A specific role of integrin Mac-1 in accelerated macrophage efflux to the lymphatics

Abstract

In response to injury, monocytes migrate to the site of inflammation, where they differentiate into macrophages and participate in various biologic processes. However, their fate during the resolution of acute inflammation is not fully understood. Here, we show that inflammatory macrophages do not die locally by apoptosis; rather, they migrate across the peritoneal mesothelium to the lymphatics, through which they further migrate to the lymph nodes and to the blood circulation. Macrophage efflux is enhanced considerably on cell activation, and such accelerated macrophage migration is dependent specifically on integrin Mac-1, and can be blocked by addition of its antagonist. Thus, genetic inactivation of Mac-1 in mice inhibits the accelerated macrophage efflux from the inflammatory site to the lymphatics, but it does not compromise the accumulation of blood monocytes into the inflammatory site. Together, our study demonstrates that Mac-1 is involved specifically in the efflux of activated macrophages to the lymphatics, suggesting that Mac-1 may play an important role in the removal of local inflammatory macrophages and in their subsequent migration to the lymph nodes, a process that is critical to the development of the adaptive immunity.

Figure 1.

Enhanced monocyte accumulation within the peritoneum of Mac-1^-/- mice. WT (□, n = 4) and Mac-1^-/- mice (•, n = 4) were injected intraperitoneally with sterile TG to elicit monocyte infiltration into the peritoneum. At different times after TG injection, peritoneal lavages were performed. The total number of leukocytes in the lavage was determined with a Coulter counter, and the percentage of monocytes/macrophages was determined by FACS analysis using mAb F4/80 and by Hema3 staining of Cytospin smears. Monocyte accumulation in the peritoneal cavity was enhanced in Mac-1^-/- mice (•) compared to the control WT mice (□), whereas the reduction in peritoneal macrophages, possibly due to their spontaneous efflux out of the peritoneum, between day 4 and 8 was similar. The data represent the mean ± SD of 3 mice. WT versus Mac-1^-/-: ^**P < .005; ^*P < .02.

Figure 2.

The infiltrated monocytes/macrophages do not die by apoptosis. WT and Mac-1^-/- macrophages were injected intraperitoneally with TG for 4 days and then were injected intraperitoneally with PBS or LPS. Four hours later, the peritoneal macrophages were assessed for apoptosis by DNA fragmentation (A) and by dual-color FACS analysis using annexin V and 7-AAD (B-C), where annexin V stains apoptotic cells and the cell-impermeable fluorescent dye 7-AAD stains necrotic cells. Additionally, the lavaged WT (B) and Mac-1^-/- (C) macrophages were cultured in vitro for 0 (B, Ci) and 20 (B, Cii) hours in the absence or 4 (Biii) and 20 (Biv, Ciii) hours in the presence of LPS plus the mitogen-activated protein kinase (MAPK) inhibitor SB202190, and then analyzed for apoptosis. The inserts shown are the percentages of different populations. The data shown are representative of 2 independent experiments.

Figure 3.

Mac-1 deficiency inhibits the loss of leukocytes from the peritoneum. (A) The TG-conditioned WT and Mac-1^-/- mice were injected intraperitoneally with either PBS or LPS. Four hours later, the total leukocytes in peritoneum were determined. The data represent the mean ± SD (n = 6) and are representative of 3 independent experiments. (B) WT or Mac-1^-/- peritoneal macrophages were stimulated with different concentrations of LPS. Four hours later, the amount of TNFα produced was determined by ELISA. The data shown are representative of 2 independent experiments. (C-D) The GFP⁺ peritoneal macrophages were injected intraperitoneally into TG-preconditioned WT mice, followed with intraperitoneal injections of PBS or LPS. At different time points, the peritoneal membrane was washed, fixed, and photographed using a fluorescence microscope (objective × 100) (C), and the percentage of GFP⁺ cells in total peritoneal cells was analyzed by FACS (D). The number of adherent GFP⁺ cells on the peritoneal mesothelium was counted manually based on 4 randomly picked fields. The data represent the mean ± SD of 3 mice and are representative of 3 independent experiments. ^*PBS versus LPS, P < .001.

Figure 4.

Macrophage efflux to the lymphatic system and to the blood circulation. (A) The GFP⁺ peritoneal macrophages, obtained from the GFP transgenic mice, were injected into the peritoneum of TG-preconditioned WT mice (5 × 10⁶ GFP⁺ cells per mouse). At different time points after LPS stimulation, the draining lymph nodes were collected and cyrofixed. The presence of the GFP⁺ macrophages within the frozen sections of the fixed lymph nodes was visualized by fluorescence microscopy (objective lens 100 ×). (B) Additionally, leukocytes were recovered from different lymph nodes of the same mice that were treated with PBS (▪) or LPS (○) for 4 hours, and the number of the GFP⁺ cells retrieved was determined by hemocytometer. The data shown are the mean ± SD (n = 3). (C) Total blood samples were taken at different time points from mice in panel A and analyzed for the presence of the GFP⁺ macrophages by FACS analysis. Representative data of 2 independent experiments are shown. ^*PBS versus LPS P < .001.

Figure 5.

Macrophage migration in vitro requires Mac-1. Migration of LPS-stimulated WT (open bars) and Mac-1^-/- (filled bar) peritoneal macrophages was determined using transwell plates with a 5-μm pore insert in the presence or absence of anti-Mac-1 (M1/70), control IgG (IgG) or EDTA. The total number of macrophages within the lower chamber was determined manually by hemocytometer and normalized for the percentage of macrophages based on Hema3-staining of Cytospin smears. The number of migrated WT macrophages is assigned to 100%. The data represents mean ± SD of 3 to 5 independent experiments. ^*P < .001 for WT versus Mac-1^-/- and M1/70 versus IgG.

Figure 6.

Mac-1 is critical to macrophage migration in vivo to the lymphatics. The GFP⁺ WT peritoneal macrophages were injected intraperitoneally into the TG-preconditioned WT mice with and without NIF, followed with intraperitoneal injections of PBS or LPS. (A) The number of GFP⁺ cells within the peritoneum (left) or the draining lymph nodes (right) was determined 4 hours later. The data represent the mean ± SD of 2 independent experiments. (B-D) PKH26-labeled Mac-1^-/- peritoneal macrophages were injected intraperitoneally into the TG-preconditioned Mac-1^-/- mice, followed by intraperitoneal injections of PBS or LPS. The number of fluorescent Mac-1^-/- macrophages within the peritoneum (B), adherent on the peritoneal membrane (C), or within the blood circulation (D) was determined 4 hours later. The data represent the mean ± SD of 2 independent experiments. ^**P < .01; ^*P < .05.

Figure 7.

Macrophage migration from the peritoneum to the lymph nodes. A mixture of PKH67-labeled WT and PKH26-labeled Mac-1^-/- macrophages (i) or PKH67-labeled WT and PKH26-labeled WT macrophages (ii) were separately injected intraperitoneally into the WT mice, followed by intraperitoneal injections of PBS or LPS. Four hours later, the number of the adoptively transferred WT and Mac-1^-/- macrophages within the peritoneum was analyzed by dual-color FACS analysis (A), and their migration into the lymph nodes was visualized by fluorescence microscopy of the corresponding frozen sections (B). The data shown are representative of 2 independent experiments.