitably lead to new medications or biologicals to treat the disorders more effectively than agents currently available and with fewer side effects. Not only does this raise the possibility of new anti-inflammatory mechanisms to disrupt cellular immune processes, but also the possibility of preventing infection and spread of infectious agents like HIV, as will be discussed in greater detail below.

Table 2 Chemokines Associated With Disease States Disease States
Sites    /   Chemokine

Asthma
Lavage Fluid MCP-1, MIP1alpha, RANTES
Arteriosclerosis
Tissue MCP-1,MIP-1alpha/beta,RANTES,GRObeta
Cystic Fibrosis
Lavage Fluid IL-8, ENA-78, MCP-1
Cytomegalovirus Encephalomyelitis
CSF MCP-1
Dermatitis (Atopic and Contact)
Tissue RANTES, Eotaxin,IL-8,MCP-1,IP-10
Endotoxemia and Sepsis
Plasma IL-8, MIP-1alpha, MCP-1 RANTES
Gastrointestinal Inflammation
Tissue IL-8,MCP-1,MIP-1alpha/beta, RANTES, IP-10 Immune Complex Glomerulonephritis
Tissue IL-8,MCP-1 Osteoarthritis
Synovial Fluid MIP-1beta
Post-major surgery
Plasma IL-8
Psoriatic Scale
Tissue Extract IL-8,GROLalpha, beta, gamma, MCP1, IP-10, ENA-78
Pulmonary Diseases (acute)
Tissues IL-8, ENA-78, MCP-1 RANTES
Rheumatoid Arthritis
Synovial Fluid IL-8, ENA-78, MCP-1, MIP-1alpha
Tuberculoid Leprosy
Tissue IP-10
Wound Healing
Tissue MCP-1,IP-10
Uveoretinitis
Tissue IL-8,IP-10,MCP-1,RANTES,MIP-1alpha/beta

    The cell-surface integrins required by inflammatory cells to migrate through tissues are stored intracellularly in granules and released upon activation. Though activation can be endogenous from the endothelial cell itself, it is commonly effected by chemical signals (signaling molecules) from other sources that include C5a (of the complement system), leukotriene B4 (LTB4), and a plethora of chemokines, which are low-molecular-weight cytokines. These signaling molecules can be chemokinetic (stimulating the overall non-directional motility of migratory cells) or chemotactic (stimulating directional migration). Directional migration requires sensitivity on the part of the migrating cell to differential gradients in concentration of the chemotactic molecule along the cell surface. Like a bloodhound, the inflammatory cell follows the increasing “scent” of the chemokine toward its source. Arriving at the site of foreign invasion or tissue injury, the inflammatory cell is activated to make its own chemical contribution to the inflammatory effort by its effector functions of granule release and cytokine production.
    As early as the 1960’s, chemoattractants for granulocytes and monocytes were observed in supernatants from cell cultures of stimulated leukocytes. Many of these chemotactic cytokines (chemokines) have been purified or cloned as soluble proteins that selectively attract and activate leukocytes. Chemokines are classified according to position of molecular cysteine residues:
* Alpha -- A single amino acid separates the first and second of four cysteine residues. (cysteine – amino acid – cysteine – cysteine – cysteine)
* Beta -- Cysteine residues are not separated by amino acid sections, but contiguous. (cysteine – cysteine – cysteine -- cysteine)
* Gamma -- These contain only one pair of cysteine residues.

Receptors and Functions
    The chemokines, as well as other chemotactic molecules, act on a variety of transmembrane receptors on the surfaces of leukocytes. Different types of receptors are found on different populations of leukocytes, accounting for the differentially selective action of various chemokines. These entities, then regulate the timing of activation and arrival of the various inflammatory cell types at sites of inflammation. While some chemokines both attract and activate target leukocytes, others only activate or attract their targets. Over 25 chemokines have been identified, all of which play roles in controlling the selective migration of inflammatory cells on endothelial surfaces as well as through the tissues. They bind heparin and may attach to heparin sulfate groups or to DARC (Duffy blood group antigens) on the cell surfaces of vascular endothelial tissues. Such attachment can trigger a cell to alter the avidity of adhesion molecules on its surface.
    They also attach to cells via specific protein-linked transmembrane “serpentine” receptors consisting of seven segments. Alpha chemokines are observed to have four different receptors (CXCR1 through CXCR4), while five have so far been identified for beta chemokines (CCR1 through CCR5); and different cell subpopulations typically express different sets of these receptors on their surfaces. This would seem to account, at least in part, for the selective migration of different cell types at different stages in the inflammatory process. Not only do different cell types respond to different chemokines, but different chemokines are produced and released by different source cells; and a given cell may change both its production and its response with the particular chemokine stimulus to which it is exposed, depending upon the particular stage of the inflammatory process.
    For example, interleukins 1 and 6 (IL1 and IL6), released by damaged tissue cells at the site of invasion or trauma, attracting mononuclear cells and lymphocytes. Upon arrival at the site and activation, these cells then release quantities of IL-1, TNF, IL-4, and IFN-gamma to act on endothelial tissues and perpetuate the inflammatory reaction. IL-8 seems to be both chemotactic and activating, attracting large numbers of neutrophils; while MCP-1 MIP-1 alpha, and RANTES (beta chemokines) tend to mediate a more delayed mononuclear infiltration.

Other Chemotactic Molecules
    The migration of macrophages and neutrophils is influenced by a number of peptide amino acids acting on their f.Met-Leu-Phe (f.MLP) receptors. These amino acids, necessarily blocked at the N terminus by formylated methionine, are what prokaryotes (bacteria) use to initiate all protein translation. Since eukaryotic cells do not use this method of translation, the presence of these amino acids provide a beacon toward which macrophages migrate. Both of these cell types also bear surface receptors activated by LTB4 (from mast cells and macrophages) and C5a (of the complement system) produced by early inflammation to attract these cells. Fibrin peptide B and thrombin, involved in the processes of blood clotting attract phagocytes.

Other Important Effects of Chemokines
    In addition to their essential roles in the regulation of inflammatory cell migration, several of the chemokines have observable immuno-enhancing effects:
* Providing chemotaxis for antigen-presenting dendritic cells,
* Enhancing the capacity of dendritic cells to activate T cells (see NEWS YOU NEED IN HOSPITAL PHARMACY, Adoptive Immunity in this issue), and
* Providing co-stimulation of lymphocyte cytolysis.

    Not only do the chemokines play vital roles in the inflammatory processes, but they may have a variety of other functions which may or may not relate to their inflammatory effects. IL-8, often released early in the inflammatory response by activate monocytes, not only attracts neutrophils and basophils to a site of inflammation, but stimulates vascularlization and proliferation of endothelial tissue cells. IP-10, MIG, and PF-4 seem to involve antagonistic actions that may prove essential in the breakdown and rebuilding process of healing wounds. There is even evidence that tumor regression and tumor immunity can be effected by transfection of certain tumors with IP-10 and RANTES.
    Resistance of cultured CD4+ lymphocytes to HIV-1 infection has recently been markedly enhanced by a “cocktail” of RANTES, MIP-1alpha, and MIP-1beta (all beta chemokines). It is thought that CCR5, CXCR3, CCR3, and CCR2B, chemokine receptors on the surfaces of T cells and monocytes, may be co-receptors along with CD4, acting as portals of entry for HIV-1 into these cells. By blocking these receptors competitively, chemokines passively block entry of the virus to the cell as well as cell-to-cell transmission. The potential for use of this technology in those with AIDS and those with HIV infection could conceivably slow or stop the disease process and block transmission. With such profound promise, it is little wonder that research in the area of cytokines, chemokines, and other chemotactic molecules continues to escalate.

 
     The large family of over a hundred identified cytokines is comprised a bewildering array of autocrine and paracrine molecular entities. Since they have been named and characterized by numerous scientific disciplines, the names can be even more confusing than the biological actions. The confusion is furthered by the fact that cytokines can have multiple effects; and multiple cytokines can overlap in their actions, all depending on target cell type, state of activation, and stimulus. They bind receptors on cell surfaces to trigger a cascade of actions that ultimately induce, enhance, or inhibit various cytokine-regulated genes in the cell nucleus. It is seldom realistic to consider the isolate effects of a single cytokine, as migratory cells are typically exposed to a variety of these regulatory molecules acting simultaneously with varying degrees of intensity and influence.

Some Common Currently-Recognized Cytokines
* Chemokines – RANTES, MCP-1, MIP-1alpha, MIP-1beta
* Interleukins (IL) – IL-1 through IL-18
* Interferons (IFN) -- IFNalpha, IFNbeta, IFNgamma
* Tumor Necrosis Factors (TNF) – TNFalpha, TNFbeta
* Growth Factors (GF) – NGF, EGF
* Colony Stimulating Factors (CSF) – M-CSF, G-CSF, GM-CSF

References
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6. Y. Feng, et al., Science 272, 872-877 (1996).