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The Classically Activated Macrophages Biology Essay

Macrophages are essential members of the innate immunity system. Together with neutrophils, eosinophils and natural killer cells, they function as a first-line defense to identify, eradicate or contain invading bacteria and toxic macromolecules. They are key players in the maintenance of tissue homeostasis. To complete these tasks, macrophages reveal a huge degree of phenotipic and functional heterogeneity. They are activated by 2 pathways classical and alternative activation states. Activated are diverse group of cells with dissimilar physiologies and performing discrete immunological functions. The exposure of to interleukin (IL)-4 or glucocorticoids induced a population of cells that up-regulated certain phagocytic receptors. Monocytes and obtained from hematopoietic bone marrow stem cells and precursors throughout a sequence of proliferation/differentiation steps synchronized by IL-1, IL-3, IL-6, GM-CSF and M-CSF. The diagram below elaborates the traditional prototypes of Mφ activation states. Mφ type-specific markers, cytokines, chemokines and functional activities are indicated in the Table below.

Vega M.A., Corbí A.L. (Octubre-Diciembre 2006)

Activated macrophages are bigger, have raised metabolism, higher levels of lysosomal proteins and an elevated ability to phagocytosis and kill microbes. They also release proteases, neutrophil chemotatic factors; reactive oxygen species such as nitric oxide and superoxide; cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin one and eight (IL-1 and IL-8), eicosanoids, as well as growth factors. These commodities of activated macrophages result in the tissue/cell obliteration which is a characteristic of inflammation

Cytokines are vital controllers of cell growth, migration, development and differentiation. The family includes a long list of inflammatory cytokines like the interleukins and interferons, growth factors like epidermal and hepatocyte growth factor and chemokines like the macrophage inflammatory proteins, MIP-1alpha and MIP-1beta. They do not consist of the peptide and steroid hormones of the endocrine nature.

Cytokines have central roles in chemically induced tissue damage repair, in cancer expansion and succession, in the control of cell replication and apoptosis, and in the modulation of immune reactions such as sensitization. They have the potential for being sensitive markers of chemically induced perturbations in function but from a toxicological point of view, the detection of cytokine changes in the whole animal is limited by the fact that they are locally released, with plasma measures being generally unreliable or irrelevant, and they have short half lives which require precise timing to detect. Even where methodology is adequate the interpretation of the downstream effects of high, local concentrations of a particular cytokine is problematic because of their interdependence and the pleiotropism of their action. A range of techniques exist for their measurement including those dependent upon antibodies specific for the respective cytokines, but with the introduction of genomic and proteomic technology, a more complete study of cytokine changes occurring under the influence of chemical toxicity should be possible. Their further study, as markers of chemical toxicity, will undoubtedly lead to a greater understanding of how synthetic molecules perturb normal cell biology and if, and how, this can be avoided by more intuitive molecular design in the future.

Macrophage Activation

Macrophage effector function significantly influences the quality, duration, and magnitude of most inflammatory reactions. Traditionally, macrophages have been described as antigen-presenting phagocytes that secrete pro-inflammatory and antimicrobial mediators.1 Mounting evidence, however, describes a more complex model involving multiple macrophage phenotypes carrying out differential functions and eliciting divergent effects on surrounding cells and tissues. Stein et al. were the first to describe "alternatively" activated macrophages as having a phenotype distinct from what are now called "classically" activated macrophages.2 From this seminal observation, a model of two major macrophage classes has developed. Classically activated macrophages exhibit a Th1-like phenotype, promoting inflammation, extracellular matrix (ECM) destruction, and apoptosis, while alternatively activated macrophages display a Th2-like phenotype, promoting ECM construction, cell proliferation, and angiogenesis. Although both phenotypes are important components of both the innate and adaptive immune systems, the classically activated macrophage tends to elicit chronic inflammation and tissue injury whereas the alternatively activated macrophage tends to resolve inflammation and facilitate wound healing (Figure 1). [Note: for recent reviews on this topic, please see references 3-6]

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Figure 1. Schematic of classical (A) versus alternative (B) activation of macrophages depicting the priming signal (classical only) and stimuli, their effects on cellular function, and subsequent effects on surrounding tissue physiology. [Note: figure adapted from Gordon, S. (2003) Nat. Rev. Immunol. 3:23.]

Classically Activated Macrophages

Differentiation of classically activated macrophages requires a priming signal in the form of IFN-gamma7 via the IFN-gamma R.8 When the primed macrophage subsequently encounters an appropriate stimulus, such as bacterial LPS, it becomes classically activated. LPS is first bound by soluble LBP and then by either soluble or membrane-bound CD14. CD14 delivers LPS to the LPS recognition complex,9 which consists of at least TLR410 and MD-2.11 Pathogens and pathogen components are subsequently taken up by phagocytosis12 and delivered to lysosomes where they are exposed to a variety of degradation enzymes including several Cathepsin cysteine proteases.13Suitable antigens are processed and loaded onto MHC class II molecules in late endocytic compartments and antigen/MHCII complexes as well as co-stimulatory B7 family members are presented to T cells.14

These events are followed closely by a significant change in cellular morph-ology and a dramatic alteration in the secretory profile of the cell. A variety of chemokines including IL-8/CXCL8, IP-10/CXCL10, MIP-1 alpha/CCL3, MIP-1 beta/CCL4, and RANTES/CCL5, are released as chemoattractants for neutrophils, immature dendritic cells, natural killer cells, and activated T cells.15Further, several pro-inflammatory cytokines are released including IL-1 beta/IL-1F2, IL-6, and TNF-alpha/TNFSF1A.3,4,5,6 TNF-alpha also contributes to the pro-apoptotic activity of the classically activated macrophage.16,17,18 TNF-alpha is accompanied by Fas Ligand/TNFSF6 secretion16 and NO release as a result of iNOS upregulation.19,20,21,22 In addition, the classically activated macrophage releases proteolytic enzymes including MMP-1, -2, -7, -9, and -12, which degrade Collagen, Elastin, Fibronectin, and other ECM components.23,24,25

While the release of these molecules is important for host defense and direction of the adaptive immune system, when uncontrolled they can levy significant collateral damage on the microenvironment. By eliciting massive leukocyte infiltration and flooding the surrounding tissue with inflammatory mediators, pro-apoptotic factors, and matrix degrading proteases, the classically activated macrophage is capable of dismantling tissues to the point of inflicting serious injury. Tissue destruction perpetrated by chronic inflammation has been associated with the development of tumors, type 1 autoimmune diseases, and glomerulonephritis among other pathologies (Figure 1A).4,6

Alternatively Activated Macrophages

Differentiation of alternatively activated macrophages does not require any priming. IL-42 and/or IL-1326 can act as sufficient stimuli. The binding of these factors to their respective receptors is followed by fluid-phase pinocytosis of soluble antigen.27,28,29 Soluble antigen is then loaded onto MHC class II molecules and antigen/MHCII complexes and co-stimulatory B7 family members are subsequently displayed to T cells.14

Similar to the classically activated macrophage, the alternatively activated macrophage changes its cellular morphology and secretory pattern as a result of appropriate stimulation. Leukocytes are attracted by the macrophage via its release of chemokines including MDC/CCL2230,31PARC/CCL1832,33 and TARC/CCL17.31 Inflammation is counteracted by the release of factors such as IL-1ra/IL-1F3,34 Ym1, Ym2, RELMa,35,36 IL-10,6 and TGF-beta. TGF-beta also functions indirectly to promote ECM building by inducing nearby fibroblasts to produce ECM components.18 The alternatively activated macrophage itself secretes the ECM components, Fibronectin and bIG-H3,37the ECM cross-linking enzyme, Trans-glutaminase,38 and Osteopontin, which is involved in cell adhesion to the ECM.39

In addition, alternatively activated macrophages upregulate the enzyme Arginase I, which is involved in proline as well as polyamine biosynthesis. Proline promotes ECM construction while polyamines are involved in cell proliferation.19 Other factors secreted by the alternatively activated macrophage that promote cell proliferation include PDGF, IGF, and TGF-beta.18,40 These factors, along with FGF basic, TGF-alpha, and VEGF, also participate in angiogenesis.40,41

The molecules secreted by the alternatively activated macrophage work toward resolution of inflammation and promotion of wound repair due to their anti-inflammatory, fibrotic, proliferative, and angiogenic activities. This macrophage is also especially efficient at combating parasitic infections such as Schistosomiasis. In addition to its beneficial activities, the alternatively activated macrophage has been implicated in several pathologies, the most prominent of which are allergy and asthma (Figure 1B).3,4

Figure 1: Concentration of nitrite ug/ml VS OD

The values of the concentration were calculated by the given formula:

1ul of 50ug/ml standard contains 50/1000 ug sodium nitrite = 0.05 ug

0.05 ug sodium nitrite added to1ml culture medium = 0.05 ug /ml

0.05 ug sodium nitrite added to 0.5ml culture medium = 0.1 ug /ml

Table 1: Summary of the experiment treatment.

Volume of 50ug/ml standard added to 0.5ml culture medium

0

1l

2l

4l

8l

16l

Sodium nitrate present

0

0.05

0.1

0.2

0.4

0.8

Concentration

0

0.1

0.2

0.4

0.8

1.6

OD 550

0.042

0.056

0.085

0.114

0.163

0.305

Culture Conditions in 0.5ml medium

Cells only

Cells + IFN

Cells + IFN LPS

Cells + LPS

Cells + IL-10 + IFN LPS

Cells + IL-4 + IFN +LPS

OD 550 (Mean)

0.057

0.084

0.156

0.068

0.092

0.111

Concentration

0.06

0.08

0.16

0.07

0.09

0.11

Figure 2: Calibration curve of concentration of nitrite ug/ml VS OD

Figure 3: Cell stimulation results in ug/ml nitrite.

Discussion: the potential role that these cytokines may have on macrophage activation.

In the current practical the potential role of cytokines was monitored via cell/macrophage stimulation.

Here the effect of cytokines on macrophage activation (nitric oxide production) was studied. According to the data provided the calibration curve of nitric oxide production in g nitrite/ml was created. The graphical representation has helped to interpret the extent of macrophage activation by the cytokines LPS, IFNg, IL-4 and IL-10. As per the figure 3 the highest macrophage activation (nitric oxide production) was observed in cells + IFNg + LPS as compared to the others. The second best seen is in the cells+IL4+IFNg+LPS. In the case where the cells where only treated with LPS and only IFNg; the stimulation in IFNg produced my nitric oxide than the other. It indicated that macrophage respond more aggressively when treated or stimulated with IFNg. As the result is seen to be normalized by monitoring the release of nitric oxide by un-stimulated cells i.e. cells only shows that cells produce very tiny amount of nitric oxide on its own.

References suggest that in response to cytokine stimulation, the macrophages produced NO and, TNF-[alpha], IL-1[beta], and IL-10. Increased production of NO, TNF-[alpha], and IL-1 [beta] leads to inflammation-induced tissue/cell damage (Wu et.al; 2003 Sep).

Firstly to highlight and explain about cytokines; then they are various substances which are secreted/released by specific cells of the immune system which transmit signals between cells, and thus are responsible for different effect on other cells. They are also termed as key players of cellular communication. They are proteins, peptides, or glycoproteins in nature. They are also termed as the immunomodulating agents (interleukins, interferons, etc.). Literature highlights the property of cytokine that is can induce response or effect at picomolar concentrations. They are seen to rise up to 1,000-fold during trauma or infection therefore placing them as makers for diagnosis for various diseases (Nemeth et.al; 2012 Apr).

Cytokines can be classified as:

Monokines, produced by mononuclear phagocytic cells

Lymphokines, produced by activated lymphocytes, especially Th cells

Interleukins, those act as mediators between leukocytes

Chemokines, responsible for leucocyte migration

Cytokines

Cytokines are the key players of the larger inter-related system of proteins and signaling cascades. Cytokine signal in a very flexible manner they can be protective and can cause damage as well. They trigger or reduce the response of another in a cascading pattern their effects can be: antagonistic, additive, or synergistic. The cytokines act as autocrine or paracrine, but not by the endocrine. Cytokines are required in the expansion and performance of both the innate and adaptive immune responses. Their presence is not restricted to the immune system. They are the maker of immune response when the immune cells come in contact with a pathogen (Chen et al; 2010 Dec 23). They have major contribution in clinically diagnosing and treating various disorders.

figure_12-01a.jpgfigure_12-01b.jpg

As the above picture suggest the cytokines are not produced or stored as proteins but are synthesized and initiated by gene transcription and their mRNAs are short lived. They are created as required in immune responses. Genes encoding cytokines can generate variants in the course of alternative splicing to yield proteins with somewhat different but biologically significant bioactivities (Belardelli 1995 Mar).

Many cytokines are generated by different cell types occupied in both the innate and adaptive immune responses. Individual cytokines are pleiotropic in nature and at times have similar actions attributing redundancy to them and their receptors (Belardelli 1995 Mar).

Macrophages are the cells that can be activated by cytokines and on activation release cytokines. They are activated by interferon-gamma (IFN-gamma) and the commonest bacterial endotoxins, such as lipopolysaccharide (LPS). Activated macrophages transform into mechanized cells capable of killing the foreign bacteria or infected cells; due course they can release noxious chemicals and proteins which can have deadly effects on other cells. (Foster 2001 Jun)

Macrophages when activated secrete number of cytokines under various conditions:

TNFalpha`: Tumour Necrosis Factor alpha which initiates a cascade of cytokines that further are responsible for inflammation. They regulate the expression of several genes in different cell types significant in the host response to infection.

IL-1beta: Interleukin-1b is a pro-inflammatory cytokine. It is released by macrophages on activation by a stimulus of TNFa, bacterial endotoxin and IL-1b itself.

Interferon alpha/beta: They are immunomodulatory, as well as antiviral cytokines in nature.

IL-10: They are immunoregulatory cytokines. They suppress IL-2 and IFN g production by TH1 T-cells. They are potent modulator of monocyte/macrophage function and down-regulating proinflammatory cytokine release, and the respiratory burst (Belardelli 1995 Mar).

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