Macrophage markers increase during mammary gland involution in the rat. A: IHC stain for CD68+ cells (arrow) in mammary glands from nulliparous (N) or involution day 6 (Inv D6) rats and quantified per ×400 field/10 fields per gland, n = 4 rats per stage, *P < 0.0001, unpaired t test; scale bars represent 50 microns. B: IHC stain for CSF-1R+ cells (arrow) in mammary glands from nulliparous (N) or involution day 6 (Inv D6) rats and quantified per ×400 field/10 fields per gland, n = 4 rats per stage, *P = 0.0022, unpaired t test; scale bars represent 50 microns. C: Western blot for CD68 using mammary tissue lysates from nulliparous (N) or involution day 6 (Inv D6) rats, n = 6 rats per stage. IgG is used as a loading control. D: Immunoassay results for monocyte chemotactic protein (MCP-1) using rat mammary tissue lysates from nulliparous (N), lactation (L), and involution days 2, 4, and 6 stages, n = 6 rats per stage, *P < 0.03 compared with N, Unpaired t-test.

Involution macrophages display an M2-tumor promotional phenotype. A: IHC stain for M1 macrophage marker iNOS (arrow) and M2 macrophage marker Arginase-1 in involution day 6 (Inv D6) rat mammary tissue; scale bars represent 50 microns. B: Quantification of iNOS, Arginase-1, and CD68 IHC stain in rat mammary tissue per ×400 field/10 fields per gland, n = 4 rats per stage, *P < 0.005 compared with N, unpaired t test. (N indicates nulliparous; P, pregnant; L, lactation; Inv D, involution day; R, regressed). C: Immunoassay results for M2 cytokine IL-4 using rat mammary tissue lysates, n = 6 rats per stage, *P < 0.05 compared with N, unpaired t-test. D: Immunoassay results for M2 cytokine IL-13 using rat mammary tissue lysates, n = 6 rats per stage, *P < 0.05 compared with N, unpaired t test. E: Quantification of mouse macrophage marker F4/80 IHC stain per ×400 field/10 fields per gland, n = 2 for L, Inv D5; n = 3 for Inv D1, D4; n = 4 for N, Inv D2, D3 R; *P < 0.005 compared with N, unpaired t test. F: Quantification of iNOS, Arginase-1 IHC stain in mouse mammary tissue per ×400 field/10 fields per gland, n = 2 for L, Inv D5; n = 3 for Inv D1, D4; n = 4 for N, Inv D2, D3, R; *P < 0.005 compared with N, unpaired t test.

Characterization of postpartum lobule involution in human breast tissue. A: H&E-stained breast biopsy tissues from developmental stages representative of nulliparous (N), pregnant (P), lactation (L), involution (I), and regressed (R); scale bar represents 100 microns. Tissue sections Stained by IHC for: B: CD45, scale bar represents 100 microns. C: CD68, scale bar represents 100 microns. D: Quantification of CD45 staining as described in Materials and Methods section. Number (n) of cases for each developmental stage: nulliparous n = 5, pregnant n = 8, lactation n = 11, involution n = 8, regressed n = 10, * different from involution, P < 0.0001, Tukey–Kramer Multiple Comparisons Test. Black bars indicate average CD45 stain signal values for each developmental stage. E: Quantification of CD68 staining. Number (n) of cases for each developmental stage: nulliparous n = 6, pregnant n = 9, lactation n = 9, involution n = 8, regressed n = 8, * different from involution, P < 0.0001, Tukey–Kramer Multiple Comparisons Test. Black bars indicate average CD68 stain signal values for each developmental stage. F: Serial sections of human involuting breast biopsy tissue stained by IHC for CD45 (monocytes), CK18 (epithelial cells), and CD68 (macrophages) to reveal multiple cell populations present in human breast tissue, arrowheads indicate cells that are CD45- and CD68-positive but CK18-negative, arrows indicate cells that are either CD45-positive or CD68-negative and CK18-negative, scale bar represents 50 microns. G: Dual fluorescent staining of human involuting breast biopsy tissue using E-cadherin (green) to identify mammary epithelial cells and CD68 (red) to identify macrophages (arrow). Nuclei are stained blue with DAPI, the dotted line indicates the acinar lumen, scale bar represents 10 microns. H: Human involuting breast biopsy tissue stained by IHC for Macrophage Mannose Receptor (arrow) and M2 cytokine IL-13 (arrow), scale bar represents 50 microns. I: Involution (Inv) and nulliparous (N) cases from CD45 quantitation separated based on presence (+), or absence (−), of cancer in tissues examined.

Immune cells are recruited to involuting lobules in part by fibrillar collagen. A: Human breast biopsy tissue with both lactating (upper lobules) and involuting (lower lobules) stained by H&E (left panel) and IHC for CD45 (middle panel) and CD68 (right panel) showing CD45 and CD68 specifically elevated in involuting lobules; scale bars represent 200 microns. B: Mammary ECM isolated from nulliparous (N) or involution day 6 (Inv D6) rats and used as chemoattractant for J774 mouse macrophages shows increased chemotaxis toward involution matrix (upper panels); scale bars represent 200 microns. Quantification of transwell filter data, chemotaxis toward involution matrix is increased compared with nulliparous matrix, three wells per condition, four quadrants per well, n = 12, *P = 0.0002, unpaired t test. C: Human lactating/involuting breast biopsy tissue stained by Masson's trichrome method and individual lobules (n = 77 for lactation and n = 70 for involution) from four cases, scale bar represents 50 microns. Arrows indicate areas where collagen staining (blue) is evident. Images quantitated for percent composition of intralobular collagen, *P < 0.0001, unpaired t test.

Intra- and extra-lobular collagen content increases during mammary involution. A: Picro-sirius red stain for collagen in rat mammary tissue; N indicates nulliparous; P, pregnant; L, lactation; Inv D, involution day; R, regressed; scale bars represent 100 microns. B: Whole section images of picro-sirius red collagen stain with brown nuclei. C: Images in B with collagen signal converted to white and noncollagen converted to black. D: Quantification of intralobular collagen (left axis) and interlobular collagen (right axis) (n = 3 rats per reproductive stage, *P < 0.05, **P ≤ 0.005 compared with N, unpaired t test).

Increased MMP activities and proteolyzed collagen seen during mammary involution may account for macrophage chemotaxis observed in vivo. A: Gelatin zymogen assay using mammary tissue lysates from nulliparous (N) or involution days 4 to 6 (I) rats depicts increased MMP-9 and activity MMP-2 in involuting mammary tissue. B: Western blot for collagen-1 using mammary ECM from nulliparous (N) or involution days 4 to 6 (I) rats shows partial proteolysis of collagen I during involution. C: 10% FBS, fibrillar collagen, or nonfibrillar collagen (gelatin) used as chemoattractant for J774 mouse macrophages and quantified per ×50 quadrant; scale bars represent 100 microns, n = 3, *P < 0.002, unpaired t test.