A) Heat map showing the induction of serum cytokines, based on multiplex bead-based analysis, over baseline levels (0 h) in wild-type (WT) mice after PHx. Colored blocks represent changes at each time point, with the color intensity indicating the magnitude of the change, as indicated in the legend to the right. Cytokines are clustered based on similarity of post-PHx induction. Branches to the left of the heat map indicate similar groups; branches closer to the left edge of the heat map connect cytokines with stronger similarities, as compared to those farther out (n = 8–18 mice per time point). B) Same cluster analysis as in (A), but for cytokine induction following sham surgery (n = 5–9 mice per time point).

A) Graph representing the percentage of WT and IL-4-deficient (IL-4^−/−) mice showing morbidity (clinically and/or pathologically) or mortality 24–72 h after PHx. Data are presented as means ± s.e.m. (n =92 [WT] and 51 [IL-4^−/−] mice). *, p = 0.0023, Fisher’s exact test; **, p = 0.0005, Fisher’s exact test. B) Same analysis as in (A), but for WT mice treated with IL-4 antibody (α-IL-4) or control IgG antibody (α-IgG). Only morbidity is shown, as there were no differences in mortality rates (n = 8 [α-IgG] and 9 [α-IL-4] mice). C) Graph representing proliferating hepatocytes, expressed as the number of BrdU-positive nuclei stained per 10 high-powered fields per liver, in healthy WT and morbid IL-4^−/− mice following PHx. Data are presented as means + s.e.m. (n = 4–27 [WT] and 3–4 [IL-4^−/−] mice per time point). *, p = 0.0449, t-test; **, p = 0.0402, t-test; ***, p = 0.0145, t-test. D) Same analysis as in (C), but for WT mice treated with α-IL-4 and showing morbidity, or treated with α-IgG without subsequent morbidity or mortality (n = 4 [α-IgG] and 3 [α-IL-4] mice).

A-B) H&E staining of IL-4^−/− (A) and WT (B) liver sections 48 h after PHx. Necrotic foci are evident in the IL-4^−/− liver (n = 33 [WT] and 24 [IL-4^−/−] mice). C-D) Same analysis as in (A–B), but following sham surgery (n = 7 [WT] and 3 [IL-4^−/−] mice). E–F) Staining for the activated caspase 3 site of cytokeratin 18, indicating areas of apoptotic injury (brown staining) in liver sections from IL-4^−/− (E) and WT (F) mice analyzed 44 h after PHx (n = 3 mice per genotype). Scale bar = 100 μm.

A) Graph showing the number of NKT cells (CD3⁺NK1.1⁺), expressed as a percentage of total CD45⁺ leukocytes, in WT and C3-deficient (C3^−/−) mice before (resected livers, 0 h) and 3 h after PHx (remaining liver tissue from the same mice). Data are presented as means + s.e.m. (n = 19 mice per genotype). *, p < 0.0001, t-test; **, p = 0.0004, two-way ANOVA. B) Graph representing intracellular IL-4 in a portion of the NKT cell populations described in (A), expressed as the increase in mean fluorescence intensity over IgG control antibody staining (n = 9 mice per genotype). *, p = 0.0009, t-test; **, p = 0.0002, t-test.

A) Graph showing the levels of complement cleavage products (C3b/iC3b/C3c) in the plasma of WT and IL4^−/− mice after PHx. Values represent percentages of values for cobra venom factor (CVF)-activated serum, which was considered to cause 100% complement activation. Data are presented as means ± s.e.m. (n = 4–6 [WT] and 4–5 [IL-4^−/−] mice per time point). p < 0.0001, two-way ANOVA. B) Graph of IgM levels in the plasma of WT and IL-4^−/− mice before (0 h) and 2–3 h after PHx. Data are presented as means + s.e.m. (n = 11–16 [WT] and 5–9 [IL-4^−/−] mice per time point). *, p = 0.0022, t-test; **, p = 0.0326, t-test; *** p = 0.0316, t-test. C) Immunofluorescent staining for IgM deposition in liver sections from WT mice before (0 h) or 2 h after PHx. Bright green staining indicates the presence of deposited IgM. Immunofluorescence at 2 h is representative of staining seen at 2 and 3 h after PHx (n = 3–4 mice per time point). D) Same staining as in (C), but for IL-4^−/− mice (n = 3–4 mice per time point). Scale bar = 100 μm. CV, central vein.

A) Graph showing the induction of IL-6 in sera of WT and IL-4^−/− mice after PHx. Baseline (0 h) levels were set to 1, and subsequent levels measured in the same mice were adjusted to show increases over baseline. Data are presented as means ± s.e.m. (n = 5 mice per time point per genotype). p = 0.0195, two-way ANOVA. B) Graph showing the induction of IL-4 in sera of WT and C3^−/− mice after PHx. Data are presented as means ± s.e.m. (n = 13–14 [WT] and 8–9 [C3^−/−] mice per time point). p = 0.0067, two-way ANOVA.

Partial hepatectomy (removal of the two largest lobes of the liver) induces a regenerative response in the remaining liver tissue. A regulatory feedback loop exists in which complement activation, involving the cleavage of C3 and C5 to produce the C3a and C5a anaphylatoxins, leads to the recruitment of NKT cells to the liver (independently of anaphylatoxin activity), and the subsequent production of IL-4 by these cells. IL-4 maintains IgM levels, and increased IgM deposition in the liver induces further complement activation. C3a and C5a are thought to act on macrophages, leading to production of IL-6, a cytokine important for the hepatocyte proliferation and protection that is necessary for proper liver regeneration.