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A quantitative comparison of rates of phagocytosis and digestion of apoptotic cells by macrophages from normal (BALB/c) and diabetes-prone (NOD) mice

Maree, A.F.M., Komba, M., Finegood, D.T. and Edelstein-Keshet, L. 2008. A quantitative comparison of rates of phagocytosis and digestion of apoptotic cells by macrophages from normal (BALB/c) and diabetes-prone (NOD) mice. Journal of Applied Physiology 104 (1) , pp. 157-169. 10.1152/japplphysiol.00514.2007

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Abstract

Macrophages play an important role in clearing apoptotic debris from tissue. Defective or reduced clearance, seen, for instance, in non-obese diabetic (NOD) mice, has been correlated with initiation of autoimmune (Type 1) diabetes (T1D) (O'Brien BA, Huang Y, Geng X, Dutz JP, Finegood DT. Diabetes 51: 2481–2488, 2002). To validate such a link, it is essential to quantify the reduced clearance (for example, by comparison to BALB/c control mice) and to determine which elements of that clearance are impaired. Recently, we fit data for the time course of in vitro macrophage feeding experiments to basic models of macrophage clearance dynamics, thus quantifying kinetics of uptake and digestion of apoptotic cells in both mouse strains (Marée AFM, Komba M, Dyck C, Łabeçki M, Finegood DT, Edelstein-Keshet L. J Theor Biol 233: 533–551, 2005). In the cycle of modeling and experimental investigation, we identified the importance of 1) measuring short-, intermediate-, and long-time data (to increase the accuracy of parameter fits), and 2) designing experiments with distinct observable regimes, including engulfment-only and digestion-only phases. Here, we report on new results from experiments so designed. In comparing macrophages from the two strains, we find that NOD macrophage engulfment of apoptotic cells is 5.5 times slower than BALB/c controls. Significantly, our new data demonstrate that digestion is at least two times slower in NOD, in contrast with previous conclusions. Moreover, new data enable us to detect an acceleration in engulfment (after the first engulfment) in both strains, but much smaller in NOD macrophages. a variety of innate and adaptive immune system components are implicated in the destruction of β-cells in the pancreatic islets of Langerhans, causing autoimmune diabetes [Type 1 diabetes (T1D)]. Self-reactive T cells are known to be the main effectors of β-cell killing (10, 19). However, controversy exists as to the events that initiate the activation of β-cell-specific T cells (9, 35). Macrophages are among the earliest cells to infiltrate the islets. In the nonobese diabetic (NOD) mouse, a common animal model for T1D, this infiltration occurs as early as 2 wk of age and coincides with a normal wave of apoptosis of β-cells (40). This is followed by infiltration of dendritic cells and CD4+ and CD8+ T cells at 6–10 wk (8). Female NOD mice become diabetic by 12–15 wk (19) when 95% of their β-cells have been destroyed (23). Relative to BALB/c (controls), macrophages of NOD mice are identifiably different (16, 39), exhibiting abnormal cytokine secretion (2), reduced activation of other immune cells (17, 20), and reduced phagocytosis (35). Based on these observations, it has been suggested that defective macrophage clearance of the neonatal wave of β-cell apoptosis leads to self-presentation and, subsequently, to T-cell activation (27, 40). In earlier mathematical models, it has been shown that reduced macrophage clearance could be a major factor in T1D initiation (12, 25). These observations have motivated an ongoing comparative study of macrophage behavior in NOD vs. BALB/c mice. We recently showed that interstrain differences can be inferred by fitting time-course data of in vitro macrophage feeding experiments to mathematical models of macrophage dynamics (25). Our previous study demonstrated the feasibility of determining multiple kinetic parameters from such data. Moreover, it identified the importance of measuring data on multiple time scales and at different regimes: one of the improvements described here is inclusion of a “digestion only” regime to directly measure the rate that macrophages process apoptotic bodies after engulfment. Our main goal in this paper is to report on results of these redesigned experiments and to show how these facilitate more accurate determination of clearance kinetics and, hence, better inter-strain comparison. Before comparing between strains, we ask basic general questions about macrophage clearance. Question 1: After the first engulfment, do subsequent engulfments take place at higher rate? If so, we refer to the acceleration in engulfment as an “activation” step.1 1Our term “activation” should not be confused with similar term applied to transition of macrophages to antigen-presenting cells or other changes in response to inflammatory stimuli. Question 1a: If activation is observed, is that activation reversible or irreversible on the time scale of several hours? Question 2: Is there evidence for a maximal capacity of engulfed apoptotic bodies in a macrophage? Question 2a: If so, what is that maximal number? Question 3: Are the engulfed apoptotic bodies digested simultaneously, or does digestion saturate? In question 1a, we distinguish between the case that a macrophage reverts to a quiescent state (reversible activation) after fully digesting a meal or remains in an elevated engulfment mode (irreversible activation) over the time scale of a few hours. In question 3, we distinguish between 1) digestion that can handle many apoptotic bodies in parallel, 2) a saturated digestion machinery that can only break down one body at a time, and 3) a machinery that saturates only at higher numbers of apoptotic bodies. (Note that we refer to apoptotic cells preengulfment and to apoptotic bodies while still visible inside a macrophage.) By comparing the quality of fit of data to several model variants, we distinguish between competing models and answer (questions 1–3). Having identified the optimal model (and hence the most likely macrophage dynamics), we assess differences between the control (BALB/c) and diabetes-prone (NOD) mice.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Publisher: American Physiological Society
ISSN: 8750-7587
Date of Acceptance: 21 October 2007
Last Modified: 18 Feb 2019 08:46
URI: https://orca.cardiff.ac.uk/id/eprint/119506

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