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Human health is vastly dependant on the innate immune system, which is in constant battle with the invading pathogens, entering the human body from the different environments to which the body is exposed to. The environment being home to various microbes and pathogens, poses a constant threat to the human health. The immune system is tested at each and every moment of a living being. It is responsible for a healthy individual, keeping at bay 99% of the pathogens. In cases when an individual does become ill, external medication is required to provide aid to the weakening human defences, for medicines only prove to be a support for the immune system, which has to overcome the pathogens on its own. Therefore human health can only be determined by the strength of the immune system, which if impaired, will lead to increase risk of diseases.
The activation of an immune system is primarily dependant on the identification of the pathogens as a threat. Once the pathogens have been established as a hazard, the defence mechanism initiates the process of overcoming these harmful agents. The recognition of these pathogens is a vital task handled by specific proteins of the immune cells named as the 'pattern recognition receptors'. They may be found on the cell membrane called as 'membrane bound PRR's' or may be suspended in the cytoplasm called as 'cytoplasmic PRRs'. Molecular patterns associated to various pathogens are recognized by these proteins, constituting the innate immune system. These molecules can be classified as bacterial carbohydrates, bacterial nucleic acids, peptidoglycons, lipoproteins, lipotechoic acid and various others. In this paper, bacterial lipopolysaccharide (LPS); a carbohydrate present in the cell wall of gram negative bacteria, will be discussed with its complementary identifying receptor CD14.
CD14 (cluster of differentiation) is one of the pattern recognition receptors vital in the detection of LPS. Occurring in two forms named as mCD14 (found anchoring to the cell membrane by a glycosylphosphatidylinositol) and sCD14 (found suspended in the cell cytoplasm), it does not function in isolation but acts in coordination with other proteins and receptors. In fact it can only bind with the LPS in the presence of another protein called lipopolysaccharide binding protein (LBP). CD14 has a potential of recognising and binding to other molecules besides the LPS. (2). It has the ability to interact with ligands like lipoteichoic acid, peptidoglycans, muramyldipeptide, polumannouric acid and athrogenic lipoproteins, which proves to be essential owing to the importance of formation of groups by various receptors for the effective recognition of the diverse bacterial components. The alliance of different receptors means a diversified approach towards the bacteria as these receptors utilize their individual characteristic in the identification of the bacteria. Each receptor being selected in accordance with the type of protein needed to be identified. (3)
LPS recognition is a very sensitive and crucial task. CD14, being the primary innate recognition molecule, still has to involve other proteins in this task. Cytokines secretions enable the immune cells to respond to minute quantities of LPS. These secretions are controlled very precisely by the body where even a slight deviation in the amount can result in the septic shock. LBP is the main initiating agent of the immune cells. LPS binds with the LPB present in the serum which facilitates the interaction with differentiation antigens on monocytes, the CD14. CD14 has a drawback of not being able to initiate the activation signal on its own, being itself a glycosylphosphatidyl anchored protein, which is precisely why the receptor complex theory in LPS pathway was developed. Therefore numerous other proteins apart from CD14 and LBP would suffice for an effective and specific reaction. (4).
Introduction: LPS, also known as lipoglycans, are molecules constituting of a lipid and a carbohydrate moieties, fused together by covalent bonds into one single molecule. Situated in the outer cell wall of gram negative bacteria, it acts as an endotoxin creating a cellular immune response in mammals. It plays a dual role of being an integral part of the gram negative bacteria and also performs as a shielding agent against physical and chemical injuries. LPS is necessary because of the negative charge it renders on the bacterial cell wall, stabilizing the overall membrane and thus protecting its existence. In addition it also acts as an exogenous pyrogen leading to its usage in research methods. It is a much sought after molecule in the drug discovery owing to its unique role in the bacteria themselves and to its versatility as an endotoxin, exogenous pyrogen and its role in the immune system. Many drugs available have their targets against LPS of the gram negative bacteria. (5).
Identification: The detection of LPS requires an extremely sensitive mechanism by the immune system. Owing to its vitality to the bacteria and toxicity to the host, the detection is a complex and inconvenient system having cascade of numerous proteins, ions channels and chemical components, all contributing to the identification of this extraordinary molecule. LPS, may be found in two basic types, the smooth type called 'S-type' and the rough type 'R-type'. Their names have been derived on the basis of the chain length of carbohydrate present in the LPS, as mentioned earlier it contains both lipid and carbohydrate components. The immune cells have been assigned with the dual task of identifying and differentiating between the two types, simultaneously engaging in a complex process of defending the cells from invading pathogens. The immune response has to vary according to the differing types of LPS in different bacteria. And it has also been speculated that the changing immune response also owes to the arrangement of the lipid and carbohydrate chains present in the LPS. The immune system therefore has been found to produce varied reactions and processes in accordance with the diverse types of LPS. (6)
The Receptor Complex:
As an established point described earlier above, a group of receptors is involved in the identification and formulation of an effective and specific immune response, so this holds true for CD14, which combines with other receptors in response to LPS (lipopolysaccharides). Most commonly studied basic receptor complex, consists of three receptors; the CD14, the toll like receptor 4 (TLR-4) and myeloid differentiation factor 2 (MD-2). The fourth component being LBP, as mentioned earlier. In invading the main blood stream, the detection process commences with the LPS binding with the LBP in the serum, this compound then in turn is bound to CD14 on the surface of monocytes. A heterodimer is formed between CD14 and the other two receptors, TLR-4 and MD-2. This happens on the cell membrane once TLR-4 and MD-2 come in contact with CD14. This resolute structure aids in the identification of the common areas of LPS, an objective hard to obtain because of LPS molecular diversity, leading to the signalling and hence immune responses. (7)
The precise role of TLR-4 and MD-2 are yet to be determined through further research and studies. LBP on the other hand has its limited yet an important role as an efficient binding mediator between the LPS and CD14. It speeds up the process as compared to the direct interaction of CD14 with LPS but its presence is not vital as CD14 has the potential of binding directly to LPS. The LPS and CD14 binding leads to rapid intracellular Ca++ mobilization, cellular tyrosine kinase phosphorylation, nuclear factor (NF)-kB activation, cytokine and chemokine production. The blockage of CD14 on the cell surface with the help of CD14 antibodies will disable the binding of LPS with CD14 thus resulting in the impairment of LPS mediated cellular response, failure of cytokine production and prevention of cellular adhesion molecule receptor PMNs which are normally expressed in response to LPS. (8)(9).
Many receptor complexes have been found to play important roles in the LPS signalling. For instance various proteins and receptors like decay accelerating factor (CD55), chemokine receptor 4 (CXCR4), heat shock protein 70 (HSP-70), heat shock protein 90 (HSP-90), CD18 and CD81 have been found to be part of the LPS detecting receptor complex. All of these proteins were found to react with CD14 on the cell surface as a result of LPS stimulation, which led to speculations of their role in the signalling. Therefore it is imperative that these associations should also be discussed.
CD14 and Associated Receptors:
As discussed earlier, the formation of a particular receptor group is largely dependant on the type of LPS which also determines the type of immune response. Various researches have been conducted on LPS and analogues of LPS with their complementary receptor associations. These have involved the stimulation of LPS with CD14-TLR4-MD2 receptor complex and other proteins like CD55, CD18, CD81, HSP 70 and HSP 90 and CXCR4. In case of analogues of LPS, CD55 was still found but proteins like CXCR4 and CD18 and CD81 seemed to be omitted form signaling, leading to the conclusion that the type of LPS determines the association CD14 forms with other proteins. (10)
Many surface proteins have been the subject of various researches, but CD55 has been the most popular due to its multiple observations. Multiple signalling pathways might be required for a complete LPS response. The idea being supported by observations made on CD14 deficient mice responding normally to LPS in the induction of acute phase protein expression. CD55, identified as an LPS binding protein present in almost all types of cell, is a complement regulatory molecule causing rapid decay of C3 and C5 convertase. Its existence is found ubiquitous and on all cells coming in contact with the blood stream. Immunoprecipitation experiments have indicated LPS interaction with CD55 presence on the cell surface, the complex including CD14 as one of its components. LPS hyporesponsive Chinese hamster ovary cells were transfected with human CD55 which caused the translocation of NF-kB when stimulated with LPS and free lipid A. Comparison of CHO-CD55 and CHO-CD14 cell lines showed, the activation of p38, JNK and ERK MAP kinase upon stimulation with LPS in both types of cells. In a study utilising Fret analysis, observations were made of LPS stimulating the clustering of CD55 and CD11/CD18 in monocytes. The functionality of CD55 in LPS signalling and instigation of appropriate immune responses has been significantly brought to light. CD55 has been revealed to be a receptor for many virus families alongwith other GPI linked molecules, found inside large insoluble glycolipid-enriched domains (DIG), also known as rafts present on cell surface. These DIGs are also linked to the src-family protein tyrosine kinase and various other signal inducting proteins, speculated to also play a role in T-cell receptor signalling. (11)(12).
Chemokine receptor 4, another receptor extensively studied, has been shown for its association with CD14 as a result of LPS presence on the cell surface. LAP proteins, observed to be an agent in the LPS activation, in the immunoaffinity chromatography and peptide mass fingerprinting experiments combines with HSP 70, HSP 90, GDF-5 and the chemokine receptor 4 (CXCR4), forming a heterogeneous complex of receptors needed in the LPS detection. CXCR4 previously known to be present only on leucocytes is also present on nonhematopoietic cells and its expression has been found on endothelial cells surface. On exposure to specific bacterial agents like LPS, the expression of CXCR4 increased. This was also true in case of CXCR4 transfected cells. The direct association of CD14 to CXCR4 in case of LPS exposure was observed in fluorescence spectroscopy.CXCR4 is also vital in the LPS signalling specially mitogen activated protein kinases (MAP kinases). CXCR4 has been seen to co-localise with CD14 on cell surface, which is an important indicator of other receptors being involved in the identification of LPS. (13)(14)
The association patterns of CD14 with other receptors are unique and diverse. It is a vital characteristic which helps it to become a mechanism in its entirety. A study in which chemotypes of LPS were used, revealed the inability of CD14 to identify these types, hence initiating the signal without differentiating these types. But because of this interesting aspect of association of CD14, it effectively overcomes this flaw, as was explained in the case of TLR4 and MD2. The complex compound formed as a result, enables the effective identification and signalling of various LPS types thus responding accordingly. Differentiated signals are not initiated without recognition of the LPS types. Therefore the combination of CD14 with various other receptors becomes exceedingly significant. (15)
Researchers have pointed out to the requirement of additional cell membrane proteins in the binding of CD14 to LPS to initiate the signalling. But many factors have still to be explored in the association of CD14 with its receptors. For instance the functionality of many toll like receptors (TLRs) have yet to be established, though their vitality in the process has been known, still researchers have been unable to fill the gaps. Different combinations of TLRs in association with CD14 might be responsible for the inflammatory responses to the various pathogens disrupting the innate immune system. While its association with sCD14 initiate responses in the non-leucocytes and being a significant signalling component of the LPS receptor complex, it has yet to be explored as the most significant receptor. (16)
The stimulation of substances similar to LPS with monocytes reveals an interesting aspect. Ceramide, an important constituent of athrogenic lipoproteins, binds with CD14 to cause the formation of similar receptor clusters like that of LPS. These clusters includes CD55, CD11, CD18 and CD36, with the striking exclusion of TRL4. The reaction helps to understand that CD14 also reacts with ligands and other proteins. But its association pattern is variable on the pathogenic molecules to which it is coming in contact, each of which can hold a use of significant importance. (17)
The association characteristics are important to the innate immune system's defending abilities and its timely recovery. It holds the key to defeating pathogens as in it lies the strength of the immune system and its control over the pathogens in totality. As demonstrated, CD14 is a very important receptor, but without its alliance with various other receptors and proteins it cannot be rendered useful against the varying pathogens. The complex compounds that it forms enables in the effective signalling and thus initiating the accurate responses against pathogen identifying molecules like the LPS. The association of these receptors is significant in comprehending the sensitive and intricate immune system of our bodies