Selective Down-Regulation of Neutrophil Mac-1 in Endotoxemic Hepatic Microcirculation via IL-10

Gustavo Batista Menezes, Woo-Yong Lee, Hong Zhou, Christopher Curtis Matchett Waterhouse, Denise Carmona Cara and Paul Kubes
J Immunol December 1, 2009, 183 (11) 7557-7568; DOI: https://doi.org/10.4049/jimmunol.0901786

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    A local fMLP stimulus induces hepatic neutrophil adhesion. A and B, Three-dimensional reconstruction of liver Z-stacks showing GFP-expressing neutrophils (in green) accumulated in sinusoids (red) due to a local stimulus (A, saline; B, fMLP; ×10 objective). Neutrophil accumulation due to fMLP stimulus is observed within 15 min (C) and progressively increased over 2 h. The majority of neutrophils accumulated adjacent to the filter (D). Neutrophil accumulation was assessed by counting the number of cells adhered in liver sinusoids in each half of the field of view, being one-half immediately adjacent to the filter and the other the distant field of view, labeled as distal (E and F; ×10 objective). ∗, Indicates statistically significant difference compared with controls (saline). p < 0.05.

  • FIGURE 2.
    FIGURE 2.

    fMLP induces neutrophil adhesion, polarization, and emigration. A and B, Lower magnification (×4 objective) showing filter positioning on liver surface. Neutrophil (in green) accumulation in sinusoids due to local stimulus (A, saline; B, fMLP) was mainly observed adjacent to the filter in fMLP-treated mice. fMLP-stimulated neutrophils exhibited polarized cell shape in comparison with controls (C, D, and G), with elongated axis (D, white arrow). E and F, Histological confirmation of neutrophil accumulation by esterase staining (Leder). Neutrophils (F, black arrows) were significantly increased in liver parenchyma following 2 h of fMLP local stimulus (H). ∗, Indicates statistically significant difference compared with controls (saline). p < 0.05.

  • FIGURE 3.
    FIGURE 3.

    Local fMLP stimulus induces Mac-1/ICAM-1-dependent, CD44-independent neutrophil adhesion and crawling in liver sinusoids. Neutrophil adhesion (A), percentage of crawling cells (B), and crawling velocity (C) within sinusoids were assessed following 2 h of fMLP local stimulus. ∗, Indicates statistically significant difference compared with controls (saline filter), and †, in comparison with vehicle-injected fMLP-treated mice (C). p < 0.05.

  • FIGURE 4.
    FIGURE 4.

    Effects of LPS systemic injection and neutrophil accumulation in the liver and brain microvasculature. A–D, Systemic administration of LPS (0.5 mg/Kg) caused integrin-independent, CD44-dependent neutrophil accumulation in liver sinusoids. Neutrophils (in green) accumulated in liver sinusoids (red) following 4 h of i.p. LPS injection, displaying a very low crawling velocity (×10 objective). E–G, Integrin-dependent neutrophil (red) accumulation in brain vessels following i.p. LPS injection. ∗, Indicates statistically significant difference compared with controls (saline injection), and †, in comparison with vehicle-injected, LPS-treated mice (C). p < 0.05.

  • FIGURE 5.
    FIGURE 5.

    A, Integrin-independent, CD44-dependent E. coli induced neutrophil accumulation in liver sinusoids. E. coli (107 CFU) induces neutrophil (green) accumulation 4 h after i.p. administration. This is blocked with anti-CD44 mAb (B and C), but not with anti-Mac-1 mAb (C). ∗, Indicates statistically significant difference compared with saline (UT; E. coli injection alone), and †, in comparison with vehicle-injected mice (UT). p < 0.05.

  • FIGURE 6.
    FIGURE 6.

    LPS decreases Mac-1-dependent neutrophil adhesion in liver. A, Neutrophil adhesion following 2 h of local fMLP or 4 h of i.p. LPS. Treatments were indicated by the + sign. ∗, Indicates statistically significant difference compared with controls (saline injection), and †, in comparison with LPS injection alone. B and C, Flow cytometry analysis of blood and liver neutrophils showing selective down-regulation of Mac-1 in neutrophils that home to the liver. Intracellular staining (D) for Mac-1 and LFA-1 in liver neutrophils revealed that these molecules are not internalized during systemic (LPS) inflammation. ∗, Indicates statistically significant difference compared with controls (saline injection). p < 0.05.

  • FIGURE 7.
    FIGURE 7.

    LPS does not affect ICAM-1 and ICAM-2 expression in liver sinusoids. PE-coupled anti-ICAM-1 (red; 2 μg/mouse) and Alexa Fluor 488-coupled anti-ICAM-2 (green; 15 μg/mouse; Invitrogen) were used to quantify the expression of these adhesion molecules during endotoxemia. The expression of ICAM-1 and ICAM-2 during endotoxemia was assessed by measuring the integrated density of the fluorescence in liver three-dimensional reconstructions using ImageJ software. Iso = isotype controls of mAb; C = control 4 h; LPS = inflammatory protein LPS injection 4 h.

  • FIGURE 8.
    FIGURE 8.

    IL-10 as a candidate for Mac-1 down-regulation in liver neutrophils. Dosage of IL-10, TNF-α, and IL-6 levels in liver tissue (A) and blood (B). Disproportionate amounts of IL-10 are expressed in liver tissue, in contrast to the lower levels in blood. C, Flow cytometry analysis of Mac-1 expression on neutrophils following LPS incubation (100 ng/ml) in the presence and absence of IL-10 (10 ng/ml). ∗, Indicates statistically significant difference compared with incubation with LPS alone. p < 0.05. D, Flow cytometry analysis of Mac-1 adhesivity. Blood samples were incubated with LPS (100 ng/ml), fMLP (0.1 μM), or IL-10 (100 ng/ml) for 4 h. Alexa Fluor 488-coupled fibrinogen was added to the samples at the end of the incubation process for measurement of Mac-1 adhesivity. ∗, Indicates statistically significant difference compared with incubation with saline, and †, compared with fMLP plus LPS incubation. p < 0.05.

  • FIGURE 9.
    FIGURE 9.

    Down-regulation of Mac-1 in liver neutrophils during LPS challenge is not observed in the absence of IL-10. Blood (A) and liver (B) neutrophils from wild-type (WT) or IL-10−/− mice were harvested for flow cytometry analysis of Mac-1 and CD44 expression following LPS i.p. injection. ∗, Indicates statistically significant difference compared with controls (saline injected). C, Effect of Ab blockade of Mac-1 and CD44 during LPS systemic inflammation in IL-10−/− mice. ∗, Indicates statistically significant difference compared with controls (saline injected), and †, in comparison with vehicle (UT)-treated mice. p < 0.05.

  • FIGURE 10.
    FIGURE 10.

    Proposed mechanism for Mac-1 down-regulation in liver neutrophils during endotoxemia. Local liver stimulus (fMLP) displays a conventional integrin-dependent neutrophil adhesion mechanism, which is abolished by LPS systemic inflammation. Kupffer cells, liver sinusoidal endothelial cells, and other liver parenchymal cells may be large sources of IL-10, creating an environment where neutrophils would be exposed to high amounts of this anti-inflammatory cytokine, down-regulating Mac-1 expression on the neutrophil surface, while permitting CD44 to retain neutrophils in the liver sinusoids, where they may enhance bacterial trapping.

Additional Files

  • Data Supplement

    Files in this Data Supplement:

    • Movie 1 (MOV, 1.1 Mb) - Three dimensional reconstruction of liver sinusoids (red) with adhered GFPexpressing neutrophils (green) due to 2 hours fMLP local stimulus. Note that the majority of the cells are adhered inside sinusoids and emigrated adjacent to the filter (right side of the screen).
    • Movie 2 (MOV, 937 Kb) - Time lapse movie (total time: 2 hours) showing GFP-expressing neutrophils (in green) crawling toward fMLP impregnated filter in liver sinusoids (X10 increase objective). Note the directional crawling movement to the right side of the field of view (fMLP filter).
    • Movie 3 (MOV, 968 Kb) - Time lapse movie (total time: 2 hours) of liver sinusoids with GFP-expressing neutrophils exposed to a control filter impregnated with sterile saline (X10 increase objective).
    • Movie 4 (MOV, 1.44 Mb) - Time lapse movie (total time: 1 hour) of GFP-expressing neutrophils (in green) adhered after 4 hours of LPS i.p. injection. Note that the majority of the cells are stopped, with no crawling movement (X10 increase objective).
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