Biologic roles of cytokines and growth factors

Growth factors are polypeptide proteins produced by the body to regulate division, proliferation, and growth of cells by binding to receptors on the cell surface. Specifically, proliferation and differentiation of nonimmune system cells are regulated primarily by growth factors. In contrast, cytokines are protein molecules that primarily regulate the interactions between cells that participate in the immune response (Frenette and Wagner, 1996a, 1996b; Springer, 1990).

Cytokines are a unique family of growth factors that are small signaling proteins that mediate and regulate immunity, inflammation, and hematopoiesis. Produced in response to an immune stimulus, cytokines are secreted primarily from leukocytes. Cytokines function at very low concentration and act over short distances. After the cytokines bind to specific membrane receptors, second messengers are triggered that signal the cell to alter its behavior by increasing or decreasing membrane proteins, proliferation, and secretion of molecules. The same cytokine may be secreted by different types of cells, and the same cytokine may act on several different cell types.

Adhesion molecules and adhesion receptors in inflammation

The chemotactic attraction of leukocytes to a wound and the movement of leukocytes from the blood into wounded tissue (extravasation) involve expression and activation of adhesion molecules and adhesion receptors on leukocytes, platelets, and vascular endothelial cells. Cytokines and growth factors play key roles in these processes (Arai et al, 1990; Frenette and Wagner, 1996a, 1996b; Springer, 1990). Among the many types of adhesion molecules and receptors on the cell surface, four major families of transmembrane proteins stand out in the process of inflammation: integrins, selectins, cell adhesion molecules, and cadherins (Figure 20-1).

FIGURE 20-1 Four major classes of adhesion proteins and adhesion receptors embedded in a theoretic plasma membrane: integrins, selectins, cell adhesion molecules (platelet-endothelial cell adhesion molecule [PECAM-1] and vascular cell adhesion molecule VCAM-1]), and cadherins.

(From Frenette PS, Wagner DD: Adhesion molecules. Part I, N Engl J Med 334:1526, 1996.)

Integrins are glycoproteins composed of two different types of subunits, designated α and β. In simple terms, integrins are cellular receptors for extracellular matrix proteins, as shown with α₅β₁, which is a receptor for fibronectin. A short amino acid sequence, such as arginine-glycine-aspartate (RGD), is often the site of recognition by the integrin receptor. Integrins are important because they are capable of generating signals inside cells when the integrin receptor binds to a specific extracellular matrix protein, in much the same way the insulin receptor generates intracellular signals, which regulate glucose transport into a cell when insulin binds to its cellular receptor. Expression of β₂ integrins is limited to leukocytes, whereas β₁ integrins are expressed on most cell types. β₁ integrins primarily bind to extracellular matrix components such as fibronectin, laminin, and collagens. (These substances are discussed in more detail in Chapter 4.)

Selectins are proteins that have a unique structure called a lectin domain at the distal end, which can bind specific carbohydrate groups of glycoproteins or mucins on adjacent cells. Thus, unlike other adhesion proteins, which recognize specific protein structures, selectins recognize and bind to carbohydrate ligands on leukocytes and vascular endothelial cells. E-selectin appears on endothelial cells after they have been activated by inflammatory cytokines, and P-selectin is stored in the α-granules of platelets and the storage granules of endothelial cells (Weibel-Palade bodies).

Cell adhesion molecules (CAMs) are members of the immunoglobulin superfamily of proteins. CAMs can bind to other CAMs or to integrins on cells. CAMs that are important in inflammation include the platelet-endothelial cell adhesion molecule (PECAM), vascular cell adhesion molecule (VCAM), and intercellular adhesion molecule-1 (ICAM-1).

Cadherins are important in establishing molecular links between adjacent cells, especially during embryonic development. They form zipperlike structures of dimers at specialized regions of contact between neighboring cells called adherens junctions. Cadherins are linked to the cytoskeleton through molecules called catenins, which associate with actin microfilaments.

During the process of extravasation of inflammatory cells into a wound, important interactions occur between blood vessels and blood cells (Arai et al, 1990; Frenette and Wagner, 1996a, 1996b; Springer, 1990). Initially, circulating leukocytes begin rolling on endothelial cells through the binding of glycoproteins expressed on their cell surface to selectins, transiently expressed by activated endothelial cells of venules (Figure 20-2). The binding affinity of selectins is relatively low but is enough to serve as a biologic brake, making leukocytes quickly decelerate by rolling on endothelial cells. While rolling, leukocytes can become activated by chemoattractants (cytokines, growth factors, or bacterial products). After activation, leukocytes firmly adhere to endothelial cells as a result of the binding between their β₂ class of integrins and ligands, such as VCAM and ICAM expressed on activated endothelial cells. Chemotactic signals present outside the venule induce leukocytes to squeeze between endothelial cells of the venule and migrate into the inflammatory center by using their β₁ class of integrins to recognize and bind to extracellular matrix components.

FIGURE 20-2 Interactions between blood cells and a stimulated or injured venule.

(From Frenette PS, Wagner DD: Adhesion molecules. Part II: Blood vessels and blood cells, N Engl J Med 335:43, 1996.)

Adhesion and degranulation of platelets at sites of vascular injury also use a system of adhesion molecules and adhesion receptor proteins. Vascular injury immediately induces endothelial cells to release the contents of their storage granules (Weibel-Palade bodies), including the proteins P-selectin and von Willebrand factor. P-selectin promptly moves to the plasma membrane of endothelial cells, where it induces rolling of platelets on endothelial cells, and von Willebrand factor is quickly deposited on the exposed extracellular matrix, where it plays a crucial role in the adhesion of platelets to the damaged site.

Inflammatory cell cytokines and growth factors in proliferation and repair

The growth factors released by platelets diffuse away from a wound within a few hours, but they are replaced by growth factors and cytokines that are produced by neutrophils, macrophages, activated fibroblasts, vascular endothelial cells, and epidermal cells that are drawn into the wound area. For example, activated macrophages secrete several important cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), which have a variety of actions on different cells. TNF-α and IL-1β are potent inflammatory cytokines, which further stimulate inflammation. TNF-α also induces macrophages to produce IL-1β, which is mitogenic for fibroblasts and up-regulates expression of MMPs. Both TNF-α and IL-1β directly influence deposition of collagen in the wound by inducing synthesis of collagen by fibroblasts and by up-regulating expression of MMPs. In addition, these cytokines down-regulate expression of the tissue inhibitors of metalloproteinases (TIMPs), which are the natural inhibitors of MMPs. Interferon-γ (IFN-γ), produced by lymphocytes attracted into the wound, inhibits fibroblast migration and down-regulates collagen synthesis (Table 20-1).

TABLE 20-1 Growth Factor Families

Inflammatory cells secrete other growth factors, including TGF-β, TGF-α, heparin-binding epidermal growth factor (HB-EGF), and basic fibroblast growth factor (bFGF). The growth factors secreted by macrophages continue to stimulate migration of fibroblasts, epithelial cells, and vascular endothelial cells into the wound. As the fibroblasts, epithelial cells, and vascular endothelial cells migrate into the site of injury, they begin to proliferate, and the cellularity of the wound increases. This begins the proliferative and repair phase, which often lasts several weeks. If the wound is not infected, the number of inflammatory cells in a wound begins to decrease after a few days. Other types of cells, such as fibroblasts, endothelial cells, and keratinocytes, are drawn into the wound and begin to synthesize growth factors. Fibroblasts secrete IGF-I, bFGF, TGF-β, PDGF, and keratinocyte growth factor (KGF). Endothelial cells produce vascular endothelial cell growth factor (VEGF), bFGF, and PDGF. Keratinocytes synthesize TGF-α, TGF-β, and IL-1β. These growth factors continue to stimulate cell proliferation and synthesis of extracellular matrix proteins and to promote formation of new capillaries.

Cytokines

Cytokines are produced extensively by activated T cells and macrophages, although nonimmune system cells such as keratinocytes and vascular endothelial cells also produce some cytokines. Studies have revealed that cytokines generally induce multiple biologic activities (pleiotropic) and that a single cytokine can act as both a positive signal and a negative signal, depending on the type of the target cell. Cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, and IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), IFN-γ, and TNF-α are key mediators of immune and inflammatory responses. A cytokine is also referred to as a lymphokine (cytokine made by lymphocytes), monokine (cytokine made by monocytes), chemokine (cytokine with chemotactic action), and interleukin (cytokines made by one leukocyte and acting on other leukocytes). Two cytokines in particular, TNF-α and IL-1β, have activities that substantially influence skin wound healing through their ability to increase production of MMPs and suppress production of TIMPs. Table 20-2 lists the cytokines involved in wound healing along with cell source, biologic activity, and their subclassification as proinflammatory or antiinflammatory.

TABLE 20-2 Cytokines Involved in Wound Healing

Cytokines have not been investigated extensively in human wound healing studies. IL-1β was evaluated in a prospective, randomized, double-blind, placebo-controlled trial performed on 26 patients with Stage III and IV pressure ulcers (Robson et al, 1994). No statistically significant differences were seen in the percentage decrease of wound volumes between the treatment groups.