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This study deals with two related aspects of macrophage function, surface recognition and signal transmission across the plasma membrane. The introduction reviews the properties and functions of macrophages with particular reference to specific and non-specific recognition and activation as expressed by enhanced effector function. This review is set against the background of the structure and function of plasma membranes, biological recognition and mechanisms of cellular activation. The ability of macrophages to recognize a variety of foreign particles without the mediation of specific recognition molecules was investigated. In a binding assay performed at 4°C using non-opsonized bacteria, it was found that several types of Gram-positive and Gram-negative bacteria bound to normal mouse peritoneal macrophages. The binding could be inhibited by pre-incubating the macrophages at 4°C with various monosaccharides at a concentration of 10 aM. There was a very close correlation between the ability of a sugar to inhibit binding of a particular type of bacterium and the presence of that sugar in the bacterial cell wall. It was, therefore, postulated that the binding of non-opsonized bacteria by macrophages was based on the recognition of cell wall carbohydrates. The nature of the binding reaction was further studied using Corvnebacterium parvum. It was found that binding at 4°C depended on the presence of both Ca++- and MgtLions whilst binding at 20°C occurred to some degree when only Mg++-ions were present. The binding was not mediated by cell-bound antibody as shown by experiments using mild trypsin-treatment and specific antibody. Pro-treatment of the macrophages with trypain, process, /3-galactosidase and phospholipases A, C and D caused a marked reduction in binding whilst treatment with neuraminidase resulted in some increase in binding. Exposure of the macrophages to periodate also led to a decrease in binding of C. parvum, an effect largely reversed by subsequent treatment with borohydride. Recovery from the effects of enzyme treatment was rapid, but was inhibited by EDT& in the case of trypsin and β3-galactosidase. These results suggested that plasma membrane glycoproteins played an important part in the binding reaction which might involve a bridging action of divalent cations. The effect of neuraminidase was most easily explained by a reduction in cell surface negative charge. The enhancement of phosphatidyl inositol turnover was investigated as a possible mechanism of signal transmission initiating the intracellular effects of an activating agent following contact with the macrophage surface. The rate of phosphatidyl inositol turnover was assayed by measuring the uptake of tritiated Wo-iuositol into macrophage phosphatidyl inositol during one hour, It was shown that two macrophage activating agents, endotoxin and C. parvum caused an increase in phosphatidyl inositol turnover after 4 hours of incubation whilst exposure to three inert particles, Staphylococcus albus, latex and collol 1 carbon, had no such effect. All the particles tested were phagooytoses, and it was concluded that enhanced turnover of phosphatidyl inolitol was an ear1y event following exposure to activating agents that was not linked to the process of phagocytosis.

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No description available.

Improvements in the purification of <i>Galleria mellonella </i> haemocytes using Percoll gradients are described. This procedure resulted in 95% purity for plasmatocytes, 89% for granular cells and 69% for spherule cells. Using unwashed haemocytes taken directly off the Percoll gradients for making monolayers showed that Ca<SUP>2+</SUP> ion concentration, incubation time and temperature affect the number of cells attaching to the coverslips. The inability of these unwashed cells to undertake phagocytosis is discussed. In contrast, separated and washed haemocytes were highly phagocytic although ingestion was confined to the plasmatocyte cell type. Maximal ingestion by the plasmatocyte was, however, obtained only after the readdition of the granular cells to the plasmatocyte monolayer, illustrating the necessity for cell-cell co-operation in this process. Phenoloxidase activity was also tested in separated cells and it has been confirmed that prophenoloxidase is present in the granular cell type which indicates that this system may be involved in the haemocyte co-operation process. Haemolymph from <i>G. mellonella</i> was tested for the presence of lectins using a range of vertebrate erythrocytes. Only French Press-treated haemolymph with calf and rabbit erythrocytes yielded positive results, however, the levels of haemagglutination were low. Analysis of the physical and chemical properties of <i>G. mellonella</i> lectin showed it to be Ca<SUP>2+</SUP> ion dependent, heat-labile and precipitated by ammonium sulphate. Also, the haemagglutination could be inhibited maximally by lactose and the titre was unchanged in the presence of 2% BSA. <i>G. mellonella</i> lectin was purified via a three-step procedure utilizing ammonium sulphate precipitation, ion exchange chromatography with DEAE Sephadex, and affinity chromatography with Sepharose 4B. One band with an estimated molecular mass of 40 kDa in the lactose fraction is probably the isolated lectin.

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When a cell gets infected with a virus, the innate immune system swings into action within minutes. The rapid production of pro-inflammatory cytokines and antivirally active type-I Interferons (IFN-a/p) is the most significant mechanism to limit virus spread. Two conceptually different pathogen recognition mechanisms are known that lead to antiviral responses through production of IFN-a/p: Specialised immune cells possess Toll-like receptors (TLRs), which sense incoming viruses in endosomes. Most other cells rely on the cytoplasmic RNA-helicases RIG-I and MDA5 that sense the presence of viruses within the cell. However, although proteins and signalling networks involved in innate recognition of viruses are well known, the exact molecular details of their interactions with the virus are only marginally understood. During my PHD thesis I dedicated myself to aid our understanding of virus recognition. I could show that recombinant lentiviruses are weak inducers of IFN-ct/p* in murine immune cells. Standard preparations of lentiviral vectors, however, are strong activators of the innate immune system. This activity is contained in tubulo-vesicular structures that are present within standard lentiviral preparations and have the ability to activate TLR9. Tubulo-vesicular structures can serve as adjuvant to facilitate adaptive immune responses and may therefore be important when considering lentiviral vectors for clinical applications. In my second project I focused on cytoplasmic virus recognition. Surprisingly, viral genomic single-stranded RNA from influenza virus can activate the cytoplasmic virus recognition receptor RIG-I. Unlike most cellular RNA species, single-stranded RNA from influenza and other viruses bear a 5' triphosphate group, which marks this RNA as 'foreign' and thereby induces interferon responses. Importantly, influenza virus codes for an interferon antagonist, the non-structural protein 1 (NS1), which forms a complex with RIG-I, suggesting that influenza virus specifically interferes with this pathway. In conclusion, the innate immune system employs diverse mechanisms to sense the presence of a virus through recognising diverse forms of viral nucleic acid.

Macrophages play an important role in both innate and adaptive immunity by engulfing intruding pathogens and damaged or infected cells, antigen presentation, and by secretion of immune mediators. Interleukin 10 (IL-10) is a potent anti-inflammatory cytokine mainly produced by macrophages in response to cell activation, which serves to suppress immune reactions and inflammation. Hypoxia refers to oxygen deprivation. It is a common condition found in pathological tissues. The relationship between hypoxia and the production of IL-10 by macrophage is not yet thoroughly understood. In previous unpublished work by Staples and Burke et. al., it was found that both basal and LPS-induced IL-10 mRNA and protein are reduced in hypoxia. In the present study, it was shown that the transcription factor Hypoxia Inducible Factor 1 (HIF-1) appears to be involved in this reduction of IL-10 production in hypoxia. Although the regulatory elements on the IL-10 promoter responsible for this blockage effect were not definitively identified, our results indicated that the activity of a -4kb IL-10 luciferase reporter adenovirus was significantly reduced in cells treated with the HIF-1 inducing agents, cobalt chloride and desferrioxamine. The activity of a -195bp IL-10 luciferase reporter adenovirus was also decreased in the HIF-1inducing agent treated samples. These data imply that either by indirect interaction or physically binding to the IL-10 promoter or gene, HIF-1 does play a role in blocking IL-10 expression in hypoxia. Sequence and transcription factor analysis indicated the presence of a HIF-1 consensus sequence hypoxia responsive element (HRE) located in the -2,171bp to -2,187bp position on the IL-10 promoter. This result suggests that HIF-1 may affect IL-10 production, at least in part, by physically binding to its promoter. Lastly, we showed that the effect of hypoxia on IL-10 expression is observed with a range of different toll-like receptor ligands, and is not limited to induction by LPS.

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No description available.