Identification and characterization of infiltrating macrophages in acetaminophen-induced liver injury

Abstract

The role of macrophages in the pathogenesis of acetaminophen (APAP)-induced liver injury remains controversial, as it has been demonstrated that these cells display pro-toxicant and hepato-protective functions. This controversy may stem from the heterogeneity and/or plasticity of macrophages and the difficulty in distinguishing and differentially studying subpopulations of macrophages in the liver. In the present study, using flow cytometric analysis and fluorescence-labeled antibodies against specific cell surface macrophage markers, we were able to, for the first time, identify an APAP-induced macrophage (IM) population distinct from resident Kupffer cells. The data demonstrated that the IMs were derived from circulating monocytes that infiltrated the liver following APAP-induced liver injury. The IMs exhibited a phenotype consistent with that of alternatively activated macrophages and demonstrated the ability to phagocytize apoptotic cells and induce apoptosis of neutrophils. Furthermore, in the absence of the IMs, the resolution of hepatic damage following APAP-induced hepatotoxicity was delayed in CCR2(-/-) mice compared with wild-type mice. These findings likely contribute to the role of the IMs in the processes of tissue repair, including counteracting inflammation and promoting angiogenesis. The present study also demonstrated the ability of separating populations of macrophages and delineating distinct functions of each group in future studies of inflammatory disease in the liver and other tissues.

Fig. 1.

Identification of macrophage populations in the liver of mice after APAP challenge. NPCs were isolated from the livers of female and male Balb/cJ and C57Bl/6J mice at 24 h following PBS or APAP challenge. The cells were stained with anti-CD45-FITC, anti-CD11b-PE, and anti-F4/80-APC antibodies, as well as 7-AAD, and analyzed by flow cytometry. 7-AAD^– and CD45⁺ cells were gated, and their expression of CD11b and F4/80 is demonstrated in the dot plots. The percentages of IMs and resident KCs among total 7-AAD^– and CD45⁺ cells are shown. Data shown are representative of six mice per group.

Fig. 2.

Investigation of the time course for the appearance and disappearance of the IMs after APAP challenge. NPCs were isolated from the livers of female C57Bl/6J mice after PBS or at 12 h, 24 h, 48 h, 72 h, and 5 days following APAP challenge. The cells were stained with anti-CD45-FITC, anti-CD11b-PE, and anti-F4/80-APC antibodies, as well as 7-AAD, and analyzed by flow cytometry. 7-AAD^– and CD45⁺ cells were gated, and their expression of CD11b and F4/80 is demonstrated in the dot plots. The percentages of IMs and resident KCs among total 7-AAD^– and CD45⁺ cells are shown. Data shown are representative of three mice per time-point.

Fig. 3.

Expression of various surface markers by the IMs. NPCs were isolated from livers of male Balb/cJ mice at 24 h following APAP challenge. The cells were stained with anti-CD11b-FITC, anti-F4/80-APC, and anti-CD3-, -CD19-, -CD11c-, or -NK1.1-PE antibodies, as well as 7-AAD, and analyzed by flow cytometry. IMs (CD11b^high F4/80^low) were gated, and their expression of CD3, CD19, CD11c, or NK-1.1 is demonstrated in the dot plots. Data shown are representative of two mice.

Fig. 4.

IMs are recruited from the circulation rather than derived from resident KCs. (A) Female Balb/cJ mice were injected (i.v.) with empty liposome (Empty Lipo) or liposome/clodronate (Lipo/Cld) 2 days prior to APAP challenge. Liver NPCs were isolated 24 h after APAP challenge and stained with anti-CD45-FITC, anti-CD11b-PE, and anti-F4/80-APC antibodies, as well as 7-AAD, and analyzed by flow cytometry. 7-AAD^– and CD45⁺ cells were gated, and their expression of CD11b and F4/80 is demonstrated in the dot plots. The percentages of IMs and resident KCs among total 7-AAD^– and CD45⁺ cells are shown. Data shown are representative of four mice per group. (B) Liver NPCs were isolated from female Balb/cJ-nonirradiated (control) and -irradiated mice at 24 h following APAP challenge, stained as described above, and analyzed by flow cytometry. The percentages of IMs and resident KCs among total 7-AAD^– and CD45⁺ cells are shown. Data shown are representative of six mice per group.

Fig. 5.

IMs express genes characteristic of M2. (A) IMs and resident KCs were purified via FACS from the livers of male Balb/cJ mice at 24 h following APAP challenge. RNA was extracted, and the message level of various genes was determined by RT-PCR, including chemokine receptors (CX₃CR1, CCR2) and macrophage markers characteristic of an alternative activation (Ym1, MMP-12, MMP-9, Fizz1, Arg-1, Mgl1, and MMR). (B) CX₃CR1^– and CX₃CR1⁺ subpopulations of the IMs were purified via FACS from the livers of heterozygous C57Bl/6 CX₃CR1^GFP/+ mice at 24 h following APAP challenge. RNA was extracted, and the message level of CCR2 was determined by RT-PCR.

Fig. 6.

In vitro phagocytosis of apoptotic Jurkat T cells and in vivo phagocytosis of red latex beads by the IMs. Viable or apoptotic (UV-irradiated) Jurkat T cells were cocultured for 90 min with IMs (isolated via FACS from male Balb/cJ mice at 24 h following APAP challenge) at a ratio of 3:1 (Jurkat T cell:IM). (A) Representative image of the IMs following coculture with apoptotic Jurkat T cells. Arrows indicate ingested, apoptotic Jurkat T cells and asterisks indicate IMs that have not phagocytized. (B) PI of the IMs following coculture with viable (open bar) or apoptotic (solid bar) Jurkat T cells. The PI was calculated as the number of Jurkat T cells ingested divided by the total number of macrophages counted × 100. *, P < 0.05, compared with + viable cells. Data shown are representative of three separate experiments with one to two replicates per experiment. (C) Liver NPCs were isolated from male Balb/cJ mice (injected i.v. with red latex beads 2 h prior to sacrifice) at 24 h following APAP challenge. The cells were stained with anti-CD11b-PE and anti-F4/80-APC and analyzed by flow cytometry. The IMs and resident KCs were then gated, and their respective red fluorescence is demonstrated in the dot plots. Data shown are representative of two mice.

Fig. 7.

Morphological analysis of hepatic NPCs in WT and CCR2^−/− mice. Liver NPCs were isolated from male Balb/cJ (A) WT and (D) CCR2^−/− mice at 24 h following APAP challenge. The cells were stained with anti-CD45-FITC, anti-CD11b-PE, and anti-F4/80-APC antibodies, as well as 7-AAD, and analyzed by flow cytometry. 7-AAD^– and CD45⁺ cells were gated, and their expression of CD11b and F4/80 is demonstrated in the dot plots. The percentages of IMs and resident KCs among total 7-AAD^–and CD45⁺ cells are shown. Data shown are representative of six mice per group. Liver NPC populations were sorted via FACS at 24 h following APAP challenge. The purified populations, labeled 1 (B) and 2 (C) from the WT mice and 3 (E) and 4 (F) from the CCR2^−/− mice, were cytospun individually and stained with Hema 3 manual staining system. Each population of cells was examined using light microscopy (original magnification, 400×). Arrows indicate PMNs. *, Macrophages. Data shown are representative of two separate experiments.

Fig. 8.

The ability of the IMs to induce apoptosis of PMNs. Thioglycolate-elicited peritoneal exudate PMNs were cultured alone and in contact with or separated from IMs (isolated via MACS selection from KC-depleted male Balb/cJ mice at 24 h following APAP challenge) by transwell inserts at a ratio of 1:3 (PMN:IM). Following 18 h coculture, the supernatant was removed and stained with anti-Gr-1-PE antibody. (A) The total number of Gr-1⁺ cells remaining within each well was calculated by multiplying the percentage of Gr-1⁺ cells by the percentage of total cells gated from FSC versus SSC times the total number of cells collected in the supernatant for analysis [PMN alone (open bar), PMN+IM in direct contact (solid bar), PMN+IM separated via transwell insert (lined bar)]. *, P < 0.05, compared with PMN alone and PMN + IM (Transwell). (B) PMNs were analyzed for early apoptosis using Annexin V-FITC by flow cytometry. Data shown are representative of two separate experiments with four replicates per experiment.

Fig. 9.

Resolution of hepatic damage is delayed in the absence of the IMs. Liver sections were obtained from female Balb/cJ WT and CCR2^−/− mice at 24 h, 48 h, and 72 h after APAP challenge and stained with H&E. Representative liver sections from (A) WT and (B) CCR2^−/− mice at 48 h following APAP challenge (original magnification, 100×). *, Area of necrosis and hemorrhage. (C) Hepatic necrosis was quantified using light microscopy by measuring the total area (mm²) of necrosis within six separate fields per tissue section. *, P < 0.05, compared with WT. Data are the mean area of necrosis among the six fields from three to seven mice per group per time-point.