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Dependence of macrophage phagocytic efficacy on antibody concentration.

ABSTRACT: Macrophages ingest the fungus Cryptococcus neoformans only in the presence of opsonins, and this provides a remarkably clean system for the detailed analysis of phagocytosis. This system is also unusual in that antibody-mediated phagocytosis involves ingestion through both Fc and complement receptors in the absence of complement. Mathematical modeling was used to analyze and explain the experimental data that the macrophage phagocytic index increased with increasing doses of antibody despite saturating concentrations and declined at high concentrations. A model was developed that explains the increase in phagocytic index with increasing antibody doses, differentiates among the contributions from Fc and complement receptors, and provides a tool for estimating antibody concentrations that optimize efficacy of phagocytosis. Experimental results and model calculations revealed that blocking of Fc receptors by excess antibody caused a reduction in phagocytic index but increased phagocytosis through complement receptors rapidly compensated for this effect. At high antibody concentrations, a further reduction in phagocytic index was caused by interference with complement receptor ingestion as a consequence of saturation of the fungal capsule. The ability of our model to predict the antibody dose dependence of the macrophage phagocytic efficacy for C. neoformans strongly suggest that the major variables that determine the efficacy of this process have been identified. The model predicts that the affinity constant of the opsonic antibody for the Fc receptor and the association-dissociation constant of antibody from the microbial antigen are critical parameters determining the efficacy of phagocytosis.

SUBMITTER: Macura N

PROVIDER: S-EPMC1865677 | biostudies-literature | 2007 Apr

REPOSITORIES: biostudies-literature

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Dependence of macrophage phagocytic efficacy on antibody concentration.

Macura Natasa N Zhang Tong T Casadevall Arturo A

Infection and immunity 20070205 4

Macrophages ingest the fungus Cryptococcus neoformans only in the presence of opsonins, and this provides a remarkably clean system for the detailed analysis of phagocytosis. This system is also unusual in that antibody-mediated phagocytosis involves ingestion through both Fc and complement receptors in the absence of complement. Mathematical modeling was used to analyze and explain the experimental data that the macrophage phagocytic index increased with increasing doses of antibody despite sat ...[more]

PMID: 17283107

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Project description:Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination(1-3). Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 297(4-6). Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome. Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis(7). Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response(8). Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 ?m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc?Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc?Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection(9).

Project description:Inflammation plays a major role in the pathogenesis of several vascular pathologies, including abdominal aortic aneurysm (AAA). Evaluating the role of inflammation in AAA pathobiology and potentially outcome in vivo requires non-invasive tools for high-resolution imaging. We investigated the feasibility of X-ray computed tomography (CT) imaging of phagocytic activity using nanoparticle contrast agents to predict AAA outcome. Methods: Uptake of several nanoparticle CT contrast agents was evaluated in a macrophage cell line. The most promising agent, Exitron nano 12000, was further characterized in vitro and used for subsequent in vivo testing. AAA was induced in Apoe -/- mice through angiotensin II (Ang II) infusion for up to 4 weeks. Nanoparticle biodistribution and uptake in AAA were evaluated by CT imaging in Ang II-infused Apoe -/- mice. After imaging, the aortic tissue was harvested and used from morphometry, transmission electron microscopy and gene expression analysis. A group of Ang II-infused Apoe -/- mice underwent nanoparticle-enhanced CT imaging within the first week of Ang II infusion, and their survival and aortic external diameter were evaluated at 4 weeks to address the value of vessel wall CT enhancement in predicting AAA outcome. Results: Exitron nano 12000 showed specific uptake in macrophages in vitro. Nanoparticle accumulation was observed by CT imaging in tissues rich in mononuclear phagocytes. Aortic wall enhancement was detectable on delayed CT images following nanoparticle administration and correlated with vessel wall CD68 expression. Transmission electron microscopy ascertained the presence of nanoparticles in AAA adventitial macrophages. Nanoparticle-induced CT enhancement on images obtained within one week of AAA induction was predictive of AAA outcome at 4 weeks. Conclusions: By establishing the feasibility of CT-based molecular imaging of phagocytic activity in AAA, this study links the inflammatory signal on early time point images to AAA evolution. This readily available technology overcomes an important barrier to cross-sectional, longitudinal and outcome studies, not only in AAA, but also in other cardiovascular pathologies and facilitates the evaluation of modulatory interventions, and ultimately upon clinical translation, patient management.

Project description:The phagocytic NADPH oxidase generates superoxide by transferring electrons from cytosolic NADPH to extracellular O2. The activity of the oxidase at the plasma membrane can be measured as electron current (I(e)), and the voltage dependence of I(e) was recently reported to exhibit a strong rectification in human eosinophils, with the currents being nearly voltage independent at negative potentials. To investigate the underlying mechanism, we performed voltage-clamp experiments on inside-out patches from human eosinophils activated with PMA. Electron current was evoked by bath application of different concentrations of NADPH, whereas slow voltage ramps (0.8 mV/ms), ranging from -120 to 200 mV, were applied to obtain 'steady-state' current-voltage relationships (I-V). The amplitude of I(e) recorded at -40 mV was minimal at 8 microM NADPH and saturated above 1 mM, with half-maximal activity (K(m)) observed at approx. 110 microM NADPH. Comparison of I-V values obtained at different NADPH concentrations revealed that the voltage-dependence of I(e) is strongly influenced by the substrate concentration. Above 0.1 mM NADPH, I(e) was markedly voltage-dependent and steeply decreased with depolarization within the physiological membrane potential range (-60 to 60 mV), the I-V curve strongly rectifying only below -100 mV. At lower NADPH concentrations the I-V curve was progressively shifted to more positive potentials and I(e) became voltage-independent also within the physiological range. Consequently, the K(m) of the oxidase decreased by approx. 40% (from 100 to 60 microM) when the membrane potential increased from -60 to 60 mV. We concluded that the oxidase activity depends on both membrane potential and [NADPH], and that the shape of the I(e)-V curve is influenced by the concentration of NADPH in the submillimolar range. The surprising voltage-independence of I(e) reported in whole-cell perforated patch recordings was most likely due to substrate limitation and is not an intrinsic property of the oxidase.

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Dependence of macrophage phagocytic efficacy on antibody concentration.

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Dependence of macrophage phagocytic efficacy on antibody concentration.

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