Article Figures & Data
Figures
- Figure 1.
Immune cells are distributed throughout the mouse adrenal gland. A, CD45-immunoreactive myeloid cells are present in the cortex (Crtx) and medulla (Med) of the adrenal gland. B, In the medulla, the majority of CD45-IR cells are elongated with fine processes. C, An example of a rare spherical CD45-IR cell in the medulla. ZG, zona glomerulosa; ZF, zona fasciculata. Scale bar: 200 µm (A); 20 µm (B, C).
- Figure 2.
Cells in the adrenal medulla are labeled with a variety of macrophage markers. A, F4/80-IR cells are present in the adrenal cortex (Crtx) and medulla (Med). F4/80-IR labels a band of cells in the cortical X-zone (xz; open arrowhead) and cells throughout the medulla (closed arrow heads). B, Higher magnification of F4/80-IR cells in the medulla indicates the cells are elongate with fine processes. C, CD68-IR cells are prominent in the X-zone and also present in the medulla. D, CD301-IR cells are located in the capsule/zona glomerulosa and medulla. E, CD115-IR cells are located in the cortex and sparse in the medulla. F, MHC II-IR cells are primarily located in the outer cortex. G, Macrophages that are Iba1-IR are present throughout the adrenal including the medulla. H, Lysozyme2-expressing cells (Lyz2) labeled with tdTomato (Lyz2cre-GCaMP6f mouse) are present in an inner cortical region and throughout the adrenal medulla. Scale bar: 100 µm (A, C–H); 20 µm (B). See also Extended Data Figure 2-1 for more details.
- Figure 3.
Macrophages in the adrenal medulla are closely associated with multiple cell types. A, B, F4/80-IR cells in the medulla are intermingled with tyrosine hydroxylase-IR chromaffin cells. C, D, F4/80-IR macrophages are juxtaposed to PNMT-IR (white arrowheads) and PNMT-negative chromaffin cells (red arrowheads). E, F, F4/80-IR cells are located next to chromaffin cells that are NPY-IR. G, H, F4/80-IR cells (white arrowheads) are close to GFAP-IR satellite glial cells in the medulla. Note also a band of GFAP-IR cells is located in the adrenal capsule (green arrowheads). I, J, Some Lyz2-expressing cells (white arrowheads) are close to CD31-IR endothelial cells in the medulla. Scale bar: 100 µm (A, C, E, G, I); 20 µm (B, D, F, H, J). See also Extended Data Figure 3-1 for more details.
- Figure 4.
Nerve-associated macrophages in the adrenal medulla. A, B, Staining for an axonal marker (acetylated tubulin, acTub) reveals dense labeling throughout the medulla. Colocalization with F4/80-IR shows that macrophages are associated with acTub-IR axonal processes and often aligned (white circles). C, D, Synapsin-IR synaptic varicosities are densely distributed throughout the adrenal medulla. Higher magnification reveals potential sites of interaction with F4/80-IR macrophages in the medulla (white circles). Scale bar: 100 µm (A, C); 20 µm (B, D).
- Figure 5.
Macrophages are present in the adrenal medulla throughout postnatal development. A−D. Examples of adrenal cryosections showing that F4/80-IR macrophages (arrow heads) are present throughout the adrenal cortex and medulla (TH-IR zone) in male mice at postnatal days 1 to 50. E, F, F4/80-IR macrophages in the cortex and medulla of female mice. G, Group data showing the relative density of F4/80-IR cells in the adrenal medulla at P25 and P50 (mean #/0.01 mm2 ± SD, n = 3–4 mice). Scale bar, 100 µm.
- Figure 6.
Adrenal macrophages express P2Y purinergic receptors. A, Adrenal cryosections from a Lyz2cre-GCaMP6f-tdTomato mouse, costained for RFP and F4/80 (top panel) and RFP and CD45 (bottom panel). Quantification of immunoreactivity within the medulla (n = 3 mice, 67–171 cells per mouse) shows that RFP-IR colocalizes with F4/80- and CD45-IR (R+/F+ and R+/C+, respectively). Most F4/80- and CD45-IR also colocalizes with RFP-IR (F+/R+ and C+/R+, respectively). B, Example of GCaMP6f fluorescent signal in medulla macrophages in a single adrenal slice from a Lyz2cre-GCaMP6f-tdTomato mouse (left panel, IU, intensity units). Application of 100 µM UDP-glucose, UDP, and UTP is indicated by the green bars. Group data (right panel) showing the response to a panel of P2Y agonists (each applied at 100 µM, mean ± SEM, n = 4–10 mice, each open symbol is the average of 15 cells per mouse). C, Calcium increase in response to 100 µM UDP is blocked following application of 100 µM reactive blue-2 (left panel). Group data (right panel; mean ± SEM, n = 4 mice, 15 cells per mouse). *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar, 100 µm. See also Extended Data Figures 6-1, 6-2 for more details.
- Figure 7.
Multiple GPCR agonists increase calcium levels in adrenal macrophages. A, Examples of GCaMP6f fluorescence changes in medulla macrophages in response to 100 µM ACh, 100 µM histamine, 100 µM norepinephrine, and 1 µM bradykinin (left panel). Group data quantifying the change in GCaMP6f signal in response to multiple agonists (right panel; mean ± SEM, n = 3–9 mice, 15 cells per mouse, 100 µM ACh, norepinephrine, oxotremorine-M, histamine; 50 µM DMPP; 10 µM LPA; 1 µM NPY, substance P, angiotensin II, bradykinin, PACAP, met-enkephalin). B, GCaMP6f fluorescence signal in macrophages in an adrenal slice in response to histamine application (left panel). Group data (right panel; mean ± SEM, n = 3–5 mice, 15 cells per mouse; EC50 ∼2.7 µM). C, Calcium increase in medulla macrophages in response to 10 µM histamine is blocked by 0.1 µM pyrilamine maleate (H1 receptor antagonist), but not by 10 µM cimetidine (H2 receptor antagonist). Left panel, each trace is the average of 15 cells in a single adrenal slice). Group data quantifying the effect (right panel; mean ± SEM, n = 3–4 mice, 15 cells per mouse). D, Sample records showing the response to 100 µM histamine is reduced in the absence of external calcium (left panel, 15 cells in a single adrenal slice, red and blue arrows indicate the rapid and slow phases of the histamine-evoked increase in GCaMP6f signal). Group data quantifying the histamine-evoked increase in GCaMP6f signal (right panel; mean ± SEM, n = 5 mice, 15 cells per mouse). *p < 0.05, **p < 0.01. See also Extended Data Figure 7-1 for more details.
- Figure 8.
Isolation of adrenal macrophages alters the calcium increase in response to GPCR agonists. A, Quantification of the change in GCaMP6f signal in adrenal macrophages in vitro, in response to 100 µM histamine, ACh, UDP, and ATP. In contrast to the response in slices, isolated macrophages do not respond to histamine and ACh. B, Lung macrophages in slices and C, peritoneal macrophages in vitro respond to UDP and ATP but not to histamine or ACh. Top images in A−C show corresponding phase contrast images and RFP fluorescence, respectively. Mean ± SEM, n = 3–5 mice, 15 cells per mouse. Scale bar 50 µm.
Tables
Data structure Type of test Power Figure 5G Non-normal; Kruskal–Wallis ANOVA NS Figure 6B Normal distrib; one-way ANOVA 0.05 Figure 6C Normal distrib; one-way ANOVA 0.01–0.001 Figure 7C, pyrilamine Normal distrib; one-way ANOVA 0.5–0.01 Figure 7C, cimetidine Non-normal; Kruskal–Wallis ANOVA NS Figure 7D, rapid Normal distrib; one-way ANOVA 0.5 Figure 7D, slow Non-normal; Kruskal–Wallis ANOVA NS
Figure 2-1
Macrophages in the adrenal medulla. A-C. Adrenal cryosections from a wild type mouse co-stained for Iba1 and F4/80. D. Distribution of fluorescence intensities for Iba1-ir cells fit with a single Gaussian distribution. E. Distribution of F4/80-ir fluorescent signal. F. Group data showing that Iba1- and F4/80-ir cells in the medulla are co-localized (mean ± SEM, n = 6 mice). Scale bar 50 µm. Download Figure 2-1, TIF file.
Figure 3-1
Macrophages in the adrenal medulla are close to multiple adrenal cell types. A. F4/80-ir macrophages intermingle with TH-ir chromaffin cells (left panel). In the orthogonal view (middle panel), areas of close juxtaposition (arrow heads) are seen. Line scans of fluorescence intensity normalized to peak intensity in each channel (right panel). B. F4/80-ir macrophages and GFAP-ir satellite glial cells. C. Some RFP-ir macrophages in a Lys2-cre-GCaMP6f-tdTomato mouse are close to CD31-ir endothelial cells. D. Some F4/80-ir macrophages are aligned along acTub-ir neuronal processes. In A-D, orthogonal views are taken from the white boxed areas shown in the corresponding left panels. Line scans were measured along the yellow vertical line shown in each Y-Z projection. Scale bar 20 µm (left panels); 5 µm (z axis, middle panels). Download Figure 3-1, TIF file.
Figure 6-1
Controls for specific labelling of immune cells in the mouse adrenal gland. A. RFP-immunoreactive cells in the adrenal gland of a Lys2-cre-GCaMP6f-tdTomato mouse. B. Higher power image showing RFP-ir cells (arrow heads) in the adrenal medulla. C. RFP-ir in the adrenal gland of a wild type mouse. D. Higher power view of the adrenal medulla showing that background levels of fluorescence are low. Thus, fluorescence signal in A,B does not arise from non-specific secondary antibody staining. E. RFP-ir in the adrenal gland of a NPYcre-GCaMP6f-tdTomato mouse. F. RFP-ir cells in the adrenal medulla with the characteristic morphology of chromaffin cells (arrow heads). No cells with a macrophage-like morphology (compare with B) are seen. Scale bar 200 µm (A,C,E); 20 µm (B,D,F). Download Figure 6-1, TIF file.
Figure 6-2
No neuronal expression of GCaMP6f is seen in the adrenal medulla of Lyz2cre-GCaMP6f-tdTomato mice. A. RFP-ir in the adrenal medulla of a Lyz2cre-GCaMP6f-tdTomato mouse. B. MAP2-ir (a neuronal marker) in the same cryosection is present in cells throughout the adrenal medulla (presumably chromaffin cells and intra-adrenal ganglion neurons) and in cell processes in the capsule and cortex (arrow heads). C. Merged image. D-F. In the adrenal medulla, higher power images show that RFP-ir cells (white arrowhead) do not co-localize with MAP2-ir cells (filled arrowhead) or MAP2-ir processes (open arrowhead). Combined with other data (Fig 2–4, 6) this indicates that RFP (and thus GCaMP6f) is expressed in macrophages and not neuronal cells. Scale bar 200 µm (A,B,C); 20 µm (D,E,F). Download Figure 6-2, TIF file.
Figure 7-1
Agonists increase calcium levels in adrenal macrophages. A. Examples of GCaMP6f fluorescence changes in medulla macrophages in response to varying concentrations of ACh. B. Group data quantifying the change in GCaMP6f signal in response to ACh (mean ± SEM, n = 3 - 9 mice, 15 cells per mouse, 100 µM data is also shown in Figure 7A). C. The biphasic increase in GCaMP6f fluorescence evoked by the nicotinic agonist DMPP (50 µM) is blocked by the antagonist mecamylamine (100 µM). Application of histamine (100 µM) shows that the cells remain responsive. D. Group data showing the change in GCaMP6f fluorescence in response to 5 µM and 50 µM DMPP; 50 µM DMPP ± 100 µM mecamylamine; 100 and 1000 µM GABA and glutamate (mean ± SEM, n = 3 - 6 mice, 15 cells per mouse). * P < 0.05, *** P < 0.001. Download Figure 7-1, TIF file.
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