Cytokine Effector Functions in Tissues

France

Preface

Maria Foti and Massimo Locati

Cytokines are important mediators of diverse biological processes and are therefore highly regulated in any organism. Cytokines assist the initial recognition of danger or nonself-antigens as they are rapidly induced during host responses to infection, inflammation, and damage. They launch innate host protections and form the bridge that allows a nonspecific response to develop into an acquired immune response. Clearly, any event that affects the biology of these important mediators is likely to influence the overall immune competence of the host. In Cytokine Effector Functions in Tissues, experts of cytokine biology bring together their knowledge on various aspects of how modulation of cytokines can affect tissue regulation in the context of health and disease. First, the basic biology of cytokines is reviewed, particularly as this relates to their biological function on tissue regulation.

Various chapters define how differential cytokine modulation determines the type of immune response generated during a specific disease settings and how cytokines may regulate tissue homeostatic interactions.

This sets the step for practical preclinical understanding of the effect of cytokines, including how they function in specific pathological conditions such osteoarthritis, atherosclerosis, diabetes, sarcoidosis, nerve degeneration, skeletal muscle disease, allergies, asthma, and COPD. This group of chapters provides the reader with a broad understanding of the range of cytokine activity in human diseases.

Although cytokines show great promise as therapeutic agents, they exhibit a sustained level of toxicity that need to be taken into account when a cytokine trial is designed. Therefore, we summarized the promising advantages in using cytokines in cancer therapies, their role in the field of transplantation, and the possibility to exploit specific cell types such as mesenchymal stem cells (MSCs) for their ability to produce cytokines that may affect the outcome of cellular therapies. The field is far from being completely covered as the issue is heavily complex and novel aspects of the cytokine biology are rapidly evolving. Nevertheless, it is our truthful hope that this collection of expert reviews will help as a starting point for a more complete investigation by the reader of the role these charming biological regulators play in human health and disease.

Part I

Cytokine Biology Overview

Outline

Chapter 1 Introduction to Cytokines as Tissue Regulators in Health and Disease

Chapter 1

Introduction to Cytokines as Tissue Regulators in Health and Disease

Maria Foti,    University of Milano-Bicocca, Milan, Italy

Abstract

Cytokines are powerful mediators of several biological processes and are highly regulated in the body. Chronic uncontrolled levels of such proteins can initiate and potentiate many pathologies, including autoimmunity and cancer. Cytokines shares basic properties: they are pleiotropic—a given cytokine may drive proliferation of a cell type and induce growth arrest in another cell type, they are redundant, and the cytokine network is tightly regulated and its alteration may lead to impaired tissue and cellular physiology. Currently, there is an emerging understanding of the role of cytokine in tissue homeostatic functional regulation and it is becoming clear that pathological conditions may develop from dysregulation of cytokines. Some organs seem more vulnerable to dysregulated cytokines networks than others, so it is becoming more and more important to study the cytokine network in the contest of tissue specificity. In addition, cytokines can act in concert with other tissue-specific signals. Therefore, unraveling the complexity of such network is extremely challenging as different organs have developed diverse strategies to cope with immune cell migration. Here, I summarize the basic knowledge on functional groups of cytokines and the role played in the context of tissue regulation in health and disease.

Keywords

Cytokines families; inflammation; immunomodulation; tissue homeostasis; cytokine therapeutics

Outline

Introduction 3

Biological Characteristics of Cytokines 4

Proinflammatory Cytokines 5

Regulatory Cytokines 10

Cytokines Important in T Lymphocyte Differentiation (Th1, Th2, Th22) 12

Cytokines Important in B Cells Differentiation and Activation 14

Cytokines and Tissue Homeostasis 15

Cytokines as Growth Factors 20

Immunomodulation by Cytokines in Human Diseases 21

Conclusion 24

References 25

Introduction

Cytokines are a complex of soluble, cell-signaling proteins that affect the biological function of cells and process such as inflammation, a variety of immune responses, and the wound healing activity. Cytokines include interleukins, chemokines, interferons, and growth factors. Although similar to hormones in that they act at low levels either systemically or locally, cytokines are unique in the large number of cells they are able to target. Cytokines are not restricted to any single organ or tissue but instead they are synthesized by a variety of hematopoietic cells, including myeloid (e.g., macrophages, dendritic cells (DCs), granulocytes, mast cells), lymphoid cells (e.g., B cell, T cells, natural killer cells) and by nonhematopoietic cells such as endothelial and epithelial cells. Different immune cells can secrete the same cytokines building up complex microenvironments that are functionally pro or antiinflammatory depending on the specific cytokine and cellular target present in that organ. In fact, a role of cytokines in certain organs is not yet fully determined, for example, the role of interleukin-1β (IL-1β) in normal physiology of the central nervous system still remains unclear. Therefore, understanding the basic of this network of interactions in a tissue-specific manner will advance our knowledge on cytokine biology to better exploit them in the context of clinical immunotherapeutic settings.

Biological Characteristics of Cytokines

There are over 100 cytokines and many exist in families that share receptor components and signal transduction pathways. Many diseases are characterized by complex cytokine networks that may have a positive or a negative impact on the disease.¹ Cytokines are low molecular weight molecule that possess some common properties: They have a short half-life, they are de novo synthesized and secreted during cellular activation; they are produced by different cells of the immune system and have multiple biological activities depending on the cell type to which they bind, for this reason they are defined as pleiotropic molecules; their biological functions can be redundant, as two different cytokines can have overlapping effects on the target cells; many cytokines can induce or inhibit the production of other cytokines by creating complex network of regulators that are able to finely modulate the biological effects; they may influence the activity of other cytokines, inducing antagonistic or synergistic effects.

They induce their biological effects by activating specific receptors on the cell surface and they can act on the same cells that secreted them (autocrine activity) or on neighboring cells (paracrine activity). In general, cytokines act mainly in the neighboring microenvironment in which they are produced; however, some cytokines (IL-1β and tumor necrosis factor α (TNFα)) may be present in the circulating blood and exert systemic and endocrine effects. Generally, the cellular responses to cytokines are expressed in several hours and require de novo production of mRNA and the synthesis of specific proteins. Cytokines play a critical role in cellular interactions and immune responses. These molecules exert many biological activities which ultimately coordinate multiple aspects of innate and acquired immunity as well as the control of the organs physiology. An important characteristic of cytokines is represented by their ability to regulate various aspects of the host immune response. The study of immunological characteristics of cytokines is complicated by the properties of pleiotropism, redundancy, regulation of the production of other cytokines, synergism, and antagonism. For examples, IL-1 is important for T-cell differentiation and at the same time together with TNFα is able to control the body temperature.

Most cytokines are not produced and do not act alone but they work as part of a complex network in which they regulate both the production activities of other cytokines and their effector activities. For this reason, their study is particularly complicated. The synergism is another important characteristic of certain cytokines. IL-2 and IL-12 act together in modulating the activity of cytotoxic lymphocytes (CTL) and the antagonism is another important characteristic of cytokine appearance. For example, the IL-10 produced by Th2 cells inhibits the activity of Th1 cells. Therefore, it is the overall balance between the different cytokines activities to determine the beginning, the maintenance, and the resolution of the immune and inflammatory responses.

It follows that the study of these mediators in vivo is extremely challenging since it is difficult to determine the exact sequence of events in which the cytokines are produced during the tissue interactions in which cells and mediators cooperate in the different phases of the inflammatory processes.

Cytokine Families

The number of known cytokines is wide and constantly expanding and therefore it is difficult to describe them all individually. However, the biological activity of cytokines is important in terms of molecular and cellular interactions between cells of the immune system and the parenchymal cells. Here, I summarize the main functional class of cytokines categorized based on the biological process they participate in. The key cytokines for each biological process will be described.

Proinflammatory Cytokines

Proinflammatory cytokines are produced by several cell types following interaction with microbes or from tissue-damaging events. These cytokines play a very important role as they are responsible for the activation of the innate and acquired immune response by enhancing the activity of immune system cells. These activities are mainly carried out by altering the expression of molecule important for the presentation of antigens such as Major Histocompatibility Complex (MHC) molecules and the costimulatory molecules such as CD80, CD86, and CD40. The cells most involved in these processes are the neutrophils, macrophages, dendritic cells (DCs) and mast cells. In general, proinflammatory cytokines possess pleiotropic functions in innate immunity. They orchestrate acute inflammatory processes locally and, in severe cases, systemically. In addition, they participate in shaping adaptive immunity by skewing the differentiation of naive helper T lymphocytes and by directly affecting the effector functions of different subsets of T and B lymphocytes.

The first cytokines to be produced in this class are IL-1β, IL-6, TNFα, and Interferons gamma (IFNγ). These cytokines are produced and act both on cells that produced them and on other cells of the immune system or the parenchyma.

IL-1α and IL-1β are ubiquitously expressed although monocytes/macrophages are the main producers. Both cytokines display an immune-stimulating and inflammatory activity and the mature form of the proteins as well as its extracellular release requires caspase-1 activity. Currently, IL-1β is considered the master regulator of the innate immunity. Different microbial ligands are able to induce the IL-1β gene either directly or indirectly through the secretion of other cytokines such as TNFα, IFNγ, or IL1 itself. IL-1β, IL-6, and TNFα participate to the induction of the acute phase response. Among different functions, IL-1β is able to induce other cytokines such as IL-6, adhesion molecules to promote cell–cell contact and transendothelial migration of inflammatory cells. IL1α and IL-1β exert similar effect by binding the same receptor complex, the IL1 type I and type II receptor. The IL1 receptors are present in different cells types.

IL-6 is a pleiotropic molecule produced by monocytes, endothelial cells, and from fibroblasts in response to IL-1β and TNFα. It is one of the major players in the induction of the acute phase response by stimulating hepatocytes to release plasmatic proteins important to fight bacterial infections. In addition, IL-6 induce B-cell differentiation and survival, T cells and thymocytes proliferation, and increase NK cell activity. IL-6 release is induced during inflammatory conditions upon stimulation of Toll-like receptors (TLRs) or upon stimulation of cells by IL-1β or TNFα. It binds to interleukin-6 receptor (IL-6R) which is not signaling competent on target cells; however, signaling is initiated upon association of the IL-6/IL-6R complex with a second receptor protein, the glycoprotein (gp) 130. gp130 dimerization leads to the activation of the tyrosine kinase Janus kinase 1 (JAK1).² An important characteristic of IL-6 is that it binds to its receptor gp130 only when the complex IL-6/IL-6R is formed. This has the consequence that whereas gp130 is expressed by all cells of the body, IL-6R expression is restricted to few cell types such as hepatocytes, some leukocytes, and some epithelial (e.g., biliary epithelial cells) and nonepithelial cells (e.g., hepatic stellate cells). Therefore, only IL-6R positive cells can respond to IL-6. Nevertheless, it has been shown that IL-6R can be cleaved by different proteases during a process called shedding³ and still being able to bind IL-6, broadening the spectrum of target cells. This IL-6 activity was defined IL-6 trans-signaling. As already mentioned, the major inducer of the hepatic acute phase proteins is IL-6 that is secreted by neutrophils, monocytes, and macrophages upon TLR stimulation.⁴ Activation of myeloid cells induces the release of IL-1β and TNFα that lead to a massive production of IL-6 from other cells. It is generally believed that while IL-6 classical signaling is crucial for the induction of the acute phase response, IL-6 trans-signaling mediates strong mitogenic signals for T, B cells and hepatocytes, function that it is particular important during liver regeneration. Nevertheless, it needs to be mentioned that there is a growing body of evidence that IL-6 maybe important for the control of metabolic functions.⁵ It has been observed that a correlation exists between serum levels of IL-6 and degree of obesity⁶ and the development of type 2 diabetes.⁷ These data show that in the liver, IL-6 not only regulates glucose metabolism but is also necessary to maintain tissue homeostasis for proper control of metabolic functions. The metabolic function of IL-6 should be further investigated also in other organs and indeed and future studies should consider that IL-6 classic and IL-6 trans-signaling might differentially regulate metabolism in a tissue-specific manner. In summary, IL-6 is a cytokine with pleiotropic functions. Under physiological conditions, it is essential for proper hepatic tissue homeostasis, liver regeneration, infection defense, and fine tuning of metabolic functions. However, persistent activation of the IL-6 pathway seems to be detrimental and can even lead to the development of liver cancer. Although much advancement has been made, there are still many open questions concerning the implication of IL-6 in physiology and pathology. In order to efficiently target only the detrimental effects of IL-6, we need to better understand the effects of IL-6 of different cell types of the liver and other organs.

TNF alpha (TNFα) is a key cytokine with a broad spectrum of biological activities with a key role in a variety of pathological processes. The biologically active native form of TNFα is a homotrimer. TNFα is produced by macrophages, DCs, B cells, and T cells as well as by other types of somatic cells such as endothelial cells, mast cells, neuronal tissue cells, and tumor cells.⁸,⁹

TNFα operates in various parts of the body as a key cytokine in inflammatory and immune processes.¹⁰ At low concentrations, TNFα has multiple biological properties; it increases the expression of some adhesion molecules (e.g., Intercellular Adhesion Molecule (ICAM-I)), important for the transendothelial migration of lymphocytes toward the site of tissue damage. It stimulates the release of IL-8, IL-1, and IL-6 by macrophages. In the course of bacterial infections, high TNFα concentrations may be present in the blood and they are a powerful mediator of the inflammatory response to bacteria. From this point of view, TNFα shares many of the biological properties of IL-1. TNFα is important in the control of the body temperature and induce the acute phase response proteins. TNFα has a crucial role in host immunity, preventing infection, and the growth of malignant tumors.¹¹,¹² TNFα acts by binding to two receptors, TNFR1 (known as p55TNFR) and TNFR2 (known as p75TNFR). TNFR1 is ubiquitously expressed by a broad array of cell types and contains a death domain in its intracellular portion. The two receptors are differentially expressed in various cells and also differ in terms of their functionally. TNFR1 contains an intracellular ‘death domain’ that activates Nuclear Factor kappa B (NF-kB) signaling pathways leading to apoptosis.¹³ TNFR2 does not contain a death domain and it is expressed in lymphocytes and in different anatomic regions of the brain cells. Its effects include T-cell activation and proliferation via signaling pathways that involve NF-kB, Activator Protein 1 (AP-1), and the Mitogen-Activated Protein Kinase (MAPKs).¹⁴ Knockdown studies have shown that TNFR2 has a trophic or protective role in neuronal survival.¹⁵

Similar to most cytokines, TNFα is expressed at very low levels in a healthy adult brain but highly expressed in neurodegenerative brains, and neuroinflammation can be detected years before neuronal death which is partially mediated through TNFR1.¹⁶ High concentrations of TNFα can cause severe disease and therefore TNFα synthesis should be kept at homeostatic levels to combat infections avoiding overproduction of the cytokine that may cause adverse reactions. Indeed, extremely high blood concentrations of TNFα cause disseminated intravascular coagulation, shock, and severe tissue damage. In conclusion, the biology of TNFα whose effects depend on whether it acts in soluble or membrane-bound form and through which of two distinct receptors it signals, underlies the complex nature of this cytokine. More understanding on the regulation of expression of the two TNFα receptors and signaling molecule in primary cells will allow to understand how to control the duality of function of TNFα in organs such the Central Nervous System (CNS) in physiological conditions and during disease state.

Type I IFNs were described as proteins that are secreted by virus-infected cells and interfere with virus replication in autocrine and paracrine ways.¹⁷ It is now more appreciated that type I IFNs are cytokines produced in response to a wide variety of microbes such as viral, bacterial, and fungal pathogens as well as parasites.¹⁸ It was later discovered that these molecules can also activate inflammatory cells. There are two forms of interferons, the type I IFNs and type II IFNs. The type I IFNs are produced primarily during viral and microbial infections and include IFNα produced by leukocytes, IFNβ produced by fibroblasts, and IFNk produced by keratinocytes. Type II IFN, better known as IFN-γ, is produced by activated T and NK cells and share many of the antiviral activity of type I IFNs. However, IFNγ is not structurally related to IFNα and IFNβ as it interacts with specific receptor and possesses immune-regulatory properties that are distinct from type I IFNs.

IFNγ is a key mediator of inflammatory immune responses and it activates mononuclear phagocytes to phagocytose and kill microorganisms. This cytokine belongs to a class of molecules that induce the differentiation of monocytes into macrophages. IFNγ increases the expression of MHC class I and II molecules on the antigen-presenting cells (APC) surface, enhancing the capacity of recognition by the T cells during antigen presentation. IFNγ possesses numerous other biological properties: It directly promotes T and B-cell differentiation and can activate neutrophils and NK cells. Also, IFNγ stimulates the expression of adhesion molecules on different immune cells and increase transendothelial migration to the inflammatory sites. IFNγ is widely distributed playing a pivotal role in host defense.¹⁹–²¹ The binding of IFNγ to its receptor activates the JAK–signal transducer and activator of transcription (STAT) pathway which modulates the transcriptional activation of several genes and mediates diverse biological responses. The IFNγ receptor consists of two subunits, IFNγ receptor (IFNΓR)1 and IFNΓR2 and each molecule interacts with a member of the JAK family. JAKs phosphorylate receptors and transcriptional coactivators named STATs.²² IFNγ acts as a master regulator of many inflammatory pathways as it is involved in regulating the production of several molecules involved in the inflammatory response. For example, IFNγ upregulates the production of IL-12, TNFα, and several chemokines in macrophages induced by lipopolysaccharide (LPS).²³ The pleiotropic effects of IFNγ are mediated by modulating a large collection of genes and highlight the importance of identifying and studying IFNγ-regulated genes. Thus, it would be interesting to identify novel genes that are the direct targets of the IFNγ response. Further investigations into the functions of IFNγ-modulated genes will help in understanding their functional effects. Such studies may identify therapeutic targets and interventions during injury or disease.

IL-18 is closely related to IL-1β in that both are first synthesized as inactive precursors, both require caspase-1 for cleavage, and both have decoy receptors. IL-18 is an important component of polarized Th1 cell and NK cell responses. IL-18 primarily induces Th1 responses due to the induction of IFNγ in combination with IL-12. However, in the absence of IL-12, there is a role for Th2²⁴ and NK cell maturation, cytokine production, and cytotoxicity.²⁵

IL-33 is a member of the IL-1 family of cytokines with a growing number of target cells and a plethora of biological functions. The cytokine acts on many different target cells in many organs. Its main effects on innate and adaptive cells, Th2 and innate lymphoid cell-2 (ILC2) cells, and alternatively activated M2 polarized macrophages are consistent with its general function. It can be expressed in a constitutive or inducible manner by several stromal, parenchymal, and hematopoietic cell types. Mature IL-33 signals through the suppression of tumorigenicity 2 (ST2) receptor, which associates with IL-1 accessory protein (IL-1AcP) to induce myeloid differentiation primary response gene MyD88-dependent signaling. IL-33 amplifies innate immunity and inflammatory responses.²⁶

IL-36 family members IL-36α (IL-1F6), IL-36β (IL-1F8), and IL-36γ (IL-1F9) bind to IL-1Rrp2 and use IL-1RAcP as a coreceptor. IL-36 is produced by innate immune cells and lymphocytes inducing the production of proinflammatory cytokines, chemokines, and costimulatory molecules, thus promoting Th1 and Th17 cell polarization.²⁷,²⁸ In general, IL-36 mirrors IL-1 and may serve as an amplifier of innate immunity and a mediator of inflammation in selected tissues such as skin and lung.

Regulatory Cytokines

Interleukin-10 (IL-10) and transforming growth factor-beta (TGF-β) are the two best studied regulatory cytokines. In humans, IL-10 suppresses the expression of MHC class II, costimulatory, and adhesion molecules on monocytes. IL-10 also inhibits the production of proinflammatory cytokines and the activation of T cells via APCs. It possesses important anti-inflammatory factor that controls the response of lymphoid and myeloid cells and thus inhibits immunopathology. This role of IL-10 is extremely important in protecting the host from infection-associated immunopathology, autoimmunity, allergy, sepsis, arthritis, insulitis, and inflammatory bowel disease (IBD). In addition to these activities, IL-10 regulates growth or differentiation of B cells, NK cells, cytotoxic and helper T cells, mast cells, granulocytes, DCs, keratinocytes, and endothelial cells.²⁹

IL-10 production was originally ascribed to CD4+ Th2 cells.³⁰ However, it is now known that IL-10 is released by a wide variety of immune cells, including Th1 and regulatory T (Treg) cells, CD8+ T cells, B cells, macrophages, DCs, neutrophils and eosinophils. The cytokine presents a complex transcriptional regulation and indeed mechanisms such as epigenetic regulation, activation of specific transcription factors and triggering of signaling pathways have been all described.³¹ It is clear that deregulation of these tightly regulated processes is associated with detrimental effects of IL-10. Therefore, understanding the regulation of IL-10 expression by different cells is instrumental for the targeted design of immune intervention strategies. Binding of IL-10 to its receptor triggers a series of signaling cascades mediated by the JAK signal transducer and STAT pathway, particularly by STAT3.³² Signaling through the IL-10 receptor regulates several steps of the immune response, from decreasing cytokine gene expression to downregulating the expression of MHC-II and thus antigen presentation to T cells.²⁹,³² Moreover, IL-10 has been shown to prevent apoptosis by activating the phosphatidylinositol-4,5-bisphosphate-3-kinase (PI3K)/Akt cascade and enhancing the expression of antiapoptotic factors as B cell lymphoma-xl (Bcl-xl) and Bcl-2, while attenuating that of caspase-3. It is important to note that the cellular source of IL-10 production is critical to its immunological activities in a cell-specific manner. For example, a critical role of B cell-derived IL-10 has been demonstrated in the mouse model of experimental autoimmune encephalomyelitis (EAE). Mice with a disruption in the Igμ heavy chain (μMT), which results in a lack of B cells, develop a non-remitting form of EAE. Transfer of Wild Type (WT) B cells restored remission, whereas B lymphocytes from IL-10-deficient mice were unable to suppress the disease progression.³³ Therefore, these studies highlight the importance of understanding the biology of IL-10 derived from different cellular origins as it determines its unique range of