VII.  The Immune System: Current Concepts  (Part II of II)   

                                                                                                   Dr. S.G. Deodhare

     

OUTLINE

            Cytokines

                        General aspects

                        Defective cytokine production

                        Cytokines as markers of bacterial sepsis in the newborn infant

                        Cytokines and chronic mucocutaneous candidiasis

                        Transforming growth factor-b in health and disease

                        Interleukins

                        Interferons and their therapeutic applications

            Transplantation

                        Transplantation of thymus tissue in complete DiGeorge syndrome

                        Umbilical cord blood transplantation

References

Graphics

            Table 1

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CYTOKINES: General Aspects

            Innate responses frequently involve cytokines, complement and acute phase proteins. All these constitute as a group of soluble mediatoris.

            When cells and tissues in complex organisms need to communicate over distances greater than one cell diameter, soluble factors must be employed. Cytokines are soluble peptide mediators that play pivotal roles in communication between cells of the haemopoietic system and other cells in the body. Cytokines influence many aspects of leucocyte function, including differentiation, growth activation and migration.

          Cytokines are pleotropic regulatory peptides that can be produced by virtually every nucleated cell in the body. In most tissues, constitutive productivity of cytokines is absent or minimal. However, the physiological stimuli activate cells, the production of these autocrine, paracrine and endocrine effector molecules increases, and they in turn, orchestrate the tissues response to the stimulus.

            Cytokines are comparatively low molecular weight glycoproteins that are produced in small amounts by cells in the immune system. They orchestrate many functions of the immune system and inflammatory responses. They act as messengers both within the immune system and between the immune system and other systems of the body forming an integrated network that is highly evolved in the regulation of immune responses

            The cytokine family consists of several subfamilies: the interleukins, tumour necrosis factors (TNF) family, interleukin-6 (IL-6) and related cytokines, interferons, chemokines such as IL-8, transforming growth factor (b), colony stimulating factor (CSF) and others.

            Cytokines can be measured in plasma, serum and various body fluids. For many cytokines, ELISA technology is available. The assays use specific monoclonal or polyclonal antibodies.

            The presence of cytokine is sensed by a cell by means of specific cytokine receptors. In order to mediate their effects, cytokines must first bind to receptors on the cell surface of responding cells.

            A simple concept that continued to be useful for discussions on cytofunction is the concept of primary and secondary cytokines. Primary cytokines are those that can by themselves, initiate all the events required to bring about leucocyte infiltration of the tissues. Of the large number of cytokines that have now been described, only IL-1 (both a and b forms) tumour necrosis factor (TNF, includes both TNF-a and TNF-b) truly function as primary cytokines. IL-1 and TNF are able to induce cell adhesion molecules (CAM) expression on endothelial cells, to induce a variety of cells to produce a host of additional cytokines, and to induce expression of chemokines that provide a chemotactic gradient allowing the directed migration of specific leucocyte subsets into a site of inflammation. In spite of potent inflammatory activity, other cytokines do not duplicate this full repertoire of activities. Many qualify as secondary cytokines whose production is induced following stimulation by IL-1 and/or TNF. The term secondary does not imply that they are less important and less active than primary cytokines; rather it indicates that their spectrum of activity is more restricted.

            Another valuable concept that has withstood the test of time is the assignment of many T-cell derived cytokines into one of two groups, depending on which of the two helper T cell subsets (Th1 and Th2) produce them. For example, the secretion INF-gamma by Th1 cells inhibits Th2 cells, and the secretion of IL-10 by Th2 cells reciprocally inhibits Th1 cells. Exploitation of such regulatory interactions between subgroups of T cells may lead to new therapeutic approaches for a variety of diseases. For example, the secretion of IL-4 by Th2 cells stimulates the production of IgE, so that devising a way of shifting the system to induce a Th1-cell-mediated response could ameliorate atopic allergy.

            Th1 cells secrete INF-gamma and IL-2, which activate macrophages and cytotoxic T cells to kill intracellular organisms; Type Th2 cells secrete IL-4, IL-5, IL-6 and IL-10, which help B cells to secrete protective antibodies.

            Dominance of Type 1 and Type 2 cytokine patterns in a T cell immune response has profound consequences for the outcome of immune responses to certain pathogens and extrinsic proteins capable of serving as allergens.

            In addition to acting as messengers, some cytokines have a direct role in defence; for example, the interferons that are released by virally infected cells establish a state of viral resistance in the surrounding cells. Cytokines and their antagonists (anti-cytokines) are increasingly being used as therapeutic agents. For example, a combination of interleukin-2 (IL-2) and interferon-a has proved valuable in the treatment of melanoma (Keilholz, U et al 1998). Infliximab, a chimeric monoclonal antibody against tumour necrosis factor a, has had strikingly beneficial effects in patients with rheumatoid arthritis (Friedmann M et al, 1997).

Cytokine-mediated Signalling Pathways: JAK/STAT

            A major breakthrough in the analysis of cytokine-mediated signal transduction was the identification of a common cell surface to nucleus pathway used by the majority of cytokines. This JAK-STAT pathway was first elucidated through careful analysis of signalling initiated by interferon receptors, but was subsequently shown to play a role in signalling by all cytokines that bind to members of the haematopoietic receptor superfamily. The JAK-STAT pathway separates through the sequential action of a family of four nonreceptor tyrosine kinases (the Jacks or Janus family kinases) and a series of latent cytosolic transcription factors known as STATs (STAT stands for signal transducers and activators of transcription).

         

Defective Cytokine Production

            Two main defects of cytokine production are known. The first is a selective inability to produce IL-2. In the two reported cases, patients had severe recurrent infections in infancy. The IL-2 gene was present in both, but no IL-2 message or protein was produced. The second was found in a single patient who also presented during infancy with severe recurrent infections and failure to thrive. She had defective transcription of several genes, including IL-2, IL-3, IL-4 and IL-5.

 

Cytokines as Markers of Bacterial Sepsis in Newborn Infant

            Currently, C-reactive protein (CRP) or neutrophil indices such as the immature total neutrophil ratio and absolute neutrophil count have been used alone or in combination as markers of neonatal bacterial infection.

            During the past few years proinflammatory cytokines, molecules involved in inflammation and immunity, have been studied in newborn infants. Although several cytokines have been studied in newborn infants, attention has focused mainly on the contribution of tumour necrosis factor-alpha (TNF-alpha), Interleukin-6 (IL-6) and Interleukin-8 (IL-8) (Mehr S, et al, 2000).

            Why might cytokines, such as TNF-alpha, IL-6 and IL-8 be more useful as early markers of infection than other commonly used laboratory indices? TNF-alpha is a principal initiator of systemic inflammation, rising early in human models of endotoxaemia, and together with IL-6 induces CRP (Moshage H, 1997). Elevated TNF-alpha and IL-6 concentration might therefore be detectable before any measurable increase in the CRP. IL-8 is also likely to be an earlier marker of sepsis because of its involvement in neutrophil bone marrow release and subsequent neutrophil activation and chemotaxis.

            Data regarding the predictive value of measuring IL-6 in early and late onset infection conflict, but studies have consistently shown that IL-6 is a better marker than CRP in the first 24 hours of life. The sensitivity improves when IL-6 and CRP are combined. The limited data available on TNF-alpha and IL-8 suggest that both markers are valuable, but not infallible, as markers of sepsis in newborn infants. TNF-alpha, IL-8 and IL-6 improve in accuracy when combined with CRP. However, further studies are required with larger sample sizes before the contribution of cytokines to the diagnosis of infection beyond the more traditional markers of CRP and neutrophil indices can be determined.

Cytokines and Chronic Mucocutaneous Candidiasis

          Chronic mucocutaneous candidiasis is a group of disorders in which the common clinical manifestation is recurrent and persistent infection of the skin, nails and mucous membrane with Candida albicans. Patients with chronic mucocutaneous candidiasis rarely develop Candida sepsis or cadidiasis of parenchymal organs. For this reason, much of the research into host defence mechanisms in patients with this disorder has focussed on immune mechanisms in the skin and mucous membranes.

            A common immunologic abnormality is failure of the patients T lymphocytes to produce cytokines such as interferon gamma and macrophage migration inhibitory factor. A subgroup of patients is predisposed to development autoimmune endocrinopathies including acute adrenal insufficiency (Kirkpatric C M, 2001). Antifungal drugs are effective in clearing the infections, and treatment that restores cellural immunity has produced long-term remissions.

Transforming Growth Factor-b in Health and Disease       

          Transforming growth factor-b1 (TGF-b1) was first isolated as a secreted product of virally transformed tumour cells capable of inducing normal cells in vitro to show phenotypic characteristics associated with transformation. Over thirty additional members of the TGF-b superfamily have now been identified; these can be grouped into several families: the protypic TGF-bs (TGF-b1 to TGF-b3), the bone morphogenetic proteins (BMPs), the growth/differentiation factors (GDFs) and the activins. The TGF name for this family of molecules is somewhat of a misnomer, since TGF-b has antiproliferative rather than proliferative effects on most cell types. Many of the TGF-b family members play an important role in development, influencing the differentiation of uncommitted cells into specific lineages. TGF-b superfamily members are made as precursor proteins that are biologically inactive until a large prodomain is cleaved.

TGF-b Signalling

          Transmembrane receptors for TNF-b have been cloned, defining a new family of receptor molecules with an intracellular serine/threonine kinase activity. Participation of at least two cell surface receptors (type I and type II) with serine/threonine kinase activity is required for biological effects of TGF-b. Ligand binding by the type II receptor to phosphorylate and activate the type I receptor, a transducer molecule responsible for downstream signal transduction. Still other molecules such as proteoglycan betaglycan (also known as the type III TGF-b receptor) can modulate signalling by the type I and type II TGF-b receptors by dramatically improving the affinity of the type II receptor for ligand. A family of signalling molecules that play a critical role in signal transmission from the membrane bound receptors in the TGF-b receptor family to the nucleus are the SMAD proteins. Seven distinct vertebrate SMADs have been described to date that are involved in signalling mediated by different TGF-b superfamily members.

TGF-b Functions

          TGF-b has a profound influence on several types of immune and inflammatory processes. A combination of effects of TGF-b on fibroblast function make it one of the most fibrogenic of all cytokines studied. TGF-b treated-fibroblasts display enhanced production of collagen and other extracellular matrix molecules. In addition, TGF-b inhibits the production of metalloproteinases by fibroblasts and stimulates the production of inhibitors (TIMPs) of the same metalloproteinases. TGF-b effects on fibroblasts may be important in promoting wound healing. An immunoregulatory role for TGF-b1 was identified in part through analysis of TGF-b1 knockout mice that develop a wasting disease at 20 days of age associated with a mixed inflammatory cell infiltrate involving many internal organs.

TGF-b in Disease

            Cells escape from normal growth regulation when they become resistant to TGF-b action as occurs in neoplastic transformation. Disregulated expression of or response to TGF-b has also been implicated in the pathogenesis of many other disease processes (Blobe GC, et al 2000). 

            The role of TGF-b in human diseases encompasses two major aspects: one involving increased TGF-b activity as occurs in patients with fibrosis and progressive cancers and a second involving decreased TGF-b activity as occurs in early tumorigenesis, hereditary haemorrhagic telengiectasia, developmental defects and atherosclerosis.

            TGF-b regulates extracellular matrix formation, morphogenesis and inflammatory responses. Disregulated expression or response to TGF-b has been implicated in the pathogenesis of many disease processes including autoimmunal disease, fibrotic disease, chronic inflammation and neurodegenerative disease.

Role of TGF-b in Cancer

            In normal cells, TGF-b, acting through its signalling pathway, arrests the cell cycle at the G1 stage to inhibit proliferation, induce differentiation or promote apoptosis. During transformation of a cell into a cancer cell, various components of signalling pathways are mutated, making the cell resistant to the effects of TGF-b.  These TGF-b resistant cancer cells, which proliferate in an unregulated manner, as well as the surrounding stromal cells (fibroblasts) then increase their production TGF-b. This TGF-b by acting on the surrounding stromal cells, immune cells, and endothelial and smooth muscle cells, causes immunosuppression and angiogenesis and increase the invasiveness of the tumour. 100% of pancreatic cancers and 83% of colon cancers have a mutation affecting at least one component of the TGF-b pathway (Grady WM, et al 1999). In normal cells, TGF-b acts as a tumour suppressor by inhibiting cellular proliferation. In tumorigenesis, a cell loses its TGF-b-mediated growth inhibition property.

Role of TGF-b in Fibrotic Disease

            TGF-b acts as a regulator of extracellular matrix protein expression including proteases and protease inhibitors. It enhances formation of collagen, fibronectin and laminin as well as certain cell adhesion molecules. It has an essential role in wound healing and tissue repair (Singer AJ, Clark RAF 1999). Although TGF-b is essential for wound healing, overproduction of TGF-b can result in excessive deposition of scar tissue and fibrosis. In animals, overexpression of TGF-b results in fibrosis of the kidney, lung and liver. Overproduction of TGF-b is associated with keloid formation. Natural variations (polymorphisms) in the gene for TGF-b regulate its level of expression in humans. These polymorphisms may have a role in predisposing a person to fibrotic disease.

Role of TGF-b in Atherosclerosis

   TGF-b inhibits the proliferation and migration of smooth muscle and endothelial cells. Apolipoprotein A is an independent risk factor for cardiovascular disease when expressed at high levels. In mice, the expression of apolipoprotein A inhibits proteolytic activation of TGF-b, thereby promoting the proliferation of smooth muscle cells and the subsequent development of fatty lesions. Conversely, treatment of mice with the antiestrogen tamoxifen increases serum TGF-b1 levels and suppresses the formation of lipid lesions in the aorta.

TGF-b in the Diagnosis and Prognosis of Human Diseases

          The levels of TGF-b in serum and of TGF-b in RNA in tissue can be measured and used as diagnostic or prognostic markers for human disease. High levels of TGF-b1 in RNA in tissues are associated with gastric cancer.  High serum TGF-b1 levels are correlated with the development of fibrosis in patients with breast cancer who have received radiation therapy. Further, early increases in serum TGF-b2 levels predict a clinical response to tamoxifen in patients with breast cancer.

Interleukins (ILs)

           Interleukins (ILs) are part of a larger class of polypeptides known as cytokines. These are messenger molecules that transmit signals between various cells of the immune system. They are mostly secreted by macrophages and lymphocytes and their production is induced in response to injury or infection.  Their actions influence other cells of the immune system as well as other tissues and organs including the liver and brain. There are 18 ILs described up till now. IL-2, IL-4, IL-6, IL-8 and IL-10 are the important interleukins. Table 1 shows selected functions of representative interleukins.

Table 1 Selected Functions of Representative Interleukins*

*In each case, the functions of interleukins are limited to certain aspects. For example,

 IL-8 is principally chemotactic for neutrophils.

           

            Role of Interleukin-10 in Critical Illness

            IL-10 is the most potent anti-inflammatory cytokine yet identified. It has multiple actions affecting the innate immune system as well as humoral and cellular immune response. It occupies a pivotal role in the regulation of the immune response to microbial pathogens in health and disease. Knowledge gained in the molecular biology of IL-10 and the complex immune effects in the experimental infection models are leading to insights into therapeutic manipulations in patients with systemic inflammatory disease (Opel SM, 2000).

Interferons and their Therapeutic Applications

           Interferons are a family of closely related cytokines that possess potent antiviral and immunoregulatory activities.

            The antiviral activity of IFN was found to be non-specific; a fact that led to the idea that IFN might be used therapeutically against all kinds of viral infections. The revolution that antibiotics had meant for bacterial infections, it was reasoned, might be paralleled by therapeutic use of IFN for viral infections.

            The optimism regarding the potential of IFN as an antiviral therapeutic agent has not been fulfilled and for various reasons it is not until the last decade that IFN has been established as a potent antiviral agent in chronic viral infections. In parallel with being the object of antiviral research, however, IFN has also been studied with regard to its antitumour properties, and it is today becoming a standard treatment in certain malignant diseases.

Different Types of Interferons

            Although IFN was initially thought to be a single entity, later research has shown that there are multiple molecular species of IFN. Thus, there are three main classes of human IFNs called a, b and g interferons (IFN-a, IFN-b and IFN-g) and a minor class called w IFN (IFN-w). There are 13 genes, two of which are identical, for IFN-a, of which there are thus 12 subtypes, but only one gene, and no subtypes for each of IFN-b and IFN-g.

            The reason why there are so many subtypes of IFN-a remains enigmatic. However, the various subtypes of IFN-a vary markedly regarding their biological activities. Thus, for instance, the most pronounced antiviral activity on a molar basis is found in IFN-a8, and IFN-a1 has certain immunologic activities that are absent among other subtypes. It therefore seems plausible that the different IFN-a subtypes are indeed separate cytokines that share some activities, notably the antiviral capacity, but otherwise have different functional profiles.

Cellular Origin and Production of Interferons

            Although most cells in the body are capable of producing IFNs, the different classes of IFNs are preferentially produced by monocyte/macrophages and special natural interferon-producing cells that have characteristics in common with natural killer (NK) cells. IFN-b is preferentially produced by fibroblasts and IFN-g by T cells and NK cells.

            IFNs are part of the innate and adaptive immunological defence and are produced after introduction of foreign substances into the body. In particular, viruses have the ability to evoke production of IFNs but also bacteria, fungi and other non-self agents may induce IFN production. IFN-a and IFN-b are very rapidly produced upon stimulation whereas IFN-g, which is a major constituent of the antigenic-specific T cells response, may be produced at a somewhat later stage of immune responses. However, since NK cells, which are not induced in an antigen-specific manner, also produce IFN-g, the formation of this cytokine may also be an early event following stimulation with infectious agents.

Mechanisms of Action of Interferons

          After induction of IFNs, they react with cells that possess specific receptors for the various IFNs. IFN-a and IFN-b react with the same receptor, which however, is completely different from IFN-g receptor. Following this interaction, a complex series of signal transduction events take place, resulting, in the end, in the production of a multitude of proteins with different actions.

            A major breakthrough in the cytokine-mediated signal transduction was the identification of a common cell surface to nucleus pathway used y the majority of cytokines. This Jak-STAT pathway was first elucidated through careful analysis of signalling by interferon receptors but was subsequently shown to play a role in signalling by all cytokines that bind to members of the hematopoietin receptor subfamily.  The Jak-STAT pathway operates through the sequential action of a family of four nonreceptor kinases (Jak1, Jak2, Jak3 and Tyk2). The Jaks or Janus family kinases and a series of latent cytosolic transcription factors known as STATs (STAT stands for signal transducers and activators of transcription).

Therapeutic Uses of Interferons        

            As a result of their multiple biological activities recombinant IFN-a, IFN-b and IFN-g have been used as therapeutic molecules in the treatment of diseases such as viral hepatitis, cancer and multiple sclerosis.

Persistent Viral Infections

           In contrast to acute infections, chronic viral infections are often amenable to IFN therapy. Among these, hepatitis C virus (HCV) and hepatitis B virus (HBV) infections are the most important and IFN-a is currently a standard treatment in HCV and HBV infections.

Malignant Diseases

           With the advent of recombinant technology, it became possible to produce large quantities of IFN and trials in various malignant diseases were greatly extended. Excellent results using IFN-a therapy have been obtained particularly in hairy cell leukaemia and chronic myelogenous leukaemia. To the list of diseases that constitute indications for IFN-a treatment include multiple myeloma, carcinoid tumours, follicular lymphoma, polycythaemia vera and malignant melanoma.

Multiple Sclerosis

          Although multiple sclerosis (MS) is considered to be an immunologic disease, it has several traits that suggest that the causal agent may be a virus. Early trials with interferons revealed that IFN-g may aggravate and IFN-b ameliorate disease symptoms. This was taken to indicate that the beneficial effects of IFN-b are due to immunomodulatory effects of this substance. It is certainly possible, however, that the antiviral properties of IFN-b may contribute to the therapeutic effect if viruses are indeed involved in the pathogenesis of MS.

Therapeutic Effects of IFN-g

                       Good results with IFN-g therapy have been obtained, e.g. in severe mycobacterial infections and mycosis. IFN-g has also been tried in atopic dermatitis and rheumatoid arthritis, but the results have not been uniformly convincing.

Side Effects of Interferons

                        The use of interferons has been limited, however, by severe side effects. Since IFN-a has immunostimulatory properties and autoantibodies, sometimes even autoimmune disease may appear during IFN therapy.

TRANSPLANTATION

            A variety of organs and tissues are now transplanted from one individual to another, and this often is the only way of successfully treating the patients disease. Organs and tissues used in transplantation include kidney, heart (increasingly with the lung), liver (also with pancreas), pancreas, bone marrow, cornea and the skin.

Transplantation of Thymus Tissue in Complete DiGeorge Syndrome

          DiGeorge syndrome is a rare and severe form of T-cell immunodeficiency caused by failure of the thymus to develop in foetal life, associated with developmental defects. Functional T cells are absent, which leads to