Method for Isolating RNA from cells using TRI Reagent Solution

1.      Re-suspend the homogenized, cultured cells in 1ml of 1M TRI Reagent. If the cells are grown in monolayer, add 1ml of 1M TRI reagent to the wells. In case the cells are grown in suspension, centrifuge and add 1ml of 1M TRI Reagent to the pellet. The TRI reagent is used to lyse the cells. 

2.      Incubate the samples for 5 minutes at room temperature (RT).

3.      After incubation, transfer the samples to 1.5ml eppendorf tubes.

4.      Add 100μl of chloroform to the samples and shake the tubes for around 15 seconds to mix thoroughly and incubate the samples for 5-15 minutes at RT.

5.      Centrifuge the samples at 12000 x g for 8 minutes at 4°C. This will form 3 layers in the eppendorf tube. The top, clear aqueous layer contains the total RNA, followed by a layer of interphase DNA and finally, a layer of protein and lipids.

6.      Carefully pipette the top aqueous layer that contains the RNA into new eppendorf tubes.

7.      To these samples, add 500μl of isopropanal and mix thoroughly, vortexing for 5-10 seconds. Incubate the samples for 5-10 minutes at RT.

8.      Centrifuge the samples at 12000 x g for 8 minutes at 4-25°C and discard the supernatant.

9.      Wash the pellet with 1ml of 75% ethanol in the eppendorf tubes. Mix thoroughly by vortexing.

10.  Centrifuge the sample at 7500 x g for 5 minutes. Discard the ethanol. This will leave behind a white RNA pellet. Airs dry the RNA pellet briefly.

11.  Re-suspend the pellet in approximately 80μl of DEPC water. The volume of DEPC water added to dissolve the pellet is dependant on the size of pellet. If the pellet size appears big, increase the volume of DEPC water. If the pellet size appears small, decrease the volume of DEPC water according.

12.  Incubate the mixture at 55-60°C for approximately 10 minutes to completely dissolve the RNA pellet

13.  The RNA concentration can now be determined using Nanodrop.

First Draft of Introduction and Ideas for the project

The role of complement properdin upon infection with Mycobacterium marinum

Introduction

The complement system is a part of the innate immune response upon infection with a pathogen or a foreign particle. The complement activation consists of three main pathways: the classical pathway, the mannose-binding lectin pathway and the alternative pathway. Activation of the complement via the classical pathway involves the formation of antibody-antigen complexes. This recruits the C1 component which cleaves the C2 and C3 components forming a protease known as C3 convertase. The MBLP is very much similar to the CP, however, it is initiated when mannose-binding lectin binds to the mannose on the surface of pathogens.

The AP, on the contrary, is initiated by spontaneous cleavage of C3 in the fluid phase that leads to the formation of C3b segment. This segment associates with factor B in the environment which is cleaved by factor D, forming the AP C3 convertase, C3bBb. The C3 convertase from the three pathways cause the C3 cleavage which produces a small C3a fragment and a large C3b fragment. The latter has three possible functions: it can activate the alternative pathway causing amplification of C3b production, it can attach to the pathogen cell surface and act as opsonins or it can bind to the C3 convertase and form C5 convertase. Formation of C5 convertase causes the cleavage of C5 and the recruitment of C6, C7, C8 and poly-C9 components, forming a membrane attack comples (MAC) and ultimately resulting in cell lysis.

The role of properdin in the complement system was first suggested by Pillemar and collaborators (1954). It was suggested that properdin binds to a target protein and activates the complement system. After initial refutation, the work of Pillemar was vindicated (Lepow, 1980) as the AP was established (Medicus et al., 1976; Pangburn and Muller-Eberhard, 1986). When the alternative pathway was firmly established, the C3 convertase, C3bBb, was shown to be a short-lived complex (Medicus et al., 1976; Pangburn and Muller-Eberhard, 1986) which was stabilized by association with properdin by 5- fold to 10-fold (Fearon et al., 1975). Hence properdin was accepted as a positive regulator of the complement system.

In a recent research by Spitzer and colleagues (2007), surface plasmon resonance (SPR) methodology was used to conclude that bound to a target surface; complement activation can be initiated by properdin using the proteins found in serum. Furthermore, it has been also shown that properdin can recognize a variety of dangerous, nonself targets (for example, zymosan, Neisseria, and LPS-coated microtiter wells), and promotes the initiation of the C3 convertase of the AP (Spitzer et al., 2007; Kimura et al., 2008). These studies led to further studies that demonstrate the involvement of properdin in recognition and removal of altered or dangerous self cells, for example, apoptotic T cells (for review see Kemper et al., 2010).

In this study, the role played by properdin during Mycobacterium marinum infection will be studied using properdin deficient mice.  The first stage will be to compare the abundance of associated and/or internalized M. marinum in non-adherent cells in wild-type and properdin deficient mice using flow cytometry . This can be carried out by labeling M. marinum with a fluorescent dye like calcein (Barker et al., 1997).

This will then be followed by comparing the effects of hypoxia on leukocytes prepared from both wildtype and properdin deficient mice. Cells such as splenic macrophages, bone marrow derived dendritic cells and mast cells will be infected with M. marinum in hypoxic and normoxic conditions. These cells will then be lysed post-infection and the intracellular viable count (CFU) will be determined. The supernatants of the cells will be used to analyze TNF-α bioactivity using L929 indicator cells. In presence of TNF-α, L929 cells undergo cell death by either apoptosis or necrosis (Humphreys and Wilson, 1999).  Finally, gene expression will be analyzed to assess the time dependent host response in macrophage cell lines J774 and RAW.


References 

• Barker LP, George KM, Falkow S, Small, PLC. 1997. Differential Trafficking of Live and Dead . Mycobacterium marinum Organisms in Macrophages. Infection and Immunity 65: 1497-1504.
• Fearon DT, Austen KF. 1975. Properdin: binding to C3b and stabilization of the C3b-dependent C3 convertase. J. Exp. Med.142:856–63
•  Humphreys DT, Wilson MR. 1999. Modes of L929 Cell Death Induced by TNF-α and other Cytotoxic Agents.  Cytokine 11:773-782.
• Kemper, C., Atkinson, J. P., Hourcade, D.E. 2010. Properdin: Emerging roles of a Pattern-Recognition Molecule. Annu. Rev. Immunol. 28:131-55
• Kimura Y, Miwa T, Zhou L, Song WC. 2008. Activator-specific requirement of properdin in the initiation and amplification of the alternative pathway complement. Blood 11:732–40 
• Lepow IH. 1980. Presidential address to American Association of Immunologists in Anaheim, California, April 16, 1980. Louis Pillemer, Properdin, and scientific controversy. J. Immunol.125:471–75
• Medicus RG, Gotze O, Muller-Eberhard HJ. 1976. Alternative pathway of complement: recruitment of precursor properdin by the labile C3/C5 convertase and the potentiation of the pathway. J. Exp. Med. 144:1076–93
• Pangburn MK, Muller-Eberhard HJ. 1986. The C3 convertase of the alternative pathway of human complement. Enzymic properties of the bimolecular proteinase. Biochem. J. 235:723–30
• Pillemer L, Blum L, Lepow IH, Ross OA, Todd EW, Wardlaw AC. 1954. The properdin system and immunity. I. Demonstration and isolation of a new serum protein, properdin, and its role in immune phenomena. Science 120:279–85 
• Spitzer D, Mitchell LM, Atkinson JP, Hourcade DE. 2007. Properdin can initiate complement activation by binding specific target surfaces and providing a platform for de novo convertase assembly.J. Immunol. 179:2600–8

Complement System - A Review

To understand the physiological roles of properdin, complement system needs to be understood to a certain degree. As an innate immune system defense system, complement plays a very vital role in recognizing non-self or foreign cells or particles. This leads to either phagocytosis of invading bodies or cell lysis due to formation of membrane attack complex (MAC).  

In this review, the different pathways by which complement system is activated are described and the function of their components and products are discussed. Furthermore, the regulators of the complement system are briefly explored. The newly discovered and suggested roles of properdin will be discussed and the validity of current research will be commented upon.

The Complement System – Recognizing and eliminating pathogens and inducing inflammation

The complement system is an essential part of the immune system activated as a result of infection in the host. There are three pathways which consist of different molecular recognition events that activate the complement system. These pathways are classified as: the classical pathway (CP), the mannose binding lectin pathway (MBLP), and the alternative pathway (AP). 

The CP is activated by antibodies recognizing the specific antigens on the surface of the pathogens and forming antibody: antigen complexes. As a result, there complexes are recognized by C1 component of the complement system. This component is made up of several subunits: C1q subunit, two C1r subunits and two C1s subunits. This C1 component binds to the Fc regions of the antibodies bound to the surface of the pathogen via the C1q subunit. This results in the activation of the activation of the molecule by cross proteolysis by the C1r and C1s proteases. Another component C4, binds to the C1 component and the activated C1s subunit causes the cleavage of C4. This releases the small peptide fragment, C4a, into the environment, which acts as an anaphylatoxin. The larger peptide fragment, C4b, binds to the surface of the bacteria. This is followed by the attachment of C2 component to the C4b fragment. This C2 component is also cleaved by the activated C1s subunit in the C1 component, releasing another small peptide, C2b. Thus, the C4bC2a protease is formed, also known as the C3 convertase of the CP.

In case of the MBLP, the C3 convertase formation occurs when the mannose-binding lectin binds to the mannose on the surface of pathogens. This causes the activation of the system and cleavage of C4 by the MBL. This is followed by the recruitment and cleavage of C2 and ultimately the formation of C3 convertase, C4b2a. In case of AP, however, the formation of C3 convertase is initiated by the spontaneous cleave of C3 component into C3b. This large C3b fragment recruits and cleaves factor B, forming Bb peptide. This cleaving of factor B is catalysed by factor D. This results in the formation of C3bBb, the AP C3 convertase. It is important to note that this loop of AP causes the amplification of C3 convertase formation so that the amount of C3b is increased in the system and the infection can be cleared more quickly.

The function of C3 convertase from all three pathways is to cleave C3 component to C3a and C3b. This peptide fragment, C3b has three main functions: as already discussed, it activates the AP, forming the amplification loop, it can also bind to the surface of the pathogen as opsonins, and it can form membrane attack complexes (MACs) by forming C5 convertase. By binding to the surface of the pathogen, C3b causes the phagocytosis of the pathogen by phagocytes. The opsonized targets are readily recognized by complement receptors expressed on different cell populations.

The formation of MAC, however, also involved some of the other components of the complement system.  If C3b bind to the C3 convertase, it forms C5 convertase, which cleaves the C5 component forming C5a and C5b fragments. While the C5a component is a powerful chemoattractant for most leukocytes, the C5b component associates with the C6 and C7 components resulting in C5b67 complex. This C5b67 complex inserts into the pathogen membrane and recruits the C8 component, which penetrates deeper into the pathogen membrane. This is followed by the recruitment of several C9 subunits that form a pore in the pathogen known as the membrane attack complex. This MAC causes membrane disruption and ultimately cell lysis.  

Complement and the Adaptive Immune System

 The adaptive immune response depends, largely on the ability of the innate immune system to differentiate between self and non-self cells or particles. From the oldest studies, mainly work done by Eden et al and Pepys, it is evident that C3 component of the complement plays an essential role in B cell activity. This was shown by using C3-defienct mice, where the B cells response to T cell-dependent antigens was impaired. Eden et al also showed that B cells bind the C3 fragments on their surface.

It was also shown in the studies done by Fearon et al that C3d binding receptor, CD21, was a key component in complement mediated modulation of B cell response. It has been shown that CD21, with CD19 and CD81, forms a functional receptor group on B cells. This co-receptor complex engages with the C3d, and related fragments that opsonize the antigens on pathogen surface. This occurs to stimulate the B cell receptor. The main function of this signal is to act as an adjuvant and lower the threshold for B cell activation and antibody production. The threshold is lowered from 10 to 10000-fold, promoting the antibody production from B cells.  Furthermore, CD21 also facilitates the antigen localization to follicular dendritic cells (FDCs) within lymphoid follicles. It also contributes towards the promotion of an optimal B cell memory pool.

While complement influences the B cell function, it is also important for optimal T cell function. There are two mechanisms by which complement influences the T-cell function: either indirectly, the complement activation products affect antigen presenting cells (APCs), or they directly impact the T cell functions.

There is a wide range of complement receptors found on APCs. Hence, they can easily sense and respond to the complement activation in the environment. The association of C3 fragments with antigens influences the recognition and processing of antigens by APCs. This is evident as the deficiency of C3 causes a reduced antigen presentation and ultimately impaired T cell immunity. Furthermore, C3-defiecient mice also show a lack of maturation of dendritic cells upon antigen uptake.

From the different fragments produced during complement activation, the anaphylotoxins are very critical in modulation of APC functions. For example, C3a/C5a receptors are found on APCs and when bound to the particular fragments, these receptors regulate the production of cytokines (specifically IL-12). Hence they have a dramatic impact on Th1 or Th2 lineage development, allergy onset and development, and also pathogen clearance.  Moreover, in C3a/C5a receptor deficient mice, or mice treated with peptide antagonists, and in animals that are deficient in complement regulators,  it has been shown that CD4 and CD8 T cell responses are diminished. In addition, it has also been shown that C3a/C5a intracellular signaling pathways intersect with those of the Toll-like receptor (TLR) family.

Whilst complement impacts T-cell function indirectly via APCs, it also influences T cells directly. The immune reactions are suppressed by complement. Upon activation, the complement receptors located on T-cell surface has an impact on cell proliferation, cell viability, and IFN-γ production. The purpose of this immune response suppression by the complement may be vital to prevent tissue damage and autoimmunity.

Word List

Adaptive Immunity

Immune response that involved the B and T lymphocyte activation that leads to a pool of immunological memory with the generation of B lymphocyte clones having somatically recombined receptors.

Anaphylotoxins

Components of the complement system, like C3a and C5a, which mediate inflammatory response through cell activation to induce, for example, histamine release and chemotaxis.

 Innate Immunity

An instant or immediate immune response that is mediated by recognition of pathogens via germ-line coded pattern-recognition receptors.

Opsonin

Proteins like C3b, that bind to the surface of a pathogen or a particle and enhance phagocytosis by phagocytes.

Toll-Like Receptors

A family of receptors found on cell surface or bound to cell compartments that recognize dangerous particles or pathogens by detecting a wide range of pathogen-associated molecular patterns and sometimes endogenous ligands.  

References

1.      Barrington RA, Pozdnyakova O, Zafari MR, Benjamin CD, Carroll MC. 2002. B lymphocyte memory: role of stromal cell complement and FcγRIIB receptors. J. Exp. Med. 196:1189–99 

2.      Carroll MC. 2004. The complement system in regulation of adaptive immunity. Nat. Immunol. 5:981–86 

3.      Davies KA, Walport MJ. 1998. Processing and clearance of immune complexes by complement and the role of complement in immune complex diseases. See Reference 7, pp. 423–53 

4.      Dempsey PW, Allison ME, Akkaraju S, Goodnow CC, Fearon DT. 1996. C3d of complement as a molecular adjuvant: bridging innate and acquired immunity. Science 271:348–50 

5.      Drouin SM, Corry DB, Kildsgaard J, Wetsel RA. 2001. Cutting edge: the absence of C3 demonstrates a role for complement in Th2 effector functions in a murine model of pulmonary allergy. J. Immunol. 167:4141–45 

6.      Fang C, Miwa T, Shen H, Song WC. 2007. Complement-dependent enhancement of CD8⁺ T cell immunity to lymphocytic choriomeningitis virus infection in decay-accelerating factor-deficient mice. J. Immunol. 179:3178–86 

7.      Fang Y, Xu C, Fu YX, Holers VM, Molina H. 1998. Expression of complement receptors 1 and 2 on follicular dendritic cells is necessary for the generation of a strong antigen-specific IgG response. J. Immunol. 160:5273–79 

8.      Fearon DT, Carter RH. 1995. The CD19/CR2/TAPA-1 complex of B lymphocytes: linking natural to acquired immunity. Annu. Rev. Immunol. 13:127–49

9.      Fearon DT, Locksley RM. 1996. The instructive role of innate immunity in the acquired immune response. Science 272:50–54

10.  Fearon DT, Wong WW. 1983. Complement ligand-receptor interactions that mediate biological responses. Annu. Rev. Immunol. 1:243–71 

11.  Fleming SD, Shea-Donohue T, Guthridge JM, Kulik L, Waldschmidt TJ, et al. 2002. Mice deficient in complement receptors 1 and 2 lack a tissue injury-inducing subset of the natural antibody repertoire. J. Immunol. 169:2126–33 

12.  Gerard NP, Gerard C. 1991. The chemotactic receptor for human C5a anaphylatoxin. Nature 349:614–17

13.  Gerard NP, Gerard C. 2002. Complement in allergy and asthma.Curr. Opin. Immunol. 14:705–8 

14.  Grossman WJ, Verbsky JW, Barchet W, Colonna M, Atkinson JP, Ley TJ. 2004. Human T regulatory cells can use the perforin pathway to cause autologous target cell death. Immunity 21:589–601 

15.  Guo RF, Ward PA. 2005. Role of C5a in inflammatory responses.Annu. Rev. Immunol. 23:821–52 

16.  Hawlisch H, Belkaid Y, Baelder R, Hildeman D, Gerard C, Kohl J. 2005. C5a negatively regulates Toll-like receptor 4-induced immune responses. Immunity 22:415–26 

17.  Hawlisch H, Kohl J. 2006. Complement and Toll-like receptors: key regulators of adaptive immune responses. Mol. Immunol. 43:13–21

18.  Heeger PS, Lalli PN, Lin F, Valujskikh A, Liu J, et al. 2005. Decay-accelerating factor modulates induction of T cell immunity. J. Exp. Med. 201:1523–30 

19.  Helmy KY, Katschke KJ Jr, Gorgani NN, Kljavin NM, Elliott JM, et al. 2006. CRIg: a macrophage complement receptor required for phagocytosis of circulating pathogens. Cell 124:915–27 

20.  Hourcade D. 2006. The role of properdin in the assembly of the alternative pathway C3 convertases of complement. J. Biol. Chem.281:2128–32 

21.  Jacquier-Sarlin MR, Gabert FM, Villiers MB, Colomb MG. 1995. Modulation of antigen processing and presentation by covalently linked complement C3b fragment. Immunology 84:164–70 

22.  Kemper C, Atkinson JP. 2007. T-cell regulation: with complements from innate immunity. Nat. Rev. Immunol. 7:9–18 

23.  Kemper C, Chan AC, Green JM, Brett KA, Murphy KM, Atkinson JP. 2003. Activation of human CD4⁺ cells with CD3 and CD46 induces a T-regulatory cell 1 phenotype. Nature 421:388–92

24.  Kemper C, Mitchell LM, Zhang L, Hourcade DE. 2008. The complement protein properdin binds apoptotic T cells and promotes complement activation and phagocytosis. Proc. Natl. Acad. Sci. USA 105:9023–28 Shows that properdin binds apoptotic T cells and malignant cells and can promote phagocytosis without additional complement proteins. 

25.  Kerekes K, Cooper PD, Prechl J, Jozsi M, Bajtay Z, Erdei A. 2001. Adjuvant effect of gamma-inulin is mediated by C3 fragments deposited on antigen-presenting cells. J. Leukoc. Biol. 69:69–74

26.  Kim AH, Dimitriou ID, Holland MC, Mastellos D, Mueller YM, et al. 2004. Complement C5a receptor is essential for the optimal generation of antiviral CD8⁺ T cell responses. J. Immunol.173:2524–29 

27.  Klos A, Tenner AJ, Johswich KO, Ager RR, Reis ES, Kohl J. 2009. The role of the anaphylatoxins in health and disease. Mol. Immunol.46:2753–66

28.  Kohl J, Baelder R, Lewkowich IP, Pandey MK, Hawlisch H, et al. 2006. A regulatory role for the C5a anaphylatoxin in type 2 immunity in asthma. J. Clin. Invest. 116:783–96 

29.  Kopf M, Abel B, Gallimore A, Carroll MC, Bachmann MF. 2002. Complement component C3 promotes T-cell priming and lung migration to control acute influenza virus infection. Nat. Med. 8:373–78 

30.  Liu J, Miwa T, Hilliard B, Chen Y, Lambris JD, et al. 2005. The complement inhibitory protein DAF (CD55) suppresses T cell immunity in vivo. J. Exp. Med. 201:567–77 

31.  Longhi MP, Harris CL, Morgan BP, Gallimore A. 2006. Holding T cells in check—a new role for complement regulators? Trends Immunol. 27:102–8 

32.  Longhi MP, Sivasankar B, Omidvar N, Morgan BP, Gallimore A. 2005. Cutting edge: murine CD59a modulates antiviral CD4⁺ T cell activity in a complement-independent manner. J. Immunol.175:7098–102 

33.  Markiewski MM, Nilsson B, Ekdahl KN, Mollnes TE, Lambris JD. 2007. Complement and coagulation: strangers or partners in crime?Trends Immunol. 28:184–92

34.  Medzhitov R, Janeway C Jr. 2000. Innate immunity. N. Engl. J. Med. 343:338–44 

35.  Mevorach D, Mascarenhas JO, Gershov D, Elkon KB. 1998. Complement-dependent clearance of apoptotic cells by human macrophages. J. Exp. Med. 188:2313–20 

36.  Molina H, Holers VM, Li B, Fang Y-F, Mariathasan S, et al. 1996. Markedly impaired humoral immune response in mice deficient in complement receptors 1 and 2. Proc. Natl. Acad. Sci. USA93:3357–61 

37.  Morgan BP, Marchbank KJ, Longhi MP, Harris CL, Gallimore AM. 2005. Complement: central to innate immunity and bridging to adaptive responses. Immunol. Lett. 97:171–79 

38.  Nussenzweig V, Bianco C, Dukor P, Eden A. 1971. Receptors for C3 on B lymphocytes: possible role in the immune response. InProgress in Immunology, ed. DB Amos, pp. 73–81. New York: Academic

39.  Ollert MW, Kadlec JV, David K, Petrella EC, Bredehorst R, Vogel C-W. 1994. Antibody-mediated complement activation on nucleated cells. A quantitative analysis of the individual reaction steps. J. Immunol. 153:2213–21 

40.  Pappworth IY, Kulik L, Haluszczak C, Reuter JW, Holers VM, Marchbank KJ. 2009. Increased B cell deletion and significantly reduced auto-antibody titer due to premature expression of human complement receptor 2 (CR2, CD21). Mol. Immunol. 46:1042–49

41.  Pepys MB. 1972. Role of complement in induction of the allergic response. Nat. New Biol. 237:157–59 

42.  Prodeus AP, Goerg S, Shen LM, Pozdnyakova O, Chu L, et al. 1998. A critical role for complement in maintenance of self-tolerance. Immunity 9:721–31 

43.  Reid RR, Woodcock S, Shimabukuro-Vornhagen A, Austen WG Jr, Kobzik L, et al. 2002. Functional activity of natural antibody is altered in Cr2-deficient mice. J. Immunol. 169:5433–40  

44.  Spitzer D, Mitchell LM, Atkinson JP, Hourcade DE. 2007. Properdin can initiate complement activation by binding specific target surfaces and providing a platform for de novo convertase assembly.J. Immunol. 179:2600–8 Demonstrates that purified properdin binds certain microbial surfaces and provides a platform for the assembly of functional convertases. 

45.  Stager S, Alexander J, Kirby AC, Botto M, Van Rooijen N, et al. 2003. Natural antibodies and complement are endogenous adjuvants for vaccine-induced CD8⁺ T-cell responses. Nat. Med.9:1287–92 

46.  Strainic MG, Liu J, Huang D, An F, Lalli PN, et al. 2008. Locally produced complement fragments C5a and C3a provide both costimulatory and survival signals to naive CD4⁺ T cells. Immunity28:425–35 

47.  Suresh M, Molina H, Salvato MS, Mastellos D, Lambris JD, Sandor M. 2003. Complement component 3 is required for optimal expansion of CD8 T cells during a systemic viral infection. J. Immunol. 170:788–94 

48.  Verschoor A, Brockman MA, Gadjeva M, Knipe DM, Carroll MC. 2003. Myeloid C3 determines induction of humoral responses to peripheral herpes simplex virus infection. J. Immunol. 171:5363–71

49.  Volanakis JE, Frank MM. 1998. The Human Complement System in Health and Disease. New York: Marcel Dekker  

50.  Volanakis JE. 1998. Overview of the complement system. See Reference 7, pp. 9–32 

51.  Wagner C, Hansch GM. 2006. Receptors for complement C3 on T-lymphocytes: relics of evolution or functional molecules? Mol. Immunol. 43:22–30  

52.  Walport MJ. 2001. Complement. First of two parts. N. Engl. J. Med.344:1058–66 

53.  Walport MJ. 2001. Complement. Second of two parts. N. Engl. J. Med. 344:1140–44

54.  Wetsel RA. 1995. Structure, function and cellular expression of complement anaphylatoxin receptors. Curr. Opin. Immunol. 7:48–53

55.  Whaley K. 1985. An introduction to the complement system. InMethods in Complement for Clinical Immunologists, ed. K Whaley, pp. 1–20. Edinburgh: Churchill Livingstone 

56.  Whaley K. 1985. Measurement of complement. In Methods in Complement for Clinical Immunologists, ed. K Whaley, pp. 77–139. New York: Churchill Livingstone 

57.  Wright SD, Silverstein SC. 1983. Receptors for C3b and C3bi promote phagocytosis but not the release of toxic oxygen from human phagocytes. J. Exp. Med. 158:2016–23 

58.  Zhang X, Kimura Y, Fang C, Zhou L, Sfyroera G, et al. 2007. Regulation of Toll-like receptor-mediated inflammatory response by complement in vivo. Blood 110:228–36 

59.  Zhou W, Patel H, Li K, Peng Q, Villiers MB, Sacks SH. 2006. Macrophages from C3-deficient mice have impaired potency to stimulate alloreactive T cells. Blood 107:2461–69 

Initial Project Idea

Properdin acts as an amplifier of complement activation in the fluid phase.
Deficiency of properdin leads to a measurable reduction in complement
activation in the fluid-phase due to a decrease in the half-life of the
complement converting enzyme complexes. In addition, however, macrophages
and dendritic cells, which are deficient of properdin, appear to behave
differently from the wildtype on a cellular level: they produce more TNF-a
after stimulation with LPS and differ in their numbers of intracellular
Listeria monocytogenes. Current work aims to characterise the role of
properdin in the cellular response to infection with Mycobacterium marinum,
a category 2 model microorganism for M. tuberculosis.

At the moment, the envisaged project is thought to analyse these sets of
experiments: 

Cells (such as splenic macrophages, bone marrow derived dendritic cells or
mast cells) will be prepared from properdin deficient and wildtype mice.
They will be infected with M.marinum using normoxic and hypoxic conditions.
Cells will be lysed after infection to determine intracellular viable counts
(CFU) and supernatants will be analysed for TNF-a bioactivity using L929
indicator cells.   

Non-adherent cells such as murine mast cells may be analysed by flow
cytometry for the abundance of associated and internalised, labelled
M.marinum. Macrophage cell lines such as J774 and RAW can be used to assess
the time dependent host cell response by analysing gene expression.