The Immune System - Lab Report Example

Schwandner R, Dziarsk R, Wesche, Rothe M, Kirschning C J. (1999) Peptidoglycan- and Lipoteichoic Acid-induced Cell Activation is mediated by toll-like Receptor 2 Journal of Biological Chemistry 274: 17406-17409. Infection of a host cell with bacteria can result in the fatal condition known as septic shock or blood poisoning. When certain bacterial types invade an organism they bind to the host's cell membranes stimulating a cascade of events eventually resulting in NFB activation, its translocation to the nucleus and corresponding cytokine release and activity within the bloodstream. This can eventually lead to sepsis (Alberts et al 2002a). Previous work showed that in Gram negative bacteria lipopolysaccharide (LPN), a component of the outer membrane, activated a cell's immune response in this manner. It interacted with the Toll-like receptors types 2 and 4 (TLR2 and TLR4) present on host cell membranes (Kirschning et al 1998, Poltorak et al 1998, Quereshi et al 1999). The authors in this paper demonstrate how the components of Gram positive bacterial cell membranes stimulate cell activation through TLR2. Introduction The invasion of an organism's blood stream with bacteria often results in sepsis. Invasive bacteria will activate the host cell's immune system. Sepsis results from the inability of the immune system to limit bacterial spread during an infection. The inhibitory mechanisms controlling inflammation are over-ridden by the huge bacterial load on the cell. Inflammation during sepsis will then develop into a systemic syndrome with a number of clinical symptoms such as tissue injury, increased vascular permeability, dilation of blood vessels, loss of plasma volume and blood clotting, and, eventually, multi-organ failure and shock (Alberts et al 2002b, Decker 2004)). Initially bacteria bind to host cell membranes and this stimulates the systemic release of cytokines and other inflammatory signalling molecules into the blood. (Alberts et al 2002b). Bacteria can be divided into two groups Gram negative and Gram positive. Gram negative bacteria have a cell wall with an outer membrane rich in LPN, and deficient in peptidoglycan (PDG). Gram positive bacteria lack an outer membrane rich in LPN and are abundant in PDG and lipoteichoic acid (LTA) (Madigan et al 1997) (Figures 1,2 and 3). The mechanism for Gram negative bacterial mediated activation of the cell's immune response has been well documented (Alberts et al 2002). LPN within the bacterial cell wall binds to LPN binding protein present in serum and this complex in turn binds to the CD14 receptor (figure 4). The CD14 receptor is either soluble in serum or tethered to the host cell membrane through a GPI (glycophosphatidylinositol) anchor. Either way the CD14 receptor does not have an intracellular domain so it cannot transmit its signalling messages intracellularly. This observation suggested that another "co-receptor" acted in conjunction with the CD14 receptor to allow the transmission of an extracellular signal to the inside of the cell. The Toll-like receptors (TLRs) have an intracellular domain linking to the IL-1 signalling pathway. The Toll-like receptor acts to phosphorylate a cascade of kinases TRAF6, TAK-1, IKK and eventually the transcription factor NFB. NFB then translocates to the nucleus where it activates the transcription of a number of genes involved in immune and inflammatory responses (Alberts et al 2002a, figure 5). In Gram negative bacteria the Toll-like receptors 2 and 4 has been shown to be intrinsic in cell activation (Kirschning et al 1998, Poltorak et al 1998, Quereshi et al 1999). In this paper the authors attempted to determine if components isolated from Gram positive bacteria activated cells in a TLR dependent manner. HEK293 cells were transfected with a number of Toll-like receptors alongside a luciferase reporter gene. A NFB transcription element was placed upstream of the luciferase reporter gene, such that activaiton of NFB would initiate transcription of luciferase. When luciferase is expressed its activity can be measured and quantified and NFB activity calculated. The transfected cells were stimulated using either whole bacteria or solubilised PDG and lipoteichoic acid (LTA) purified from Gram positive bacteria. Review critique Introduction Components of the bacterial cell wall (figure 1) in both Gram positive and gram negative bacteria have been shown to be integral to the process of inducing septic shock. In particular Gram positive sepsis has great clinical significance and because of this much research is underway to further investigate this condition (Alberts et al 2002b; Rang et al 2000). PGN, the main component of its cell wall, and LTA (figure 3), a membrane anchored glycolipid, are both found in abundance in Gram positive bacteria. Purified PGN and LTA have previously been shown to stimulate the release of proinflammatory cytokines such as Tumour Necrosis Factor (TNF), IL1, IL6 and macrophages. Purified PGN and LTA produce a similar clinical response as do whole bacteria during infection of a host cell (Kirschning et al 1998, Poltorak et al 1998, Quereshi et al 1999). In Gram negative bacteria LPS binds firstly to LPS binding protein and then to CD14 which then results in cell activation. In Gram positive bacteria it has also been shown that PGN (Dziarski et al 1998) and LTA (Kirshning et al 1998) cell activation is CD14 dependent (figure 2 and 4). CD14 is either soluble within the serum or attached to the PM through a GPI anchor, so it does not have an intracellular domain to communicate with the inside of the cell. It was then suggested that a "co-receptor" transduced the extracellular signal to inside the cell. The Toll-like receptors (TLRs) have been implicated in this (Kirshning et al 1998, Poltorak et al 1998, Quereshi et al 1999, Yang et al, 1999) Drosophila Toll is a transmembrane protein with a leucine rich extracellular domain. It has been shown to be involved in establishing dorso-ventral polarity and also in Drosophila resistance to fungal infections through NFB activation. There are at least 10 TLRs in humans and each of them are activated by different ligands such as LPS, PGN and LTA. TLRs can be found in large numbers on the surface of macrophages and neutrophils. When they are stimulated an inflammatory immune response is initiated through the activation of NFB and subsequent release of inflammatory cytokines, including IL-1, IL-6, IL-8, IL-10, IL-12, GMCSF, TNF, reactive O2 intermediates, platelet activating factor and leukotrienes.. Indeed TLR resembles the family of receptors involved in NFB activation (Alberts et al 2002a; Rang et al 2000)(figure 5). Previous studies have implicated TLR2 and 4 as mediators of LPS induced cell activation in Gram negative bacteria (Kirshning et al 1998, Poltorak et al 1998). This study examines which TLRs are involved in Gram positive bacterial induced cell activation. Methods PGN purified from the Gram positive bacteria S Aureus and S pyogenes was used to stimulate cells. LTA was purified from the Gram positive B Subtillis, S Pyogenes and S Sanguis. Control LPS from E Coli was used. The authors comment that all different sources of molecule behave in a similar manner in all experiments, although the data showing this is not presented in the paper. Preparations of LTA and possibly PGN were treated with polymyxin B, an inhibitor of LPS, and all experimental results were said to be identical with and without inhibitor. This indicated that contaminating, residual LPS was not activating the cell. Again these results are not displayed in the paper. The Human Embryonic Kidney cell line, HEK293 cells, was used throughout the set of experiments. This is a human cell line eliciting NFB activation in response to stimuli. Although this is a well-documented model system for a variety of cellular signaling studies, to further the studies from this paper more cell lines should be investigated. Certainly discrepancies in results between cell lines have been shown, the authors comment on this in haematopoetic cells (Gupta et al 1996, Cleveland et al 1996 Hattor et al 1997) and also in transgeneic mice (Poltorak et al 1998, Quereshi et al 1999). It is likely that a receptor and/or ligand will behave differently depending on its cellular environment. One of the main techniques used in this paper is transfection. Transfection is the means whereby DNA in circular (plasmid/vector) form is delivered into a cell. After it has entered the cell it can be expressed and localised as a particular protein within the cell. There are a number of methods of transfection. The one used in this paper is calcium phosphate precipitation. Its mechanism of action is not well understood and DNA is understood to be taken up by the cell by a calcium-requiring process, most likely endocytosis. Other methods include using electroporation ie generating an electric shock which punches holes in the membrane allowing the DNA to enter; liposome transfer uses a solution of liposomes to surround the DNA and allows it to fuse and cross the plasma membrane; and microinjection of DNA across the cell membrane. The most efficient method of introducing DNA into cells needs to be determined and is dependent on cell line and DNA vector. (Sambrook and Russel, 2001). The proteins expressed in HEK293 cells in this paper are the Toll-like receptor (TLR) proteins and mutant forms of these receptors. They have been described in the introduction. The physiological relevance of over-expressing proteins in cell lines is often questioned in these types of experiments. Although accurate conclusions have been drawn from most of these results and experiments, it remains to be determined whether the results would have been similar in a cell line with "physiological" levels of receptor expression or in vivo. Cell "activation" or NFB activity is measured in this set of experiments using the luciferase reporter gene assay (figure 6). NFB is a transcription factor activated by a series of phosphorylation events. After activation it translocates to the nucleus where it binds to the promoter region of a gene activating its transcription and expression. In this paper the E-selectin promoter gene was placed upstream of the luciferase gene. This promoter will be activated by NFB, which in turn will elicit the expression of the protein luciferase. Thus the measure of enzymatic activity from the luciferase produced will be a direct measure of NFB activity. Firefly luciferase catalyzes luciferin oxidation using ATP-Mg2+ as a cosubstrate. Light is a byproduct of the reaction. This light is measured and quantified using a luminometer (Promega website). The cells are treated with various stimulatory compounds like PGN and LTA post-transfection and then lysed before the Luciferase assay is performed on them. All experiments had the negative control whereby cells had been transfected with vector only expressing no luciferase, and were then stimulated with compound. As a measure of transfection efficiency all cells were also transfected with a DNA construct which expresses -galactosidase. This method is often used to normalise individual results for transfection efficiency, so that like is compared with like. For example, in PGN stimulated cells 50% of cells may be transfected with luciferase construct whilst in LTA stimulated cells this may rise to 90%. If the results are not normalised for transfection efficiency then it would appear that there is greater NFB activity in LTA stimulated cells because of its increased luciferase activity. Although the authors do not state their method for detecting -galactosidase activity one of the most common ones uses the substrate 4-methylumbelliferyl-b -D-galactoside. -galactosidase catalyses the cleavage of 4-methylumbelliferyl-b -D-galactoside and yields the fluorescent molecule 4-methylumbelliferone (7-hydroxy-4-methylcoumarin, 4MU). 4-methylumbelliferone is fluorescent above pH 8 and when excited by 365nm light, it emits light at 460nm which can be measured on a fluorimeter (A TD-700 Laboratory Fluorimeter Method for -Galactosidase website). Thus -galactosidase cellular activity can be calculated, transfection efficiency calculated and corrected luciferase activity values made which allow a direct comparison between samples. For every experiment cells had to be transfected with several DNA constructs prior to stimulation. The minimum was two - the luciferase reporter gene and -galactosidase. However in some cases different Toll-like receptors were also expressed alongside these other two genes prior to stimulation with either whole bacteria or solubilised bacterial proteins. Results The first experiment was designed to determine if whole Gram positive bacteria would stimulate NFB activation in cells expressing individual Toll-like receptors. HEK293 cells were transfected with one of TLR1, TLR2 or the TLR4 DNA constructs. Consistent with experimental protocol the receptor DNA was co-transfected with the NFB-dependent E-selectin promoter luciferase reporter gene and the -galactosidase gene. Cells were then incubated with whole Gram positive bacteria, either S aureus or B subtillis, and normalised luciferase activity calculated corresponding to cell activation. NFB activation was observed by both species of bacteria in cells expressing TLR2, but not TLR1 or 4, indicating that TLR2 was important for cell activation by these strains of Gram positive bacteria. Negative controls were included in this experiment by using cells transfected but not incubated with bacteria and also cells transfected with control vector and then stimulated. No bacterial-induced cell stimulation was observed in these cells. The next experiment examined the effect of purified PGN and LTA on cell activation. The experimental protocol was similar to the first experiment, except whole bacteria were replaced with purified components from the bacterial cell wall, PGN and LTA. TLR1, 2, and 4, CD14 and a truncated mutant of TLR2 were all individually expressed in HEK293 cells. The mutant TLR2 did not have the intracellular domain, postulated to be necessary for signaling through the cell, to activate NFB. NFB activation was measured in all cell types. Solubilised PGN and LTA stimulated cell activation in cells expressing TLR2 only. There was no PGN or LTA mediated cell stimulation in cells expressing TLR1 and 4, CD14, truncated TLR2 or with control vector only. These results strongly suggested that a fully functional TLR2 was essential for PGN and LTA cell activation. The intracellular portion of the receptor is absolutely necessary to transmit the signalling of the ligands downstream of the receptor. Interestingly by merely expressing the TLR4 in HEK293 cells in the absence of any stimulation the basal level of receptor activity increased, however it did not increase further with PGN or LTA incubation. The negative control in this experiment was control vector stimulated with PGN, LTA or left unstimulated and transfected cells left unstimulated. The third experiment used Hek 293 cells expressing TLR2. Firstly it demonstrated that incubation of these HEK293 cells with increasing doses of PGN or LTA give a dose dependent increase in luciferase activities and thus cell activation. Secondly it compares cell activation by PGN and LTA in cells co-expressing both CD14 and TLR2 to those cells which only express TLR2. The authors concluded that coexpression of CD14 with TLR2 gave a slight increase in sPGN stimulated cell activation when compared to cells without CD14 expression. This suggested CD14 dependent cell activation by PGN. This was more apparent at lower concentrations of PGN. These conclusions correspond well with the results displayed in figure 3B. It was also concluded that LTA stimulation is independent of CD14 expression. If this were the case it would be expected to observe no significant difference between the two samples in figure 3C. There should be no difference in the luciferase activity in cells expressing CD14 and those which do not express this protein. However on examination of the results and error bars in the graph it would appear that there may be a statistically significant increase in LTA stimulation when CD14 is expressed with TLR2 at lower concentrations of LTA, although without the actual data it is impossible to determine this. This would suggest that LTA stimulation of cell activation may indeed be dependent on CD14 like PGN is. This has been indicated by a number of previous studies (Cleveland et al 1996, Hattor et al 1997). As well as performing the usual negative controls (cells transfected with control plasmid as previously described) this experiment displays an example of a positive control whereby CD14-expressing cells display a large LPS induced cell activation, whereas cells without CD14 do not. The final experiment in the paper examines the serum dependence of the PGN and LTA induced cell activation. This is important because in Gram negative bacteria LPS stimulation is dependent on serum (Kirschning et al 1998, Yang et al, 1998). This may be due to the presence of LPS binding protein and/or soluble CD14 in the serum. Again HEK293 cells expressing the TLR2 were used and cell activation measured using either PGN or LTA in the presence and absence of serum. Solubilised PGN and LTA responses were not inhibited when serum was removed before the experiment indicating that serum and its components are not essential for PGN or LTA activation. Indeed samples starved of serum, in particular PGN stimulated ones, showed a trend towards an increased stimulation. This may be due to competition of serum components inhibiting the receptor PGN interaction. Again good negative controls were present and on this occasion a good positive control was shown in this experiment. Increased LPS stimulation in the presence of serum was displayed when compared to serum starved. In general the data is well presented and displayed, showing a number of good controls within each experiment. Negative controls are prevalent, with cells transfected with vector only given the same treatment as transfected cells. They were also careful to leave an untreated set of transfected cells, such that a comparison could be made between these and treated ones. Positive controls were obvious in figures 3 and 4 where LPS activation of Gram negative bacteria was displayed. The bar charts provide an easily readable way of readily comparing results and observing the extent of a response. It would have been interesting to observe some of the results from their experiments using polymyxin B to inhibit LPN activity in preparations of LTA and PGN during cell activation, as this is an important control experiment. The paper itself was well-written for the target audience. The Journal of Biological Chemistry is read widely by technical scientific experts across the fields of, for example, Biochemistry, Cell Biology, Molecular Biology and Pharmacology. It is not ideally suitable for scientists relatively new to the field because it uses many technical terms and assumes background knowledge of a number of biological fields and experimental techniques. Conclusions The authors conclude from this set of experiments that TLR2 is likely to be intricately involved in Gram positive PGN and LTA mediated cell activation, and resulting septic shock. This study was performed in HEK293 cells overexpressing Toll-like receptors, and the authors acknowledge that much more research is required to elucidate fully the involvement of TLRs in cell activation and resulting septic shock in humans. Performing experiments with physiological levels of receptor in a number of different cellular environments would be one of the next steps for scientists. Then transgenic animals expressing mutant Toll-like receptors should be engineered and Gram positive bacterial cell activation measured. These in vivo experiments have already begun in mice, examining TLR4 mutants and the receptor's involvement in Gram negative bacterial induced septic shock (Poltorak et al 1998). The paper implies there is a differential mechanism of cell activation by Gram negative and Gram positive bacteria. Gram negative bacteria activate TLR2 and TLR4 through LPN, whereas Gram positive bacteria activate TLR2 through PGN and LTA. It appears controversial on whether CD14 is required for all Gram positive responses. In this set of experiments Gram positive bacterial components did not activate the TLR4. However the receptor expressed in HEK293 cells appeared to be constitutively active suggesting it may have mutated on cellular expression which may have ultimately affected its functionality. This would need to be verified by further transfection studies. The authors comment on previous work in transgenic mice where mutated TLR4 are still activated by Gram positive bacteria, but mice with no TLR4 expression are not. However in the latter case a mutation in another protein may be responsible for the lack of response. All this evidence clearly demonstrates how much more work has to be done by scientists to fully elucidate the signalling pathways leading to septic shock in vivo. Septic shock should be treated differently depending on the type of bacterial infection. Pharmaceutical companies will have to work hard to produce drugs treating Gram positive and Gram negative bacterial infections individually and perhaps together. Much more remains to be uncovered regarding the individual signaling pathways involved, and their mode of action before the best protein in the pathway(s) can be chosen and targeted by potential drug candidates. This is very clearly an important area of research for scientists, drug companies and the general public. Septic shock is a relatively common condition, and bacterial infections are becoming increasingly common amongst the general public after a period of stay within a hospital and in particular post-operation. It is essential for scientists and pharmaceutical companies to work together on developing a treatment for it as quickly as possible. It is comforting for the general public to know that ongoing research like this advances scientific progress one small step further. References A TD-700 Laboratory Fluorimeter Method for -Galactosidase. [Internet], Available from http://www.thelabrat.com/protocols/b-galactosidase.shtml Accessed 1 February, 2005. Alberts B., Johnson A., Lewis J., Raff M., Roberts K., Walter P. (2002a) Molecular Biology of the Cell, Fourth Edition. Publisher Garland Science page 1458. Alberts B., Johnson A., Lewis J., Raff M., Roberts K., Walter P. (2002b) Molecular Biology of the Cell, Fourth Edition. Publisher Garland Science page 1460. Cleveland M..G., Gorham J.D., Murphy T.L., Tuomanen E., and Murphy K.M. (1996) Lipoteichoic acid preparations of gram-positive bacteria induce interleukin-12 through a CD14-dependent pathway Infect. Immun. 64, 1906-1912. Decker T. (2004) Sepsis: avoiding its deadly toll. J Clin. Invest. 113:1387-1389. Dziarski R., Tapping R. I., and Tobias P. S. (1998) Binding of Bacterial Peptidoglycan to CD14 J. Biol. Chem. 273, 8680-8690. Gupta D., Kirkland T. N., Viriyakosol S., and Dziarski, R. (1996) CD14 Is a Cell-activating Receptor for Bacterial Peptidoglycan J. Biol. Chem. 271, 23310-23316. Hattor Y., Kasai K., Akimoto,K., and Thiemermann C. (1997) Induction of NO Synthesis by Lipoteichoic Acid from Staphylococcus aureus in J774 Macrophages: Involvement of a CD14-Dependent Pathway Biochem. Biophys. Res. Commun. 233, 375-379. Kirschning C. J., Wesche H., Ayres M. T., and Rothe M. (1998) Human Toll-like Receptor 2 Confers Responsiveness to Bacterial Lipopolysaccharide J. Exp. Med. 188, 2091-2097. Madigan M.T., Martinko J. M., Parker J. (1997) Brock Biology of Microorganisms Eighth edition, Publisher Prentice Hall International Inc. page 53. Poltorak A., He X., Smirnova I., Liu, M. Y., Huffel C. V., Du, X., Birdwell D., Alejos E., Silva M., Galanos C., Freudenberg M., Ricciardi-Castagnoli P., Layton B., and Beutler B. (1998) Defective LPS Signaling in C3H/HeJ and C57BL/10ScCr Mice: Mutations in Tlr4 Gene Science 282, 2085-2088. Promega Technical Resources. Protocols.[internet], Available from http://www.promega.com/tbs/tb281/tb281.html Accessed 1 February, 2005. Qureshi S. T., Larivitre L., Leveque G., Clermont S., Moore K. J., Gros P., and Malo D. (1999) Endotoxin-tolerant Mice Have Mutations in Toll-like Receptor 4 (Tlr4) J. Exp. Med. 189, 615-625. Rang H.P., Dale M.M., Ritter J.M., Moore P.K. (2000) Pharmacology Fourth edition, Publisher Churchill Livingston page 224-225. Sambrook J., Russel D.W. (2001) Molecular Cloning Publisher Cold Spring Harbor Publishing, vol 3 chapter 16, part 16.7-16.43. Yang, R. B., Mark, M. R., Gray, A., Huang, A., Xie, M. H., Zhang, M., Goddard, A., Wood, W. I., Gurney, A. L., and Godowski, P. J. (1998) Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling Nature 395, 284-288 Figure 1. Cell wall structure of bacteria. All types of bacteria contain a cell membrane surrounded by a PGN-containing layer. LTA and LAM are inserted into the cell membrane of gram-positive bacteria. LPS forms the outer layer of the outer membrane of gram-negative bacteria. The mycobacteria also contain a carbohydrate shell, but not all bacteria contain a capsule. This figure and legend was directly reproduced from Van Amersfoort E.S., Van Berkel T.J.C., and Kuiper J. (2003) Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock Clinical Microbiology Reviews,Vol. 16, No. 3 p. 379-414, Figure 2. Structure of lipid A (443) (A) and whole LPS (B). The composition and length of several LPS serotypes are indicated. This figure and legendwas directly reproduced from Van Amersfoort E.S., Van Berkel T.J.C., and Kuiper J. (2003) Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock Clinical Microbiology Reviews,Vol. 16, No. 3 p. 379-414, Figure 3. Structure of LTA from S. aureus This figure and legend was directly reproduced from Van Amersfoort E.S., Van Berkel T.J.C., and Kuiper J. (2003) Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock Clinical Microbiology Reviews,Vol. 16, No. 3 p. 379-414, Figure 4. Binding of bacterial ligands to CD14 and sCD14. The involvement of LBP, (s)CD14, and TLR2 and TLR4 in the activation of CD14-expressing cells (e.g., macrophages) and of cells that do not express CD14 (e.g., endothelial cells) is shown. LPS (left) and PGN (right) represent TLR4- and TLR2-specific ligands, respectively. This figure and legend was directly reproduced from Van Amersfoort E.S., Van Berkel T.J.C., and Kuiper J. (2003) Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock Clinical Microbiology Reviews,Vol. 16, No. 3 p. 379-414, Figure 5 TLR signaling pathways. The shared signaling pathway for TLR2 and TLR4 is depicted. IRAK, IL-1R-associated kinase; TRAF6, tumor necrosis factor receptor-associated factor 6; TAK1, transforming growth factor -activated kinase; TAB1, TAK1-binding protein; NIK, NF-B-inducing kinase; MKK, mitogen-activated protein kinase kinase; JNK, c-Jun N-terminal kinase; IKK, IB kinase; AP-1, activator protein 1. This figure and legend was directly reproduced from Van Amersfoort E.S., Van Berkel T.J.C., and Kuiper J. (2003) Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock Clinical Microbiology Reviews,Vol. 16, No. 3 p. 379-414, Figure 6. Schematic diagram of reporter gene constructs used in transient transfection assays. These constructs include the transcriptional start site of promoter I.1 of the CYP19 gene. In these luciferase constructs, deletion mutations of the genomic region flanking the 5'-end of exon I.1 have been fused to the firefly luciferase reporter gene This figure and legend has been directly reproduced from Sun T., Zhao Y, Mangelsdorf D. J., Simpson E.R., (1998) Characterization of a Region Upstream of Exon I.1 of the Human CYP19 (Aromatase) Gene That Mediates Regulation by Retinoids in Human Choriocarcinoma Cells Endocrinology Vol. 139, No. 4 1684-1691 Read More