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LPS is one of an important endotoxin in outer membrane of gram negative bacteria acts as a mediator of inflammatory responses. LPS has a sugar component on extracellular part of cell membrane bind to lipid A that is responsible for the toxicity of LPS and causes septic shock. (Wilson et al, 2007). LPS by activating two cell surfaces, TLR4 (toll like receptor 4) and TLR5, leads to production of TNF-Î± and result in systemic shock. (Murphy et al, 2008)
The crucial role of the innate immunity system is the detection of the invaders result in protection of the host organism. Because LPS released from bacteria is in very small amount as a result the innate immune system has to be very sensitive and potent to response to this tiny amount of LPS.
Pattern recognition toll like receptors are essential molecules for transmitting signals induced by bacterial compounds into the cell. With regard to LPS in gram negative bacteria, LBP and membrane CD14 present at the surface of monocytes and myelocytes have a critical role in response to bacterial compounds in innate system. (Heumann et al, 2003)
The purpose of this essay is to assess the interaction between innate recognition CD14 and receptors in physiological recognition pathway and define how LBP, CD14 and MD2 together give potent response to LPS.
Basis principle of innate immune recognition:
Because of directly linking receptors to a variety of defense mechanism innate immune response is rapid. The innate immune recognition causes immune response by production of important mediators that signal to other components of immune system (Kaufmann et al, 2004). The innate immune system acts by providing signals to the host that microbial pathogens are present. This response can directly kill the pathogen or keep the infection until the adaptive immune response is completely active (Marshall& Cohen, 1999).
Pattern recognition, mediated by pattern recognition receptors (PRRs).The targets of this system are product of microbial metabolism such as lipoteichoic acid in gram positive bacteria, peptidoglycan and lipopolysaccharide (LPS) that is an endotoxin in outer cell wall of gram negative bacteria (Kaufmann et al, 2004).
The innate immune system is able to detect LPS by having LPS receptor that called CD14 which is expressed on the surface of macrophages and monocytes. Interaction CD14 with LPS, leading the activation of cells which release protein inflammatory mediators such as TNFÎ± (tumor necrosis factor) (Jack, R.S, 2000)
LPS interact with the cell membrane of macrophages, as a result LPS stimulates macrophages rapidly that can lead to up regulating gene transcription and translation in TNFÎ±, result in increase in TNFÎ± expression. (Baue et al, 2007).
Structure of LPS:
In determining how LPS stimulates the innate immune system the first need to consider its structure. LPS is a complex glycolipid composed of O-antigen repeats, hydrophilic polysaccharide liked to hydrophobic domain known as lipid A. The toxic principle of LPS is lipid A and it is responsible for the biological activity of LPS. Over activation of macrophages by large amount of LPS in the blood lead to collapse of the respiratory and circulatory result in septic shock. (Dixon, F.J, 2001).
Different species of gram negative bacteria bear different structure features within lipid A and oligosaccharide portions in their LPS (Brigham. K.L, 1994).
LPS recognition via the interaction between CD14 and associated receptors:
The literature reveals several important aspects of CD14 interactions with LPS as a stimulator of the innate immune response. CD14 has an important impact on the ability of the host cells to respond to low concentration of LPS. (Jack .R.S, 2000).
CD14 is the key cell surface receptor for LPS on macrophage. CD14 exists both on the surface of macrophages and PMN. Binding of LPS to CD14 is significantly increased in the presence of LPS binding protein (LBP) (Dixon, F. j, 2001). LBP in respond to LPS enhances the ability of CD14 bearing macrophages (Jack, R.S, 2000)
CD14 was characterized as a cell surface differentiation marker. CD14 exists as two kinds; glycosyl phosphatidyl inositol (GPI) anchored cell surface receptor that called membrane CD14 also as a soluble molecule. CD14 plays a key role as a soluble cofactor mediating activation of a variety of myeloid and non myeloid cell types in response to LPS. CD14 has ability to mediate LPS induced activation of cells as both a membrane receptor and soluble cofactor. By the presence of LBP, the responses of CD14 to LPS is improved but in the absence of LBP the binding of LPS and CD14 is slow process. (Jack, R.S, 2000).
CD14 plays a dual role as both activator and inhibitor of LPS mediated activation (Jack, R.S, 2000). The responses of CD14 to LPS can restrict or activate by diverting LPS from or transferring LPS to membrane CD14, respectively. (Duggan et al, 2008)
LBP and CD14 act as amplifiers for LPS recognition. By a complex of TLR4 and MD2 that is a (TLR4 binding extracellular protein) the presence of LPS on the target cell surface is detected. In the presence of CD14, LPS binds to TLR 4 and MD2. In signaling pathway co -expression of TLR4 and MD 2 leading to the activation of nuclear factor kappa Î² (NF-kÎ²) (Wong & Arsequell, 2003).
On the cell surface, LPS is recognized by a complex mechanism. LPS is first captured by LPS binding protein (LBP) and then on the cell surface the LPS- LBP complex links to CD14, LPS is then transferred from the CD14-LBP complex to a TLR4-MD2 complex in the cell surface (MD2:TLR4 complex interacts with LPS bound to CD14 complex (figure1) (Jack, R.S, 2000).For correct targeting of TLR4 to the cell surface for recognition of LPS, MD2 binds to TLR4 within the cell. (Murphy et al, 2008).
As a result of TLR activation by different Kinase enzymes a phosphorylation cascade induced and then transcription factor NF-kÎ² release from its inhibitor IKB that can up regulate TNF in the nucleus (Roitt & Delves, 2001). Then send a signal to the cell nucleus which actives the transcription factor (NF-kÎ²) and also transduces intracellular signaling.
Other receptors include in LPS such as SR-A may attend to decrease regulate responses induced by the CD14 pathway (Male et al, 2006).
Statistical data demonstrates that LPS bind to MD2 with a higher affinity than to CD14; also there is a difference in the tendency of CD14 and MD2 in binding to TLR4. Different binding affinity enables transmission of LPS from CD14 to MD2. Furthermore, CD14 in comparison with MD2 can binds to various ligands while MD2 seems to be more ligand selective in binding with TLR4 (Shin et al, 2007).
In addition to this, the differences between the complex of LPS binding to TLR4, MD2 and CD14 show that the association phase of LPS binding to MD2 rise more rapidly than those of LPS binding to CD14 or TLR4. (Shin et al, 2007).
Figure 1: figure 1 demonstrates the recognition of LPS by CD14 and associated receptors:
LPS binds to LBP then LBP transfers the LPS to CD14. CD14 contributes in binding of LPS to TLR4-MD2 complex. MD2 has 2 receptors one of them for binding to LPS on CD14 and another one for linking to TLR4. MD2 linked to the extracellular domain of TLR4. MD2-TLR4 complex bind to CD14-LPS complex.
MD2 with 2 arms
LPS binds to LBP
LPS in gram negative bacteria
CD14 binds to LPS
Structural basis of LBP and BPI in response to gram negative bacteria:
There are two host endotoxin binding protein such as bactericidal permeability increasing protein (BPI) and lipopolysaccharide binding protein (LBP). Both of these host endotoxin binding proteins confer wide range of innate immune recognition in gram negative bacteria by interacting with sites within the core reign and lipid A in LPS. (Weiss, J, 2003)
LBP is a glycoprotein that is synthesized by hepatocytes. The recognition of tiny amount of LPS is done by LBP. LBP extracts LPS monomer and make them available to the cell surface. LBP acts by exchanging monomers of LPS in site for other lipids. LBP binds to the outer membrane of gram negative bacteria to extracted endotoxin and catalysis the transformation of endotoxin to membrane CD14 and soluble CD14. (Weiss, J, 2003)
There are two roles of the C- terminal domain of LBP: 1. extention of interaction between CD14 and membranes containing LPS. 2. Destabilization of endotoxin, make the purification easy and also cause delivery of endotoxin to CD14. In addition to this, Comparisons of the functional properties of LBP and BPI have shown that there is a specific role in C-terminal domain of LBP in the binding of CD14 to LBP-endotoxin and then in the formation of endotoxin CD14 complexes. (Weiss, J, 2003)
Because LBP and BPI have been identified as closely related LPS so information from BPI structure is likely to be informative for understanding LBP, However it must be emphasized that BPI and LBP have different impact on LPS and there is a competition of activating and inhibitory ligands for CD14; LBP disaggregates LPS and promotes LPS delivery to monocyte CD14 but BPI compress LPS aggregates and by inhibiting LPS delivery to monocyte CD14 shows its contribution in respond to gram negative bacteria. (Marshall & Cohen, 1999)
TLR 4 in LPS recognition:
The best pattern recognition receptors on macrophages are the Toll Like receptors. Much of recent data about TLRs comes from bacterial infections by gram negative specifically LPS. TLRs recognize the nature of the pathogen and turn on an effective response for dealing with it.
TLR4 the receptor for LPS, was the first mammalian homology of Drosophila toll discovered and is a type 1 transmembrane protein (Meager, A, 2006).
TLR4 is consisting of an intracellular signaling domain and extracellular leucin rich repeats (Akashi et al, 2003).
LPS in outer membrane of gram negative bacteria is the best example of PAMPs. PAMPs derived from various species of pathogens differ from each other in details of chemical structure but have common molecular pattern. Recently, studied have revealed that TLR4 is essential for LPS recognition as a signal transducing receptor (Paul, W.E, 2008).
TLRs exist as both membrane bound and cytosolic receptors. TLRs signal through different molecular pathways that result in the translocation of NFKÎ² into the nucleus and activation of many protein inflammatory genes. (Ronco et al, 2009)
In 1997, scientists initiated a new era of immunological research into innate immune receptors. These researchers discover that (NF -KB) activation is induced by over expression of active TLR4 mutant and production of inflammatory cytokines is dependent on (NF -KB). Also, in the absence and presence of LPS, co-transfection of TLR4 and MD2 intensify NF -KB activation. By using other method such as cross linking combined with radioimmunoprecipitation shows that LPS directly binds to CD14, which then associates with the TLR4/ MD-2 complex in a CD14 dependent manner. (Chan et al, 2006)
LPS recognition is complicated with another molecule that expressed on B.cell molecule which called RP 105. RP 105 and TLR4 connected through its ectodomain with an accessory protein known as MD1 which is homolog of MD-2. RP 105 has co-operation with TLR2 and TLR4 in lipoprotein and LPS recognition by B.cell. (Paul, W.E, 2008). Also, the complex of RP 105 /MD 1 shows that to be involved in the recognition mechanism for response to LPS.(Koraha et al, 2005)
The function of MD2 in response to infection caused by gram negative bacteria:
MD2 is a glycoprotein that expressed on the cell surface and in a complex with TLR-4 both are required for LPS recognition. MD2 acts as an adaptor protein that binds LPS and is essential for TLR4 signaling to occur (Meager, A, 2006). MD2 is associated with the extracellular leucin rich repeats of TLR4. (Akashi et al, 2003)
MD2 acts as an adaptor molecule that linking different TLR together. The stimulatory action on macrophages of combinations of gram negative bacteria and gram positive bacteria components are explained by a combinational model of TLRs. (Friedland & Lightstone, 2003).
Endotoxin can be transferred from MD2 to CD14 but not to LPS (in reverse). The activation of cell expressing in TLR4 without MD2 is reduced by transformation of MD2 to soluble CD14, but by transferring of endotoxin from MD2 to CD14 in membrane leads cell expressing MD2: TLR4 to be sensitive to activation by the endotoxin MD2 complex. Expression of cells TLR4 without MD2 can be happened by MD2 complex but not by CD14. (Teghanemt et al, 2007)
LPS as a complex with CD14 is stabilized, as a result the interaction between LPS and CD14 could be stable and also there is a strong binding of LPS to cell surface of TLR4 and MD2. For the stable binding of MD2, interaction between the hydrophobic residues and secondary chain is required. The findings show that LPS binds directly to MD2, as a consequence MD2 rather than TLR4 has contribution to binding in the complex. LPS binding to MD2 seems is more specific and stronger in comparison with CD14. As a result of these differences in binding function of CD14 and MD2 impressive response can be observe from host organism to pathogens (Koraha et al, 2005)
To sum up, innate immune system can respond to endotoxin LPS as a mediator of inflammatory responses. TLRs play important roles in the PAMPs and among TLRs; TLR4 because of having MD2 (associated to TLR4) is the major receptor for LPS and induce signal transduction in cells. (Koracha et al, 2005)
Signaling via TLR is enhanced by binding PAMPs to CD14. It is also becoming clear that for initiating signaling and determining ligand specificity the combination of TLRs is important TLR4 is a critical receptor necessary for LPS signal initiation (Friedland & Lightstone, 2003).
TLR4 recognizes LPS in the outer membranes of gram negative bacteria. Recognition of LPS requires not only TLR4 but also MD2 (as a TLR4 accessory molecule).It is revealed that MD2 has two host acceptors, TLR4 and CD14, and MD2 physically associated with TLR4 on the cell surface.
In response of innate immunity to infection caused by LPS in gram negative bacteria, circulating LPS in the blood interacts with the LBP, and then LBP transfers LPS to soluble CD14 or membrane CD14 (Shin et al, 2007). Then CD14 carries LPS to the TLR4-MD2 complex. LPS binds to MD2 that is associated with TLR4 on the cell surface and it has a contribution with TLR4 in response to infection caused by gram negative bacteria. (Chan et al, 2006).
Also, it is become obvious that TLR4 only in response to systemic infection is the recognizing molecule for gram negative bacteria components, where with CD14 have more complex role in the proinflammatory signal transduction events (Berczi & Gorczynski, 2001).
The basic pattern of innate immune response to LPS, can characterized as a microbial invaders, host recognition by LBP, CD14 and activation of TLR by cross binding with MD2, resulting transcription of response factor by NFKB in production of cytokines (Williams, K.L 2007).
It has become apparent that in response to infection by gram negative bacteria each of these proteins has an important role. For example, MD2 is needed for TLR4 dependent cell activation. LBP and CD14 are required for sensitivity in response and permit to host organism to show reaction to small numbers of endotoxins. (Teghanemt et al, 2007)