The resting spleen contains a large reservoir of bona fide monocytes. (A) Total number of Ly-6C^high and Ly-6C^low monocytes in the infarcted myocardium and (2 ml) peripheral blood (means ± SEM, n = 9 to 15). Monocytes were identified as CD11b^high, Lin^low, and (F4/80, I–A^b, CD11c)^low. Lin refers to the combination of CD90, B220, CD49b, NK1.1, and Ly-6G monoclonal antibodies. (B) Total monocyte number retrieved from different tissues (means ± SEM, n = 3 to 5). (C) Flow cytometric analysis of blood and splenic Ly-6C^high and Ly-6C^low monocytes, as well as CD11b⁺ macrophages and DCs (Mø/DCs) (n = 5). (D) Cytospin preparations of Ly-6C^high and Ly-6C^low monocytes from blood and spleen. Insets indicate forward (FSC) and side (SSC) scatters identified by flow cytometry (means ± SEM, n=3). (E) Microarray analysis of Ly-6C^high monocytes from blood and spleen. Data depict the average log₂– based intensity of the same probe across four replicates. Also refer to tables S1 and S2. (F) Expression of 45 genes (by quantitative reverse transcription polymerase chain reaction) and 11 proteins (by flow cytometry) for Ly-6C^high and Ly-6C^low monocytes in blood and spleen (n = 4). Also refer to table S3. (G) Ex vivo phagocytosis of beads by Ly-6C^high and Ly-6C^low monocytes and T cells (control) from blood and spleen [mean fluorescent intensity (MFI) ± SEM, n=3]. (H) In vitro differentiation of blood and splenic Ly-6C^high and Ly-6C^low monocytes to macrophages (F4/80^high) and DCs (CD11c^high) in response to macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage CSF (GM-CSF) + interleukin-4 (IL-4), respectively (MFI ± SEM, n = 3 to 9). Data pool at least three independent experiments [(A) and (H)], or are from one experiment representative of at least three independent experiments [(C) and (D)].

Splenic monocytes cluster in the subcapsular red pulp. (A) Histograms depict flow cytometric analysis of GFP fluorescence of splenic leukocytes in Cx₃cr1^gfp/+ mice (n = 3). The pie chart shows the relative proportions of various GFP⁺ populations. Also refer to figs. S2 and S3. (B and C) Multiphoton microscopy micrographs of Cx₃cr1^gfp/+ mice show GFP⁺ Mø/DCs (green) in the marginal zones (B) and GFP⁺ monocytes (green) in the subcapsular red pulp (C). Collagen fibers appear in blue. (D) A single optical section shows GFP⁺ monocytes (green) in the subcapsular red pulp. The dense collagen network (blue) of the splenic capsule indicates the boundary of the organ. (E) An intravital micrograph of the spleen subcapsular red pulp shows GFP⁺ monocytes (green) organized in clusters and topographically distinct from blood vessels (red). All data are from one experiment representative of at least two independent experiments.

Splenic reservoir monocytes emigrate from the subcapsular red pulp and populate inflammatory sites. (A) Splenic sections from mice without MI and 1 day after MI stained with hematoxylin and eosin. (B) Splenic sections stained with CD11b-specific antibodies (red) and 4′,6′-diamidino-2-phenylindole (DAPI) (blue) depict the subcapsular red pulp and the marginal zone from mice without MI and 1 day after MI. (C) Enumeration of CD11b⁺ cells in the subcapsular red pulp and marginal zone (means ± SEM, n = 10 high-power fields). (D) Total number of monocytes in the spleen, blood, or bone marrow (tibia) in control mice (no MI) or 1 day after MI (means ± SEM, n = 6 to 15). (E) Total number of monocytes in the spleen or blood in wild-type (WT) and Ccr2^−/− mice in response to MI (n = 3 to 6). (F) Total number of blood monocytes in splenectomized animals (−Spleen) and sham-operated controls (+Spleen) without MI or 1 day after MI (n = 3 to 6). (G and H) Accumulation of cells in heart in the same groups of mice as measured by flow cytometry [(G), n = 3] or by counts per mg tissue [(H), means ± SEM, n = 9]. (I) Accumulation of monocyte subsets originating exclusively from spleen as measured by flow cytometry. Dot plots (CD45.1 versus CD45.2) of gated monocytes and histograms (Gr-1) of each positive population. (J) CD45.1 versus CD45.2 profile of gated monocytes in a mouse receiving a control transplant (pancreas) and in a mouse not subjected to MI. Also refer to fig. S7. (K) FMT-MRI of in vivo phagocytic and proteolytic activities in infarcts. Fluorochrome concentration is shown in the infarcted area, on the basis of MRI-derived anatomy (n = 6). FI, fluorescence intensity. *P < 0.05; **P < 0.005. Data pool at least three independent experiments (D to F, and H), or are from one experiment representative of two [(I) and (J)] or at least three independent experiments [(A) to (C), and (G)]

Ang II–AT-1 receptor signaling promotes splenic monocyte motility and tissue emigration. (A) Percentage of monocytes lost from the spleen 1 day after MI or Ang II infusion in WT and Atgr1a^−/− mice (means ± SEM, n= 4 to 9). (B) Serum Ang II concentrations in the steady-state (control), and 1 day after MI or infusion of Ang II (n = 6 to 9). (C) Total number of splenic monocytes (Ly-6C^high and Ly-6C^low) in the groups of mice mentioned above. (D) Western blot analysis of monomeric and dimeric forms of the AT-1 receptor on control splenic monocytes in the steady state (control), and 1 day after MI or infusion of Ang II, n = 3. (E) In vitro migration of splenic monocytes in response to Ang II (1 µM) (means ± SEM, n = 6). (F) Intravital microscopy of GFP⁺ cells (green) in the spleen subcapsular red pulp of an Ang II–treated Cx₃cr1^gfp/+ mouse. Images show a splenic monocyte, an Mø or DC, and a patrolling monocyte. Blood is shown in red. Tracks indicate the position of cell centroids at 15-s intervals (time in min:s). (G) (Top) Intravital micrographs of GFP⁺ cells in control mice and 1 day after MI or infusion of Ang II at the initial recording time point. (Bottom) Tracks for all GFP⁺ cells in the field of view and over 1 hour. Some cells entered or exited our imaging area during the recording and thus were followed for a shorter duration. V, vessel; P, parenchyma. (H) Average displacement over time of all GFP⁺ splenic monocytes [means ± SEM, n = 143 (control), 163 (MI), and 125 (Ang II) cells]. (I) Displacement over time of single splenic monocytes (left), splenic macrophages of DCs (middle), and patrolling monocytes (right). (J) Recording of a departing splenic monocyte. Tracks indicate the position of the cell centroid. *P < 0.05, **P < 0.01, ***P < 0.001. Data pool two [(A) to (C), and (E)] or at least three independent experiments [(H) and (I)], or are from one experiment representative of at least three independent experiments [(D), (F), and (G)].