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. 2018 Jun;61(6):1374-1383.
doi: 10.1007/s00125-018-4592-4. Epub 2018 Mar 27.

The Resident Macrophages in Murine Pancreatic Islets Are Constantly Probing Their Local Environment, Capturing Beta Cell Granules and Blood Particles

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Free PMC article

The Resident Macrophages in Murine Pancreatic Islets Are Constantly Probing Their Local Environment, Capturing Beta Cell Granules and Blood Particles

Bernd H Zinselmeyer et al. Diabetologia. .
Free PMC article

Abstract

Aims/hypothesis: We studied here the interactions between the resident macrophages of pancreatic islets with beta cells and the blood vasculature. We also examined the immunological consequences of such interactions.

Methods: Islets were isolated from C57BL/6 mice expressing CX3C motif chemokine receptor 1-green fluorescent protein (CX3CR-GFP) and examined live by two-photon microscopy. Islets were also examined by electron microscopy to study the relationship of the intra-islet macrophages with the beta cells. In NOD.Rag1-/- mice and young (non-diabetic) male mice, the acquisition of beta cell granules was tested functionally by probing with CD4+ T cells directed against insulin epitopes.

Results: Two-photon microscopy showed that the islet resident macrophages were in close contact with blood vessels and had extensive filopodial activity. Some filopodia had direct access to the vessel lumen and captured microparticles. Addition of glucose at high concentration reduced the degree of filopodia sampling of islets. This finding applied to in vivo injection of glucose or to in vitro cultures. Ultrastructural examination showed the close contacts of macrophages with beta cells. Such macrophages contained intact dense core granules. Functional studies in NOD mice indicated that the macrophages presented insulin peptides to insulin-reactive T cells. Presentation was increased after glucose challenge either ex vivo or after an in vivo pulse. In agreement with the morphological findings, presentation was not affected by insulin receptor blockade.

Conclusions/interpretation: Islet resident macrophages are highly active, sampling large areas of the islets and blood contents and capturing beta cell granules. After such interactions, macrophages present immunogenic insulin to specific autoreactive T cells.

Keywords: Beta cells; Fluorescence microscopy; Islet; Islet autoimmunity; Macrophage.

Conflict of interest statement

The authors declare that there is no duality of interest associated with this manuscript.

Figures

Fig. 1
Fig. 1
Three-dimensional two-photon microscopy of Cx3cr1+/GFP macrophages in islets and flow cytometric analysis of the macrophages. (a) Flow analysis. Islet cells were isolated and labelled with antibodies directed against the indicated markers. Cells were first gated using forward-scatter (FSC) and side-scatter (SSC) followed by forward-scatter area (FSC-A) and forward-scatter width (FSC-W) to identify single cells. The CD45+MHCII+ cells were selected for analysis. The majority of these were CD11b+/CD11c+ and Cx3cr1+/GFP/F4/80+. Thus, all the CX3CR1+ cells were macrophages. (b) Three-dimensional two-photon microscopy of Cx3cr1+/GFP macrophages in the islet. Blue, second harmonic signal and autofluorescence; green, GFP; red, vasculature. Side views show computerised optical sections (white dotted lines show location of optical sections x:z and y:z). Macrophages scan the whole islet in all directions and form filopodia to the vessel lumen (white arrows) and touch each other (pink arrow). Please compare x:z and x:y with fly-through animation in ESM Video 1. (c) Morphology of macrophages under steady-state conditions (pink arrows indicate interactions between macrophage filopodia). These images are representative of 12 mice; 10–20 islets imaged per mouse. Scale bars, 25 μm
Fig. 2
Fig. 2
Quantitative analyses of macrophage membrane dynamics. Two-photon imaging stacks were acquired every 30 s; 31 stacks representing 15 min underwent MIP. (ac) Representative macrophage images under a glucose concentration of 3.8 mmol/l (‘low glucose’) and a glucose concentration of 25 mmol/l (‘high glucose’) are shown. (a) MIP of the whole macrophage at the start of the experiment. The white 2D scale bar (60 μm × 60 μm) shows the cropped image (scale bar also applies to (b) and (c)). (b) The accumulated computerised MIPs from all 31 stacks over 15 min. (c) The computerised MIP from the first stack, in green, on top of all computerised MIPs in purple (compare with ESM Video 2). The area probed over 15 min is shown in purple. (d) The graph shows the surface sampled by the macrophages at the 15 min time point expressed as a percentage of the initial surface area at time 0. Each dot represents one macrophage taken from 20–40 islets. Horizontal lines indicate the mean±SD
Fig. 3
Fig. 3
Islet macrophages capture intravascular particles. Fifty microlitres of 50 nm microparticles together with 80 μl of DyLight594-labelled tomato lectin were injected intravenously into Cx3cr1+/GFP mice and the islets were harvested 10 min later. Islets were then placed in a customised heated chamber (as described in the Methods section and ESM Fig. 2) and examined. (a) The long filopodia of the macrophages (blue) extend towards the blood vessels (red) and acquire microparticles (green/white) over time. Time is given in hours:minutes. Scale bar, 25 μm. (b) The number of macrophages per islet (mean: 4.9). (c) The percentage of macrophages per islet that acquire microparticles. After 20 min, about 80% of the macrophages had taken up microparticles; there was no statistical difference in microparticle uptake at 20 min, 24 h or 72 h. (d) In order to semi-quantify the microparticle content per macrophage, the total 3D volume occupied by the particles was computerised. The graph shows the percentage of the macrophage volume that is occupied by microparticles. After 20 min, about 30% of the macrophage volume contained microparticles, which was similar to all other observed time points. Horizontal lines indicate the mean±SD
Fig. 4
Fig. 4
Electron microscopy of macrophages in islets of Cx3cr1+/GFP mice. (a) Macrophage in close contact with beta cells. (b) The macrophages extend some of their filopodia into the beta cells (arrows). (c) High magnification of section through a macrophage filipodia in contact with insulin granules. (d) The macrophages take up intact insulin granules with the typical dense core and surrounding halo. β, beta cell; MΦ, macrophage. (e) Capsule of the dense core granule (arrows). (a, b) Scale bar, 2 μm; (c, d, e) scale bar, 1 μm. These images are representative of electron micrographs of islets isolated from 24 mice (2–10 islets per mouse)
Fig. 5
Fig. 5
Presentation of insulin peptides by islets. (a) Islets were isolated from NOD.Rag1–/– mice at either 4 or 12 weeks of age. A portion of the islet cells were dispersed immediately after harvesting (labelled ‘no incubation’), another set was dispersed after 1 h incubation with 3.8 mmol/l glucose (‘Lo glucose’) and a third after 1 h incubation with 25 mmol/l glucose (‘Hi glucose’). Each set was dispersed and then different numbers of cells were incubated with IIT-3 T cells (which react with the B:13–21 peptide segment of the insulin B chain). Incubation with high glucose led to an increase in presentation of insulin peptides. Wk, week. (b, c) Islets of 13-week-old male B6.g7 mice were tested against both IIT-3 T cells directed to B:13–21-(13–21 T cell) or B:12–20-(12–20 T cell). The islets were tested 30 min after intravenous injection of glucose (4 g/kg, black bars) or after 1 h ex vivo exposure to 3.8 mmol/l (white bars) or 25 mmol/l glucose (grey bars). Intravenous challenge also led to an increase in presentation of insulin peptides. Shown is a composite of two experiments with similar results. (d) Islets were treated as in (a), but one set was also treated in the presence or absence of 500 nmol/l of the insulin receptor blocker S961. S961 did not inhibit the presentation of insulin peptides induced by glucose challenge. Results were pooled from two experiments. (e) The experiment tested the effects of S961 in islets not challenged with glucose, which favours granule uptake (d). An aliquot of islets was immediately dispersed (time 0) in the presence or absence of 1 μmol/l of S961. It shows the basal level of insulin peptide presentation. This presentation was not affected by S961. The remaining islets were incubated for 1 h without or with added insulin (INS) at concentrations indicated and without or with 1 μmol/l of S961. All islets were tested againts IIT-3 T cells. Incubation for 1 h increased the basal presentation, probably due to the release of free insulin. Incubation with added insulin concentrations led to further increase in insulin peptide presentation. S961 treatment inhibited presentation of insulin to the level found at time 0. Results were pooled from duplicate experiments in 6–8 mice. Data are mean ± SD; *p < 0.05

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