Regulation of macrophage function and its subversion by pathogens

Macrophages are key mediators of innate immunity to infection and are also involved in the initiation of adaptive immune responses. Their functions include phagocytosis of invading microorganisms, killing of pathogens by the generation of toxic mediators such as reactive oxygen intermediates and nitric oxide (NO), and antigen presentation to lymphocytes. Activated macrophages also release various cytokines that can induce or direct lymphocyte activation. For example, the pro-inflammatory cytokines IL-6 and TNF-alpha promote B and T cell activation, and the heterodimeric cytokine IL-12 drives the differentiation of IFN-?-producing type 1 T helper cells. Bacterial endotoxin or lipopolysaccharide (LPS), a potent activator of macrophages, has been used extensively to study macrophage function. Multiple intracellular signalling cascades are triggered following binding of LPS to the recently-identified Toll-like receptor 4 (TLR4), resulting in the induction of macrophage mediators including cytokines and inducible nitric oxide synthase (iNOS), the enzyme that catalyses NO synthesis. Pre-treatment or co-stimulation with interferon (IFN)-gamma, which is itself a potent inducer of iNOS, results in modulation of LPS-induced cytokine production, including priming for IL-12 production. The mitogen-activated protein (MAP) kinases are important intracellular regulators of a diverse range of cellular functions. In this study, the three major MAP kinase subfamilies - extracellular-regulated kinases (ERKs), p38 and c-Jun N-terminal/stress-activated protein kinases (JNK/SAPKs) - are shown to be activated by stimulation of macrophages with LPS in the presence/absence of IFN-gamma. Furthermore, regulatory roles for the ERK and p38 MAP kinases in the induction of IL-12, IL-6, TNF-alpha and iNOS are demonstrated. p38 MAP kinase activation is required for induction of all of these macrophage mediators, regulating the induction of IL-12 and iNOS at the transcriptional level, and the translation or release of TNF-alpha. Induction of TNF-alpha and iNOS, but not IL-12 p35 or IL-6, is also dependent on ERK MAP kinases. In contrast, transcription of the IL-12 p40 gene is negatively regulated by the ERKs to promote the formation of bioactive IL-12 heterodimer without excessive production of antagonistic p40/p40 homodimers. The modulation of macrophage activation by pathogens was also examined using the immunomodulatory products of two parasites: a surface glycoconjugate of intracellular Leishmania parasites and a glycoprotein secreted by an extracellular filarial nematode. Leishmania sp. parasites, which invade and parasitise macrophages, have been shown to evade NO-mediated killing by suppressing macrophage IL-12 production and thereby preventing the production of IFN-gamma by Th1 cells. In this study, the repeating phosphodisaccharide (phosphoglycan, PG) component of the Leishmania surface molecule lipophosphoglycan (LPG) is shown to be responsible for the suppression of bioactive IL-12 production and a mechanism for this inhibition is proposed. Following binding of a synthetic version of this portion of LPG (sPG) to macrophages, probably via interaction with either the mannose-fucose receptor (MFR) or complement receptor 3 (CR3), cellular levels of dual-phosphorylated ERK MAP kinases were enhanced, thereby suppressing the induction of bioactive IL-12 by preventing synthesis of the p40 subunit at the transcriptional level. The effects on macrophage function of an excretory-secretory product of the filarial nematode Acanthocheilonema viteae (ES-62) were also investigated. ES-62 has previously been shown to modulate the activation of B and T lymphocytes and the differentiation of dendritic cells. In vitro and in vivo exposure of macrophages to ES-62 is shown to inhibit induction of IL-12, IL-6 and TNF-alpha by LPS. ES-62 targets the synthesis and activation of multiple signalling molecules including tyrosine kinases, MAP kinases and transcription factors such as NF-kappaB and IRF family members. The roles of MAP kinases in the ES-62-mediated suppression of cytokine induction were also investigated. The results described provide valuable information about the mechanisms underlying the production of cytokines and NO by activated macrophages, and demonstrate how pathogens target these responses to evade detection and destruction by the host immune system