Cytokines in Healing and Rejuvenation (Part 2 of a Series)

Cytokines are small “signaling” proteins with molecular weights ranging from 8 to 40,000 daltons.  Nearly all nucleated cells are capable of synthesizing these proteins and, in turn, responding to them. Their biological activities allow us to group them into different classes. Cytokines are regulators of host responses to infection, immune responses, inflammation, and trauma.

Cytokines, along with growth factors, enzymes, and other key proteins, play regulatory roles in wound healing. As part of this process, inflammation initiates healing and its regulation is so important that homeostatic mechanisms evolved to control this activity. Normal wound inflammation involves platelet activation and recruitment of neutrophils, macrophages, and fibroblasts to the wound site in a regimented fashion. Inflammation is a normal and necessary element that initiates wound healing.   Although acute inflammation that persists for short-term mediates host defense against infections, chronic inflammation that lasts for long term can predispose the host to various chronic illnesses, including cancer.

For skin in particular, acute inflammation is necessary for the early stages of wound healing. But if left unfettered, it becomes damaging in itself, leading to poor wound healing or scarring. In youthful  healing, the inflammatory phase is rapid and effective, but is quickly balanced by an anti-inflammatory component.  We now understand the majority of this work is also accomplished by cytokines and related growth factors and enzymes.  Some cytokines are anti-inflammatory, down-regulating inflammation. Calling off the army of white cells drawn to the damaged tissues when their principal work is completed, and thereby preventing damage that can happen when white cells overstay their welcome (i.e. chronic inflammation).

As we examine the cytokine/growth factor cascade in wound healing, we again note critical similarities between wound healing and regeneration. True regeneration takes place in what could be termed an anti-inflammatory environment. Fetuses have immature cell-based immunologic responses, and scarless healing occurs only when inflammation quickly subsides. With advancing age, scar free healing becomes more difficult as healing tends to me more inflammatory than anti-inflammatory in later stages.

Proinflammatory Cytokines

Several cytokines play key roles in mediating acute inflammation, namely IL-1, TNF-a, IL-6, IL-11, IL-8 and other

Interleukin 1-beta (IL-1-b)

chemokines, GCSF, and GM-CSF (Table 1). Of these, IL-1-a, IL-1-b) and TNF are extremely potent: they are the primary cytokines that mediate acute inflammation.

Chronic inflammation is associated with additional members of the interleukin family, including IL-3, IL-5, IL-7, IL-9, IL-14, and to a lesser extent IL-2, IL-12, IL-15.  Interferons, TNF-alpha, and TGF-beta also mediate chronic inflammation. However, the TGF beta family includes three distinct members, and the third (TGF-b-3) is an inhibitor of the other two, and thus anti-inflammatory.

Anti-Inflammatory Cytokines

Interleukin-10 (IL-10)

Anti-inflammatory cytokines counteract various aspects of inflammation, for example cell activation or the production of pro-inflammatory cytokines, and thus contribute to the control of the magnitude of the inflammatory responses in skin. The major anti-inflammatory cytokines are IL-4, IL-6 IL-10, IL-11, and IL-13. Other anti-inflammatory mediators include IL16, IFN-alpha, TGF-beta, IL1ra, G-CSF, as well as soluble receptors for TNF or IL6. IL-6 is anti-inflammatory in that it induces the production of IL-1RA and soluble TNF-α receptors. IL-6 also inhibits the production of GM-CSF, IFN-γ, and MIP-2 without affecting the release of IL-10. IL-10 may be the most important anti-inflammatory cytokine in humans. It inhibits IL-1 release, major histocompatibility complexes, and is likely responsible for the anti-inflammatory effects of exercise.

Cytokine “cocktails” – pro- or anti-inflammatory?

The net effect of an inflammatory response is determined by the balance between pro-inflammatory cytokines and anti-inflammatory cytokines. The type, duration, and also the extent of cellular activities induced by one particular cytokine can be influenced considerably by the nature of the target cells, the micro-environment of a cell, depending, for example, on the growth and activation state of the cells, the type of neighboring cells, cytokine concentrations, the presence of other cytokines, and even on the temporal sequence of several cytokines acting on the same cell.

Cytokines During Wound Healing by Stage

Platelet Activation Phase

Injury damages blood vessels, and coagulation is a rapid-fire response to initiate hemostasis and protect the host from excessive blood loss. With the adhesion, aggregation and degranulation of circulating platelets within the forming fibrin clot, a plethora of mediators and cytokines are released including transforming growth factor beta (TGF-beta), platelet derived growth factor (PDGF), and vascular endothelial growth factor (VEGF), that influence tissue edema and initiate inflammation.  TGF-beta-1, released in large quantities by degranulating platelets, is activated by proteolytic and non-proteolytic mechanisms to influence wound healing . Active TGF-beta-1 elicits the rapid attraction of white cells to the wound site. These cells in turn generate additional cytokines including TNF-a, IL-1, and PDGF, as well as chemokines, as components of a cytokine cascade. These factors act to initiate the inflammatory cell response.

Inflammatory Phase

With the initial barrage of inflammatory mediators, a chain reaction is set in motion, with recruitment, proliferation and activation of the immune cellular participants. Among the first cells to respond are the vascular endothelial cells, which not only respond to cytokines, but release them as well. In addition to the powerful chemotactic activity of TGF-beta1 for white cells, multiple chemokines are released to further entice more of them into the site of tissue injury.  Migrating through the provisional matrix (scaffolding) provided by the fibrin-enriched clot, leukocytes then release proteases and engage in essential functions including phagocytosis of debris, microbes and degraded matrix components. Proteolytic activity is not constitutive, but transcriptionally driven by the cytokines, TGF-beta, IL-1beta, TNF-alpha, and,IL-6 released from multiple cellular sources.

Once the inflammatory cells are activated, they become susceptible to TGF-beta3 mediated suppression to reverse the inflammatory process. Moreover, IL-4 may also dampen the inflammatory response as the inciting agent/trauma is dealt with and promote collagen synthesis during the proliferative (repair) phase.

Proliferative Phase  

Clearance of debris, foreign agents, and/or infectious organisms promotes resolution of inflammation, apoptosis, and the ensuing repair response that encompasses overlapping events involved in granulation tissue, angiogenesis, and re-epithelialization. Immature keratinocytes produce matrix metalloproteases (MMPs) and plasmin to dissociate from the basement membrane and facilitate their migration across the open wound bed in response to chemoattractants. TGF-beta-1 stimulates migration of keratinocytes in vitro, possibly by integrin regulation and/or provisional matrix deposition. Behind the motile epidermal cells, basal cell keratinocyte proliferation is mediated by the local release of growth factors, with a parallel up-regulation of growth factor receptors including TNF-a, epidermal growth factor (EGF) and keratinocyte growth factor (KGF ). Such growth factors are released not only by keratinocytes themselves, acting in an autocrine fashion, but also by mesenchymal cells and macrophages as paracrine mediators. TGF-beta-1, and -beta2 are potent inhibitors of keratinocyte proliferation. TGF-beta-3 is required to downregulate TGF-beta-1 and TGF-beta-2.  Once contact is established with opposing keratinocytes, mitosis and migration stop, and in the skin, the cells differentiate into a stratified squamous epithelium above a newly generated basement membrane. Other factors secreted by keratinocytes may exert paracrine effects on dermal fibroblasts and macrophages.

Granulation tissue forms below the epithelium and is composed of inflammatory cells, fibroblasts and newly formed and forming vessels. This initial restructuring of the damaged tissue serves as a temporary barrier against the hostile external environment, and is aided by fibroblast growth factor 2 (FGF-2) and by the chemokines. Besides VEGF, FGF transduces signals to mediate key events involved in angiogenesis.

Remodeling Phase

With the generation of new vasculature, matrix-generating cells move into the granulation tissue. These fibroblasts degrade the provisional matrix via MMPs and respond to cytokine/growth factors by proliferating and synthesizing new extracellular matrix (ECM) to replace the injured tissue with a connective tissue scar. Although the stage is being set for tissue repair from the beginning fibroblasts migrate into the wound and matrix synthesis begins in earnest within a couple of days, continuing for several weeks to months. TGF-beta contributes to the fibrotic process by recruiting fibroblasts and stimulating their synthesis of collagens I, III, and V, proteoglycans, fibronectin and other ECM components. TGF-beta concurrently inhibits proteases while enhancing protease inhibitors, favoring matrix accumulation. In vivo studies have confirmed that exogenous TGF-beta1 increases granulation tissue, collagen formation, and wound tensile strength when applied locally or given systemically in animal models. Increased levels of TGF-beta are routinely associated with both normal reparative processes, as well as fibrotic healing (scarring).  The progressive increase in TGF-beta3 over time and its association with scarless fetal healing have implicated this member of the TGF-beta family in the cessation of matrix deposition. Other members of the TGF-beta superfamily may also contribute to the wound healing response. Also central to repair are the FGFs, which signal mitogenesis and chemotaxis  underlying granulation tissue formation, and the production of MMPs. FGF-1 (acidic FGF) and FGF-2 (basic FGF) have been the most intensely studied, but the additional members of this family may also support tissue repair and/or have clinical application.

With many overlapping functional properties with FGFs, epidermal growth factor (EGF) orchestrates recruitment and growth of fibroblasts and epithelial cells in the evolution of granulation tissue. IL-6 has also been shown to be crucial to epithelialization and influences granulation tissue formation, as shown in the wound healing.  The remodeling phase, during which collagen is synthesized, degraded and dramatically reorganized (e.g. stabilized via molecular crosslinking into a scar), is also cytokine-mediated. Although repaired tissue seldom achieves its original strength, it provides an acceptable alternative. Degradation of collagen and other matrix proteins is driven by serine proteases and MMPs under the control of the cytokine network. MMPs not only degrade matrix components, but also function as regulatory molecules by driving enzyme cascades and processing cytokines, matrix and adhesion molecules to generate biologically active fragments. TIMPs provide a natural counterbalance to the MMPs and disruption of this orderly balance can lead to excess or insufficient matrix degradation and ensuing tissue pathology. Similarly, there exists a naturally occurring inhibitor of elastase and other serine proteases. The coordinated regulation of enzymes and their inhibitors ensures tight control of local proteolytic activity. In physiologic circumstances, these molecular brakes limit tissue degradation and facilitate accumulation of matrix and repair.

Scarring

collagen scarring

Rapid clearance of the inciting agent and resolution of inflammation during healing minimizes scar formation, whereas persistence of the primary insult results in continued inflammation and chronic attempts at healing. Prolonged inflammation and proteolytic activity prevent healing. On the other hand, continued fibrosis in the skin leads to scarring and potentially, disfigurement, whereas progressive deposition of matrix in internal organs such as lungs, liver, kidney or brain compromises not only their structure, but also function, causing disease and death.

Inhibitors of TGF-beta-1 & beta-2 (e.g. TGF-beta-3) reduce scarring, and are natural antagonists of fibrogenesis. IL-10 may be considered anti-fibrotic via its anti-inflammatory activities, as are inhibitors of TNF-alpha.

Wound healing is a complex process encompassing a number of overlapping phases, including inflammation, epithelialization, angiogenesis and matrix deposition. Ultimately these processes are resolved or dampened leading to a mature wound. Although inflammation and repair mostly occur along a proscribed course, the sensitivity of the process is underscored by the consequences of disruption of the balance of regulatory cytokines. Consequently, cytokines, which are central to this constellation of events, have become targets for therapeutic intervention to modulate the wound healing process. Depending on the cytokine and its role, it may be appropriate to either enhance (recombinant cytokine, gene transfer) or inhibit (cytokine or receptor antibodies, soluble receptors, signal transduction inhibitors, antisense) the cytokine to achieve the desired outcome.

inflammatory and anti-inflammatory cytokines affect fibroblast production during healing (vs) regeneration

Fibroblasts play a key role in the maintenance of extracellular matrix (ECM) homeostasis by continuously synthesizing and degrading ECM molecules.  Healing/regeneration can follow one of two courses.  Tissue injuries, caused by infection, chemicals, mechanical stress or autoimmune reactions, activate the immune system and repair mechanisms. Effective healing is usually characterized by a rapid shift from inflammatory to anti-inflammatory response.   Failure to make this transition leads to chronic inflammation, scarring and fibrosis.

References

Wick, G., Backovic, A, Rabenstein, E., et. al.  The immunology of fibrosis: innate and adaptive responses. Trends Immunol. 2010 Mar;31(3):110-9. Epub 2010 Jan 26.

Coming Up Next

We examine mesenchymal stem cells as sources of cytokines in vivo, and in vitro. We look at the evidence for benefits of both cellular therapy and cell free therapy (paracrine cytokine cocktails) in wound healing and regeneration. We examine the use of conditioned medium of mesenchymal stem cell as  cytokine factories – pointing out the many variables that determine the end result  composition.  This sets the stage for part 4, where the real fun begins.