A complex reaction in the vascularised connective tissue due to any type of stimuli is called inflammation. Basically, it is the reaction of blood vessels leading to accumulation of fluid and leukocyte in extravascular tissues. The inflammation is considered as an important event in body for implementation of existing defence mechanisms in circulating blood to dilute, neutralize or kill the irritant causative agent. Thus, it is said that the immunity is the resistance of body, while inflammation is the implementation of that immunity. It is beneficial to body except chronic persistent and immune origin. Inflammation starts with the sublethal injury and ends with healing, which involves following events (Chauhan, 2010):
Vasodialation and Increase in permeability
Blood flow decrease
Cells in perivascular spaces
Leucocytes degranulate in perivascular tissue spaces
Irritant is removed and damaged tissue healed
There are two types of inflammation:
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Acute inflammation: relatively short duration lasting for few minutes, several hours or few days and its main characteristics are the exudation of fluid and plasma protein and the emigration of leukocytes mainly neutrophil.
Chronic inflammation: longer duration and is associated histologically with the presence of lymphocytes and macrophages and with the proliferation of blood vessels, fibrosis and tissue necrosis.
Acute inflammation with massive tissue infiltration by neutrophils is a hallmark of many inflammatory diseases. Neutrophil activation in blood vessels and their mobilization into tissues is driven by the action of chemotactic agents, peptides or lipids, which cause directional migration of leukocytes. These mediators alter blood flow, increase vascular permeability, augment adherence of circulating leukocytes to vascular endothelium, promote migration of leukocytes into tissues, and stimulate leukocytes to destroy the inciting agent. Leucocytes when reaches in tissue spaces, they release chemical mediators of inflammation, antimicrobial factors in tissues such as cationic proteins, hydrogen peroxide, hydrolytic enzymes, lysozymes, proteases, kinins, histamine, serotonin, heparin, cytokines, and complement and this cytokine play important role in inflammation and healing (Chauhan, 2010).
Current dairy farming and livestock research has focused on the role of cytokines in bacterial infections such as Tuberculosis, Brucellosis and Mastitis, all of which have a significant impact on the dairy economy worldwide. In the case of Mastitis, a recent presentation by the Genesis Faraday Partnership in 2005 suggested that 30% of cows were affected annually, resulting in reduced production, poor milk quality, veterinary treatment and culling. Additionally, Mastitis may be harmful to suckling newborns and can cause damage to the mammary gland itself. Cytokine assays are used in this respect to monitor disease progression and prognosis. A greater understanding of the inflammatory mediators involved could lead to improved preventative programs and a role for cytokines as vaccine adjuvants (Anonymous, 2008). We will discuss the role of cytokines in inflammation in bovine.
Cytokines are small proteins (less than 50 kDa) produced by many cells, mainly activated lymphocytes and macrophages that regulate the function of other cell types. Those secreted from lymphocytes are called lymphokines and from monocytes as monokines collectively they are called as cytokines. Cytokine have long been known to be involved in cellular immune responses, but they also play an important role in both acute and chronic inflammation. Almost every mammalian cell type has the capability to produce and respond to cytokines (Dinarello, 2007). Although the majority of cytokines are secreted, some are restricted to the cellular membrane. Most cytokines have more than one function and often have redundant effects with other cytokines (Taniguchi, 1995). Because of the high affinity of their receptors, cytokines are highly potent and can elicit biological responses when present in femtomolar to nanomolar concentrations (Wahl et al., 1988; Hall et al., 1989). (Femtomolar = noncomparable and nanomolar = comparable)
Cytokines are broadly classified as interleukins, interferon, cytotoxins and growth factors (Chauhan, 2010a) as follows:
Interleukins: are of 29 different types required for cell to cell interaction among immune cells.
Interferons: are of 5 different types have antiviral action and inhibit the virus replication in cells.
Cytotoxins: are produced by macrophages and T-cells and are associated with apoptosis in tumors.
Growth Factors: Many cytokines are also known as growth factors which act on cells and stimulate them to proliferate thus they play very important role in inflammation and healing.
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The two most important cytokines that mediate inflammation are interleukin-1 (IL-1) and tumour necrosis factor (TNF). IL-1 and TNF share many biological properties. IL-1 and TNF are produced by activated macrophages. Their secretion is stimulated by endotoxins, immune complexes, toxins, physical injury, or a variety of inflammatory mediators. Their most important actions are their effects on endothelium, leukocytes and fibroblasts and induction of the systemic acute phase reactions. Both IL-1 and TNF induce endothelial activation that causes increased expression of adhesion molecules that mediate leukocyte sticking, secretion of additional cytokines and growth and production of arachidonic acid metabolites and nitric oxide. TNF also causes aggregation and activation of neutrophils and release of proteolytic enzymes, thus contributing to tissue damage. Both the cytokines activate fibroblasts, resulting in fibrosis. IL-1 and TNF also induce the systemic acute phase responses associated with infection or injury, including fever, loss of appetite, lethargy, hepatic synthesis of various proteins, metabolic wasting and neutrophil release into the circulation (Reference needed).
Functions of Cytokine:
Cytokines arc produced in response to many different stimuli. The most important of these are antigens acting through the TCR (T-cell receptor) or BCR (B-cell receptor), antigen-antibody complexes acting through Fc (crystallisable fragment) receptors, super-antigens acting through the TCR, and PAMPs (pathogen associated molecular pattern) such as lipopolysaccharides acting through TLRs (toll like receptor). Cytokines act on many different cellular targets. They may, for example, bind to receptors on the cell that produced them and thus have an autocrine effect. Alternatively, they may bind only to receptors on cells in close proximity to the cell of origin and thereby have a paracrine effect. They may spread throughout the body, affecting cells in distant locations, and thus have an endocrine effect (Tizard, 1992).
Figure 1: The distinction among autocrine, paracrine, and endocrine effects. Cytokines differ from hormones in that most of their effects are autocrine or paracrine, whereas hormones act on distant cells in an endocrine fashion (Tizard, 1992).
Cytokine Response in Acute Inflammation:
When exposed to infectious agents or their PAMPs, the sentinel cells secrete many different molecules. These molecules include the major cytokines interleukin- 1 (IL-1) and tumor necrosis factor-Î± (TNF-Î±) as well as others, such as IL-6, IL- 12, and IL-18. They also secrete oxidants, such as O2, H2O2, 'OH, and NO2 and lipids, such as the leukotrienes and prostaglandins. When released in sufficient quantities these molecules can also cause a fever, sickness behaviour, and promote an acute-phase response.
Active IL-1 is a mixture of two molecules, IL-1Î± and IL-1Î². Although its major source is the macrophage, IL-1 is also produced by dendritic cells, T cells, B cells, NK cells (natural killer cell), vascular endothelium, fibroblasts, and keratinocytes. IL-1 acts on T cells, B cells, NK cells, neutrophils, eosinophils, dendritic cells, fibroblasts, endothelial cells, and hepatocytes. IL-lÎ² is produced as a large protein that must be cleaved by IL-1Î² -converting enzyme (also called caspase 1) to form the active molecule. IL-1Î± and IL-1Î² also promote inflammation and stimulate the acute phase response.
There are nine members of the IL-l receptor family, but the most significant are CD (cluster of differentiation) 121a and CD121b. CD121a is a signalling receptor, whereas CD121b is not. CD121b inhibits IL-1 functions. Soluble CD121b can bind IL-1 and acts as an IL-l antagonist.
When activated through CD14 and TLR4, macrophages produce two glycoprotein's called IL-1Î± and IL-1Î². Ten-to 50-fold more IL-1Î² is produced than IL-1Î±, and while IL-1Î² is secreted. IL-1Î± remains attached to the cell. Therefore IL-1Î± can only, act on target cells that come into direct contact with the macrophage. Transcription of IL-1 mRNA occurs within 15 minutes of exposure to a stimulus. It reaches a peak 3 to 4 hours later and levels off for several hours before declining, Like TNF-Î±, IL-1 acts on vascular endothelial cells to make them adhesive for neutrophils.
During severe infections, some IL-1 escapes into the bloodstream where, in association with TNF-Î±. It is responsible for sickness behavior. Thus it acts on the brain to cause fever, lethargy, malaise and lack of appetite. It acts on muscle cells to mobilize amino acids causing pain and fatigue. It acts on liver cells to induce the production of new proteins, called acute-phase proteins that assist in the innate defense of the body. IL-1 can activate lymphocytes (the cells that mediate the acquired immune response) and is necessary for the successful initiation of some forms of acquired immunity.
Tumor Necrosis Factor-Î±:
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The tumor necrosis factor super-family (TNFSF) contains at least 18 related proteins that regulate cellular activation, viability and proliferation through the transcription factor. Its most important member is TNF-Î±, a 25kDa trimeric protein produced by macrophages, mast cells, T cells, endothelial cells, B cells, and fibroblasts. It can occur in soluble or membrane-bound forms. The membrane-bound form is cleaved from the cell surface by a protease called TNF-Î± convertase. Its production is stimulated by nerves such as the neurotransmitter substance P. TNF-Î± mediates many immune and inflammatory functions and regulates the growth of many cell types. It is a potent pro-inflammatory molecule and many of its activities are shared with IL-1. Of major importance are its effects on vascular epithelium at sites of microbial invasion. Thus TNF-Î± enhances their expression of adhesive molecules and triggers pro-coagulant activity. It promotes fibroblast proliferation and collagen production, a feature of importance in chronic inflammation. TNF-Î± activates macrophages to increase its own synthesis together with that of IL-1, IL-6, M-CSF (macrophage colony stimulating factor), and GM-CSF (granulocyte macrophage colony stimulating factor). As its name implies, TNF-Î± can trigger killing of some tumour cells and virus-infected cells. It does so by activating caspases, the proteases that are the major mediators of apoptosis. In high doses, TNF-Î± can cause septic shock. TNF-Î± receptors are found on almost all nucleated cells. They are of two types (CD120a and CD120b). TNF-Î± is produced very early in inflammation, and this is followed by IL-1 and then by IL-6.
TNF-Î± is an essential mediator of inflammation because in combination with IL-1 it triggers critical changes in the cells that line small blood vessels (vascular endothelial cells). A local increase in TNF-Î± causes the "cardinal signs" of inflammation, including heat, swelling, pain, and redness. Systemic increases in TNF-Î± depress cardiac output, induce micro-vascular thrombosis and cause capillary leakage. TNF-Î± acts on neutrophils to enhance their ability to kill microbes. It is a potent attractant for neutrophils, drawing them to sites of tissue damage and increasing their adherence to vascular endothelium. TNF-Î± acts on macrophages to stimulate their production of IL-1 and the inflammatory lipid, prostaglandin E2 (PGE2). It also stimulates macrophage phagocytosis and oxidant production. It amplifies and prolongs inflammation by activating other cells to release IL-1, inflammatory lipids, nitric oxide, and oxidants. TNF-Î± also activates mast cells.
IL-6 is produced not only by activated macrophages but also by T and B cells, mast cells, vascular endothelial cells, fibroblasts, keratinocytes, and mesangial cells. It is also produced by muscle cells during exercise. IL-6 acts on T cells, B cells, hepatocytes, and bone marrow stromal cells. It promotes IL-2 and IL-2R production and T cell differentiation; it synergizes with IL-4 to promote Th2 cell differentiation, and it is required for the final maturation of B cells into plasma cells. IL-6 acts as a cofactor with IL-1 in IgM synthesis and with IL-5 in IgA synthesis. It is a major stimulator of the acute-phase response.
IL-6 affects many different functions, including both inflammation and acquired immunity. It is a major mediator of the acute-phase reaction and of septic shock. It has been suggested that IL-6 regulates the transition from a neutrophils-dominated process early in inflammation to a macrophage-dominated process later on.
IL-12 is a heterodimeric 70 kDa glycoprotein, consisting of 35 and 40 kDa subunits. It is produced by monocytes and macrophages, dendritic cells, B cells, and keratinocytes. IL-12 may be stored preformed in macrophage and rapidly released in large quantities on activation. IL-12 promotes Th1 (T helper) cell activity by inducing secretion of IL-2 and IFN-Î³ and enhances T and NK cell proliferation and cytotoxicity. TNF-Î± and IL-12 synergize in promoting IFN-Î³ production. As a secondary effect it reduces IgE production by suppressing IL-4 synthesis. Its receptor, IL-12 is classified as CD212 and is expressed on mononuclear cells.
Bovine ENA is a new monocyte-macrophage derived cytokine of the Interleukin-8 family. A novel bovine neutrophil-activating peptide, bovine ENA (boENA), is identified in the conditioned media of endotoxin-stimulated bovine monocytes and alveolar macrophages. The chemotactic peptide was purified to homogeneity from conditioned media by cation exchange chromatography and several steps of reversed-phase high-performance liquid chromatography. The partial amino acid sequence of boENA was: VVRELRCVCLTTTPGIHPKTVSDLQVIAAGPVCSKVEVIATLKNGXX. Its cysteine molecules are positioned identically to those of the C-X-C family of human proinflammatory peptides. BoENA shows structural (73% identity in amino acid sequence) and functional homology to human ENA- 78, a product of the human type II epithelial cell line A549, as demonstrated in assays for chemotaxis, aggregation, shape change, and a rise in intracellular free calcium. The immunohistochemical identification of boENA in the hyperplastic type II alveolar epithelial cells and in pulmonary alveolar leukocytes of pneumonic bovine lungs strongly supports a role for ENA-78 in the genesis of pulmonary inflammation.
Cytokine Response in Chronic Inflammation:
If foreign material is not destroyed but persists for long periods within the body, the inflammatory process may also persist and become chronic. Examples of such persistence include infections with bacteria such as Mycobacterium tuberculosis, fungi such as Cryptococcus, parasites such as liver fluke or the presence of inorganic material such as asbestos crystals. Macrophages, fibroblasts, and lymphocytes may accumulate in large numbers around the persistent material. Because they resemble epithelium in histological sections, these accumulated macrophages are called epithelial cells. Epithelioid cells may fuse and form multinucleated giant cells if they attempt to enclose particles too large to be ingested by a single macrophage. Epithelioid cells and giant cells are a prominent feature of tubercles, the persistent inflammatory lesions that develop in individuals suffering from tuberculosis.
In all these cases, the persistence of foreign material results in the continual arrival of new macrophages and fibroblasts and excessive deposition of collagen in affected tissues. The lesion that surrounds the foreign material is called a granuloma. Granulomas consist of granulation tissue-an accumulation of macrophages, lymphocytes, fibroblasts, loose connective tissue, and new blood vessels. The term granulation tissue is derived from the granular appearance of this tissue when cut. The "granules" are in fact new blood vessels.
If the persistent irritant is a non-antigenic "foreign body" (for example, silica, talc, or mineral oil), few neutrophils or lymphocytes will be attracted to the lesion. Epithelioid and giant cells, however, are formed in attempt to destroy the offending material. If the material is toxic for macrophages (as is asbestos), macrophage enzymes may be released, leading to excessive tissue damage and eventually to local fibrosis and severe scarring.
If the irritant is antigenic, as a result of the release of cytokines and of persistent immune stimulation, the granuloma will contain many lymphocytes as well as macrophages, fibroblasts, and probably some neutrophils, eosinophils, and basophils. The chronically activated macrophages within these granulomas will also secrete IL-1, which can stimulate collagen deposition by fibroblasts and eventually "wall-off' the lesion from the rest of the body. Antigens that provoke this form of reaction include bacteria such as the mycobacteria and Brucella abortus and parasites such as liver fluke and schistosomes. Chronic granulomatous reactions, whether due to immunological or foreign body reactions, are important since they may enlarge and destroy normal tissues. In liver fluke infestations, for example, death may result from the gradual replacement of normal liver cells by fibrous tissue as a result of chronic irritation.
Figure 2: Pathogenesis of chronic inflammation (Tizard, 1992)
The Transforming Growth Factor Î² Family (TGF-Î²):
TGF- Î²s are a family of five glycoproteins, three of which (TGF-Î²l, TGF-Î²2, and TGF-Î²3) are found in mammals. They are secreted as an inactive or latent molecule and subsequently activated. They are produced by platelets, activated macrophages, neutrophils, B cells, and T cells and act on most types, including T and B cells, dendritic cells, macrophages, neutrophils, and fibroblasts. The TGF-Î² have three fundamental activities: they regulate cell division, they enhance the deposition of extracellular matrix proteins and most important, they are immunosuppressive.
TGF-Î² regulates the growth, differentiation, and function of all classes of lymphocytes, dendritic cells and macrophages. In general, TGF-Î² inhibits T and B cell proliferation and stimulates their apoptosis, effectively acting as an immunosuppressive molecule. Apoptotic T cells release TGF-Î², thus contributing to the suppressive environment. A subset of activated CD4+ T cells can act as regulatory cells by secreting large amounts of TGF-Î². These have been called Th3 cells and probably play an important role in some forms of tolerance. TGF-Î² influences the differentiation of Th subsets. It tends to promote Th1 responses and the production of IL-2 in naive T cells, but it also antagonizes the effects of IFN-Î³ and IL- l memory cells.
TGF-Î² is required for optimal dendritic cell development and regulates the interaction between follicular dendritic cells and B cells. It also controls the development and differentiation of B cells, inhibiting their proliferation and inducing apoptosis. It also regulates the switching of B cells to IgA production.
TGF-Î² is produced by macrophages and regulates their activities. It can be either inhibitory or stimulatory, depending on the presence of other cytokines. Thus it can activate integrin expression, as well as phagocytosis by blood monocytes. On the other hand, it suppresses the respiratory burst and nitric oxide production by phagocytic cells. It blocks monocyte differentiation and the cytotoxic effects of activated macrophages. Some protozoan parasites such as Trypanosoma cruzi and Leishmania can induce infected cells to secrete TGF-Î² and so evade intracellular killing.
Role of Cytokine in Healing:
Since acute inflammation may cause severe tissue damage, it must be carefully controlled. Likewise, once the invading organisms have been destroyed, the tissue response must switch from a killing process to repair process. The timing of this switch is very important to stop killing invaders before all have been destroyed would cause problems. As inflammation progresses, the cells involved, especially macrophages, change their properties. They gradually begin to secrete SLP1, a serine protease inhibitor. This molecule inhibits the release of elastase and oxidants by TNF-stimulated neutrophils, and inhibits the activity of elastase. SLP1 also protects the anti-inflammatory cytokine TGF-Î² from breakdown. Neutrophils also change. They secrete TNF receptor fragments that bind and neutralize the TNF-Î±. TNF-Î± induces macrophages to secrete IL-12 which induces lymphocytes to secrete IFN-Î³. The IFN-Î³ acts as a macrophages-derived oxidants destroy chemotactic factors. Anti-inflammatory cytokines such as TGF-Î² and IL-10 inhibit the release of TNF-Î±. Steroids, adrenalin, and other "stress" hormones produced as a response to neuronal stimulation suppress cytokine synthesis and signal transduction.
Neutrophils are short-lived cells that usually die during an inflammatory response. If, however, the dead cells were to rupture and release their lysosomal enzymes, they could cause severe tissue damage. This does not normally occur because as neutrophils undergo programmed cells death (apoptosis), they are phagocytosed by macrophages. Even in normal healthy animals, many cells die every day and must be promptly removed. Much of this task is the function of macrophages. A good example of this is the daily removal of enormous numbers of aged neutrophils. It appears that macrophages methodically palpate any neutrophils that they encounter. If the neutrophil is healthy, it quickly detaches from the macrophage. If, however, the cell is dead or dying, as reflected in the expression of phosphatidyl serine on the plasma membrane, the macrophage remains in contact and eats the neutrophil.
The molecular basis of this interaction operates through the adhesion protein CD31. Thus CD31 on a neutrophil binds to CD31 on a macrophage. If the neutrophil is healthy, a signal sent to the macrophage causes it to disengage. On the other hand, dead and dying neutrophils fail to signal to macrophage and get eaten. It is interesting to note that failure in CD31 signalling occurs well before a neutrophil becomes so degraded that its enzyme contents can leak and cause damage. The macrophages that consume these neutrophils do not release cytokines or vasoactive lipids. Ingestion of apoptotic neutrophils causes the macrophages to secrete more TGF-Î², which in turn promotes tissue repair. Clearly, phagocytosis of apoptotic cells is an efficient way of removing unwanted cells without causing additional tissue damage or triggering unwanted inflammation.
Plasma contains several molecules that either inactivate inflammatory mediators or inhibit the enzymes that generate these mediators. Thus Î±1-antitrypsin and Î±2-macroglobulin block the enzymes released from neutrophil granules. C-reactive protein blocks platelet aggregation. Highly reactive oxidants (superoxide and hydrogen peroxide) released during the neutrophil respiratory burst are inhibited by free radical scavengers. Catalases and peroxidases scavenge peroxides while super-oxide dismutase, ceruloplasmin, and free copper ions scavenge superoxide.
Once they reach inflammatory sites, macrophages also phagocytose and destroy damaged cells and tissues. Macrophages secrete collagenases and elastases that directly destroy connective tissue. They also release plasminogen activator that generates plasmin, another potent protease. Thus macrophages can "soften-up" the local connective tissue matrix. By releasing IL-1, macrophages attract and activate fibroblasts. The fibroblasts migrate into the damaged area and secrete collagen. Initially they secrete collagen III, but this is later replaced by collagen I. This production of collagen is stimulated by multiple cytokines and by mast cell tryptase. Once sufficient collagen has been deposited, its synthesis stops. This collagen is then gradually remodelled over several weeks or months as the area returns to normal. In addition, the reduced oxygen tension in the middle of dead tissues stimulates macrophages to secrete molecules that promote the growth of new blood vessels. Once the oxygen tension is restored to normal, new blood vessel formation ceases.
The final result of this healing process depends to a large extent on the effectiveness of the inflammation. If the cause is rapidly and completely removed, healing will follow uneventfully.
IFN-Î³ is unrelated to the other interferons and is only so named because of its antiviral activity. It is produced by Th1 cells, by some CD8+ T cells, and by NK cells; it acts on B cells, T cells, NK cells, and macrophages. IFN-Î³ stimulates B cell production of IgG2a. It enhances T cell production of MHC class I molecules but not production of MHC class II molecules. It stimulates Thl cells to produce both IL-2 and IL-2R. IFN-Î³ inhibits the production of IL-4 by Th2 cells and as a result, blocks IgE production in vitro. It enhances the activities of NK cells and is thus a potent stimulator of innate immunity. NK cells respond to activation by producing IFN-Î³ so that a positive loop exists whereby activation of some NK cells results in interferon secretion and activation of other NK cells. IFN-Î³ activates macrophages and induces the production of NOS2, so increasing their ability to destroy ingested microorganisms. It also promotes antibody-mediated phagocytosis, as well as antibody-dependent cell-mediated cytotoxicity (ADCC) reactions.
IFN-Î³ increases MHC class 1 expression on tumour cells. It induces the appearance of MHC class II molecule on endothelial cells, keratinocytes, myeloid cells, some dendritic cells and fibroblasts, as well as on macrophages. It upregulates the expression of both MHC class I and II molecules on virus-infected cells. Thus when T cells secrete IFN-Î³ during graft rejection, MHC class II molecules are induced on the cells of the graft so enhancing the rejection process.
The Interleukin 10 Family:
The IL-10 family includes IL- 19, IL-20, IL-22, lL-24, and IL-26. They are all Î±-helical proteins related to IL-10. None share its immune-regulatory functions, but they have strong anti-tumor and some pro-inflammatory properties.
IL-10 is an immunosuppressive and anti-inflammatory cytokine that regulates inflammation as well as T cell, NK cell, and macrophage function. It is mainly produced by Th2 cells but may also come from activated macrophages. Its targets are Th1 cells, B cells, macrophages, NK cells, and mast cells. IL- l0 selectively inhibits co-stimulation of T cells by blocking CD28 phosphorylation. As a result it inhibits the synthesis of the Th1 cytokines, IL-2, IFN-Î³ and TNF-Î². IL-10 suppresses the secretion of IL-1, IL-6, TNF-Î± and oxidants by macrophages. It down-regulates MHC class II expression and stimulates production of IL-1RA. Its receptor is CDw210.
The other members of the IL-10 family include IL-19, produced by B cells and activated monocytes. It acts on monocytes to induce production of IL-6 and TNF-Î± and induces their apoptosis. Interleukin 20 is produced by monocytes and keratinocytes and regulates their participation in inflammation. Interleukin 22 is produced by activated Th1 cells and mast cells. It induces acute-phase protein production in the liver. IL-22 also inhibits some Th2 cytokine production. Interleukin 24 is produced by monocytes and Th2 cells. It is involved in anti-tumor activity and stimulates acute phase responses in hepatocytes. Interleukin 26 is produced by memory T cells (especially Th1 cells) and induces the proliferation of keratinocytes and T cells.
ACUTE PHASE PROTEIN IN DIFFERENT MAMMAL SPECIES
Under the influence of IL-I, TNF-Î±, and especially IL-6, liver cells increase protein synthesis and secretion. Their response begins within a few hours of injury and usually subsides within 24 to 48 hours. The levels of these new proteins may climb enormously under appropriate stimulation (Fig. 3). Because this synthesis is associated with acute infections and inflammation, these proteins are called acute-phase proteins. Acute phase proteins (APPs) are a group of blood proteins that change in concentration in animals subjected to external or internal challenges, such as infection, inflammation, surgical trauma, or stress. Many of the acute-phase proteins are important components of the innate immune system. They include complement components, clotting molecules, protease inhibitors, and metal-binding proteins. Different mammal species produce different acute-phase proteins.
Table 1: Major acute phase protein in domestic animals (Reference Needed)
SAA, haptoglobin, lipopolysaccharide binding protein
CRP, Î±1acid glycoprotein, haptoglobin
SAA, Î±-1acid glycoprotein, haptoglobin
SAA, transferrin, fibrinogen
C-reactive protein (CRP) is the major acute-phase protein in primates, pigs, rabbits, hamsters, and dogs. CRP belongs to the pentraxin family of lectins. It was first identified and named for its ability to bind and precipitate the C-polysaccharide of Streptococcus pneumonia. CRP binds to phosphatidylcholine, a molecule found in all cell membranes. As a result, it can bind to activated lymphocytes, to invading organisms, and to damaged tissues, where it activates complement. CRP is an opsonin.
It binds to neutrophils through their Fc receptors and promotes the phagocytosis and removal of damaged, dying, or dead cells and organisms. It also has an anti-inflammatory role since it inhibits neutrophil superoxide production and degranulation. CRP may therefore promote tissue healing by reducing damage and enhancing the repair of damaged tissue. (In cattle, CRP is a lactation-associated protein whose level rises two- to five-fold in lactating cows.)
Serum amyloid A (SAA) is the major acute-phase protein in cattle, cats, and horses and is also important in humans and dogs. Thus equine SAA concentrations rise several hundred-fold during noninfectious arthritis, while canine SAA concentrations increase up to 20-fold after bacterial, inoculation. Since SAA protein is immunosuppressive, it has been suggested that SAA regulates immune responses. SAA is a chemo-attractant for neutrophils, monocytes, and T cells.
In cattle lipopolysaccharide-binding protein is an acute-phase protein that rises very rapidly after infection.
Serum amyloid P (SAP) is the major acute-phase protein in rodents. It is a pentraxin structurally and functionally related to CRP. Like CRP it can bind nuclear constituents such as DNA, chromatin, and histones. It can also bind and activate C1q, the first component of the complement system.
The major acute-phase protein in pigs is called MAP (major acute-phase protein). MAP is a substrate for the proteolytic enzyme kallikrein and so can release potent inflammatory peptides called kinins. Other major acute-phase proteins in pigs include C-reactive protein, haptoglobin and ceruloplasmin.
Haptoglobin is a major acute-phase protein in ruminants, horses, and cats. It can rise from virtually undetectable levels in normal calves to as high as 1 mg/ml in calves with acute respiratory disease. Haptoglobin binds iron molecules and makes them unavailable to invading bacteria, thus inhibiting bacterial proliferation and invasion. Haptoglobin also reduces iron availability for red blood cell production so that anemia is commonly associated with severe or chronic infections. It is possible to identify animals with severe infections or inflammatory conditions by measuring serum haptoglobin levels. This may be of benefit in ante-mortem meat inspections to identify those animals that are not fit to eat. Other iron-binding acute-phase proteins include transferrin (important in birds) and hemopexin.
Some serum protease inhibitors such as Î±1-antitrypsin, Î±1-antichymotrypsin, and Î±2-macroglobulin are acute-phase proteins in many mammalian species. All of these may inhibit the tissue damage caused by neutrophil proteases in sites of acute inflammation.
Some protein levels fall during acute inflammation. These are called "negative" acute-phase proteins. In the pig, for example, these include albumin, Î±-lipoprotein, fetuin, and transferrin.
Quantification of APP concentration in plasma or serum can provide valuable diagnostic information in the detection, prognosis, and monitoring of disease in several animal species (Eckersall PD: 2000). The inclusion of APP measurements in health monitoring programs on a herd basis in livestock has been suggested, not only for the identification of individual animals with disease, but also as a means to identify animals with subclinical disease ( Eckersall PD: 2004). In addition, the use of APPs for screening in ante- or post-mortem inspection to identify animals that should be subjected to a more thorough inspection or to ensure the health of animals prior to entry to the human food chain has been suggested (Saini PK, Webert DW: 1991). Furthermore, the recent recognition that APPs are produced in the bovine mammary gland in response bacterial mastitis has exciting potential for APP measurement to detect this economically important disease in ruminants, especially if adapted for automated milking systems (Eckersall PD: 2004).
Figure 3: The acute-phase proteins. Under the influence of IL-1, IL-6, and TNF-Î±, hepatocytes secrete a large number of proteins. Almost all of these help the body control infection (Tizard, 1992).
Use of Cytokine in Diagnosis and Prognosis of Udder Health:
There is lack of efficacy of conventional strategies for the maintenance of healthy udders in domestic cattle. The adjuvant use of recombinant bovine cytokines, such as IL-2, IFN-Î³ and TNF-Î±, in normal mammary gland, mobilizes innate and acquired immunity. However, stimulated immunity does not prevent or eradicate infection, particularly in the case of Staphylococcus aureus mastitis. Cytokines do, however, improve the bactericidal efficiency of certain antibiotics. The subtle and sensitive changes in the cytokine network of normal and mastitic bovine mammary gland may encourage the use of cytokines in the diagnosis and prognosis of udder health. Numerous studies support this hypothesis, and detection and monitoring of cytokines could become an important alternative management for udder health. The use of cytokines in the immunotherapy, diagnosis and prognosis of mastitis will grow with knowledge of the cytokine network in bovine mammary glands and the development of efficient cytokine diagnostic techniques (Alluwaimi, 2004).
Interleukin-1 is crucial to the inflammatory process in the mammary gland infused with endotoxin or with natural or experimental coliform mastitis and bovine epithelial cells in vitro (Okada et al., 1999; Persson Waller, 1997; Persson Waller et al., 2003; Riollet et al., 2000b; Shuster et al., 1996, 1997; Shuster and Kehrli, 1995).
Interleukin-1Î± and 1Î² have been detected in normal bovine milk cells using the reverse transcriptase-polymerase chain reaction (RTPCR)(Ito and Kodama, 1996; Okada et al., 1997). Development of a bovine IL-1ra enzyme linked immunosorbent assay (ELISA) could prove to be a useful tool in the diagnosis of gram negative mastitis and monitoring the effectiveness of treatment (Yamanaka et al., 2000). The newly introduced ELISA has proven practical in the quantification of bovine IL-1ra in sera and whey of mastitic and healthy cows (Yamanaka et al., 2000).
Interleukin-6 has been detected in cells from mastitic gland using RT-PCR (Taylor et al., 1997) and by bioassays employing the indicator cell lines (Slebodzinski et al., 2002).