Challenge with low doses of LPS together with d-galactosamine causes severe liver injury, resulting in lethal shock (low dose LPS-induced shock). We examined the role of LFA-1 in low dose LPS-induced shock. LFA-1−/− mice were more resistant to low dose LPS-induced shock/liver injury than their heterozygous littermates, although serum levels of TNF-α and IL-12 were higher in these mice. C57BL/6 mice were not rescued from lethal effects of LPS by depletion of NK1+ cells, granulocytes, or macrophages, and susceptibility of NKT cell-deficient mice was comparable to that of controls. High numbers of platelets were detected in the liver of LFA-1+/− mice after low dose LPS challenge, whereas liver accumulation of platelets was only marginal in LFA-1−/− mice. Following low dose LPS challenge, serum levels of IL-10 were higher in LFA-1−/− mice than in LFA-1+/− mice, and susceptibility to low dose LPS-induced shock as well as platelet accumulation in the liver of LFA-1−/− mice were markedly increased by IL-10 neutralization. Serum levels of IL-10 in LFA-1+/− mice were only marginally affected by macrophage depletion. However, in LFA-1−/− mice macrophage depletion markedly reduced serum levels of IL-10, and as a corollary, susceptibility of LFA-1−/− mice to low dose LPS-induced shock was markedly elevated despite the fact that TNF-α levels were also diminished. We conclude that LFA-1 participates in LPS-induced lethal shock/liver injury by regulating IL-10 secretion from macrophages and that IL-10 plays a decisive role in resistance to shock/liver injury. Our data point to a novel role of LFA-1 in control of the proinflammatory/anti-inflammatory cytokine network.

Septic shock is mainly attributed to exaggerated proinflammatory cytokine production in response to Gram-negative bacteria and their unique cell wall component, LPS (1). Proinflammatory cytokines, notably TNF-α, are pivotal mediators of septic shock (2, 3, 4). Although mice are relatively resistant to LPS-induced shock, high dose LPS challenge induces pathophysiological reactions, including fever, hypotension, leukocyte infiltration, and inflammation in various organs, resulting in a syndrome resembling septic shock with a high mortality (2). d-galactosamine (d-GalN)3 increases the susceptibility of mice to LPS-induced shock by impairing liver metabolism (5, 6). In contrast to high dose LPS-induced shock which induces a systemic disorder including multiple organ failures (2), liver is a major target organ after challenge with low doses of LPS in conjunction with d-GalN (5, 6). Similarly to high dose LPS-induced shock, TNF-α plays a central role in low dose LPS-induced shock/liver injury (6, 7, 8, 9). In addition to TNF-α, other cytokines, including IFN-γ, participate in low dose LPS-induced shock/liver injury (10).

LFA-1 (CD11a/CD18) belongs to the β2 integrin family and is expressed on the surface of virtually all leukocytes, albeit at different levels (11). In mice, ICAM-1 (CD54) and ICAM-2 (CD102), expressed on leukocytes, epithelial cells, endothelial cells, and fibroblasts, are the ligands for LFA-1 (11). In addition to ICAM(s), LPS is considered a ligand for LFA-1 (12). Interactions of LFA-1/ICAMs promote firm adhesion of leukocytes to vascular endothelium as the initiating event for transmigration of leukocytes into sites of inflammation (11). Infiltration of granulocytes into the liver has been suggested as crucial event in low dose LPS-induced liver damage (13, 14, 15). Although the β2 integrin family member, Mac-1 (CD11b/CD18), has been suggested to participate in low dose LPS-induced shock/liver injury (16, 17), the role of LFA-1 in low dose LPS-induced shock/liver injury remains elusive.

In the present study we examined the role of LFA-1 in low dose LPS-induced shock/liver injury. Our data reveal that LFA-1 expression is a critical prerequisite for low dose LPS-induced shock/liver injury and that IL-10 produced by tissue macrophages plays a decisive role in resistance to low dose LPS-induced damage in the absence of LFA-1. Our data define a novel role for LFA-1 in regulation of the proinflammatory/anti-inflammatory cytokine network, which determines the outcome of harmful inflammatory responses, such as low dose LPS-induced shock.

Breeding pairs of LFA-1−/− mice (18) and CD1d−/− mice were provided by Drs. R. Schmits (University of Saarland, Homburg, Germany) and A. Bendelac (University of Chicago, Chicago, IL), respectively. Breeding pairs of ICAM-1−/−, β2-microglobulin (β2m)−/−, and TCRβ−/− mice were purchased from The Jackson Laboratory (Bar Harbor, ME). These mutants backcrossed onto C57BL/6 (LFA-1−/− and LFA-1+/− mice, 4th generation; CD1d−/− mice, >8th generation; ICAM-1−/−, β2m−/− and TCRβ−/− mice, >15th generation) and C57BL/6 mice were maintained under specific pathogen-free conditions at our animal facilities, and weight- and generation-matched female mice were used at 8–10 wk of age.

mAbs against IFN-γ (R4-6A2 and XMG1.2), IL-12 (p40/p70) (C17.8), IL-12 (p40) (C15.6.7), IL-10 (JES5-2A5), TNF-α (XT22), NK1.1 (PK136), and Ly6G (RB6-8C5) were purified from hybridoma culture supernatants. Anti-IFN-γ mAb (XMG1.2) and anti-IL-12 mAb (C15.6.7) were biotinylated by standard methods. F4/80 mAb (CI:A3-1) was obtained from Serotec (Oxford, U.K.). Anti-CD41 mAb (MWReg30), biotinylated anti-mouse IgG2a mAb (R19-15), FITC-conjugated-anti-mouse IgG2a mAb (R19-15), and FITC-conjugated anti-rat IgG2b mAb (G15-337) were purchased from BD PharMingen (Hamburg, Germany). Cy2-conjugated goat anti-rat IgG and Cy2-conjugated Fab of goat anti-rat IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).

Salmonella typhimurium-derived LPS and d-GalN were purchased from Sigma-Aldrich (Deisenhofen, Germany). Highly purified S. abortus equi-derived LPS was provided by Dr. M. A. Freudenberg (Max-Planck-Institute for Immunobiology, Freiburg, Germany). Mice received (i.v.) various doses of LPS and/or d-GalN (8 mg) dissolved in sterile PBS in a total volume of 200 μl.

For histology, specimens were embedded in Tissue-Tek (Sakura Finetek Europe, Zoeterwoude, The Netherlands), frozen, and cut on a Cryotome (Leica Microsystems, Bensheim, Germany). Sections (3–5 μm) were air-dried, fixed with acetone, rehydrated, and stained with H&E (Merck, Haar, Germany). For TUNEL assay, formalin-fixed specimens were infiltrated with 20% sucrose (Merck) in PBS, placed in Tissue-Tek, frozen, and cut on a Cryotome. Sections (3–5 μm) were partially digested with 20 μg/ml proteinase K (Sigma-Aldrich) at room temperature for 15 min and subjected to the TUNEL reaction (kit from Roche, Mannheim, Germany) following the manufacturer’s instructions. TUNEL-positive cells are bright green due to the incorporation of FITC-labeled nucleotides.

Serum levels of IFN-γ and IL-12 (p40) were determined by ELISA as described previously (19). In brief, serum samples were incubated in immunoassay plates (Nunc, Copenhagen, Denmark) precoated with anti-IFN-γ mAb (R4-6A2) or anti-IL-12 (p40/p70) mAb (C17.8), respectively. After washing, plates were incubated with biotinylated anti-IFN-γ mAb (XMG1.2) or biotinylated anti-IL-12 (p40) mAb (C15.6.7), respectively, followed by streptavidin-conjugated alkaline phosphatase (Dianova, Hamburg, Germany) and the chromogen p-nitrophenyl phosphate (Sigma-Aldrich). The cytokine concentration in each sample was determined using serially diluted mouse rIFN-γ (R&D Systems, Wiesbaden, Germany) or mouse rIL-12 (Genzyme, Alzenau, Germany). Serum levels of TNF-α, IL-12 (p70), and IL-10 were assayed using the Quantikine M kit (R&D systems) following the manufacturer’s instruction.

Hepatic leukocytes were prepared as described previously (20). Splenocytes were prepared by standard methods. Blood samples were obtained from the axillary vein, and leukocytes were collected after hypotonic hemolysis.

The frequencies of IL-12 (p40)-producing cells were determined as described previously (21) with slight modifications. Briefly, appropriate dilutions of cells were cultured overnight in ELISPOT plates (Millipore, Eschborn, Germany) precoated with anti-IL-12 (p40/p70) mAb (C17.8). Plates were then washed and incubated with biotinylated anti-IL-12 (p40) mAb (C15.6.7) at 37°C for 2 h. For developing spots, streptavidin-conjugated alkaline phosphatase (Dianova) and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium tablets (Sigma-Aldrich) were used. The frequencies of IL-10- or TNF-α-producing cells were determined by ELISPOT assay using the mouse IL-10 or TNF-α measurement kit (R&D Systems), respectively, following the manufacturer’s instruction.

Multilamellar liposome-encapsulated dichloromethylene bisphosphonate (Cl2MBP-L) was prepared as described previously (22). Cl2MBP was a gift from Roche. To deplete tissue macrophages, mice were injected i.v. with 200 μl of Cl2MBP-L (containing 1 mg of Cl2MBP) suspended in PBS 2 days before LPS challenge as described previously (22). As a control, mice were treated i.v. with 200 μl of liposome-encapsulated PBS (PBS-L). For depletion of granulocytes, mice were treated i.p. with 150 μg of anti-Ly6G mAb 1 day before LPS challenge as described previously (23). To deplete NK1+ cells, mice were treated i.p. with 500 μg of anti-NK1.1 mAb 2 days before LPS challenge. Depletion of Kupffer cells (>99%), splenic macrophages (>95%), granulocytes (>95%), and NK1+ cells (>98%) in the liver was verified by immunohistochemistry and/or flow cytometry. For neutralization of endogenous IL-10 or TNF-α, mice were treated i.p. with 500 μg of anti-IL-10 mAb or anti-TNF-α mAb, respectively, 2 h before LPS challenge. Isotype-matched mAbs purified by the same procedure as that described for specific mAbs or PBS used for mAb purification were used as a control, and it was verified that the reaction produced by mAb treatment is not caused by LPS contamination of mAbs or PBS.

For immunohistochemistry, specimens were embedded in Tissue-Tek, frozen, and cut on a Cryotome. Sections (3–5 μm) were air-dried, fixed with acetone, rehydrated, and treated with blocking buffer (PBS containing 1% BSA and 0.05% Tween 20) for 15 min. Sections were then incubated with mAbs against Ly6G, CD41, or F4/80 diluted in blocking buffer at a concentration of 10 μg/ml at 37°C for 30 min. After washing with PBS, the sections were incubated with Cy2-conjugated goat anti-rat IgG. For immunodetection in tissues of animals pretreated with anti-IL-10 mAb, primary Abs were labeled with Cy2-conjugated Fab of anti-rat IgG and then allowed to react with tissue sections.

Statistical significance was determined by log-rank test (survival time) or post hoc multiple range test (serum cytokine levels and frequencies of cytokine-producing cells). A value of p < 0.05 was regarded as significant.

We compared LFA-1−/−, LFA-1+/−, ICAM-1−/−, and C57BL/6 mice for susceptibility to low dose LPS-induced shock. Mice were challenged with different doses of LPS together with d-GalN, and survival times were monitored thereafter. All C57BL/6 mice succumbed to challenge with 0.01 μg of LPS within 8 h (Table I). All LFA-1+/− and ICAM-1−/− mice also succumbed to 0.01-μg LPS challenge, although the survival times were slightly prolonged (mean survival time, 10 h). In contrast, LFA-1−/− mice resisted up to 1000 times higher LPS doses compared with LFA-1+/−, ICAM-1−/−, and C57BL/6 mice (Table I). Similar results were obtained with highly purified S. abortus equi-derived LPS (data not shown), verifying that the reaction was induced by LPS, but not other contaminating components such as lipoprotein, which can be present in commercially obtained LPS (24). Thus, LFA-1−/− mice were highly resistant to low dose LPS-induced lethal shock.

Table I.

Mortality rates of various mouse strains following challenge with d-GalN plus low dose LPS

d-GalN (mg)LPS (μg)Mortality Rate (%)a
LFA-1−/−ICAM-1−/−LFA-1+/−C57BL/6TCRβ−/−β2m−/−CD1d−/−
0.001 100b 
0.01 0c 100d 100d 100 100 100 100 
0.1 0c 100 100 100 100 100 100 
0c 100 100 100 100 100 100 
10 100 100 100 100 100 100 100 
10 
d-GalN (mg)LPS (μg)Mortality Rate (%)a
LFA-1−/−ICAM-1−/−LFA-1+/−C57BL/6TCRβ−/−β2m−/−CD1d−/−
0.001 100b 
0.01 0c 100d 100d 100 100 100 100 
0.1 0c 100 100 100 100 100 100 
0c 100 100 100 100 100 100 
10 100 100 100 100 100 100 100 
10 
a

Groups of five mice were challenged i.v. with various doses of LPS and/or d-GalN (8 mg) and mortality rates were scored on day 3. Determinations were performed twice with comparable results.

b

p < 0.01, β2m−/− vs C57BL/6.

c

p < 0.001, LFA-1−/− vs LFA-1+/−.

d

p < 0.05, ICAM-1−/− vs C57BL/6; LFA-1+/− vs C57BL/6.

Mice were challenged with LPS (1 μg) together with d-GalN (8 mg), liver tissues were prepared at different time points thereafter, and histopathology was analyzed by H&E and TUNEL staining. No measurable alterations were found in liver sections from C57BL/6 mice by 4.5 h after LPS challenge (data not shown). Yet, liver sections from C57BL/6 mice at 6 h after LPS challenge displayed characteristic features of hepatocyte destruction, including pyknosis and karyorrhexis of hepatocyte nuclei, as well as extensive parenchymal hemorrhage, whereas areas of necrosis were scarcely detected (Fig. 1,A). In LFA-1+/− mice, hepatocyte destruction was marginal at 6 h after challenge, but severe hepatocyte destruction was found at 8 h, comparable to that in C57BL/6 mice at 6 h after challenge (Fig. 1,A). Results in ICAM-1−/− mice were similar to those in LFA-1+/− mice (data not shown). In contrast, only marginal signs of liver injury were detected in LFA-1−/− mice at 8 h after LPS challenge (Fig. 1,A), and no exacerbation was found thereafter (data not shown). High numbers of parenchymal cell nuclei were positively stained in C57BL/6 mice at 6 h after LPS challenge by the TUNEL method, suggesting apoptotic death of these cells (Fig. 1,B). Marginal signs of apoptosis were detected in parenchymal cells from LFA-1+/− mice at 6 h after LPS challenge, but high numbers of parenchymal cell nuclei were positively stained at 8 h (Fig. 1,B), and this was also true in ICAM-1−/− mice (data not shown). In contrast, only marginal signs of apoptosis were found in the liver of LFA-1−/− mice at 8 h after LPS challenge (Fig. 1 B), and no exacerbation occurred at later time points (data not shown). LPS (1 μg) or d-GalN (8 mg) alone did not cause any alterations in livers, and treatment with both LPS (1 μg) and d-GalN (8 mg) did not affect other organs, including spleen (data not shown). Thus, LFA-1−/− mice were highly resistant to low dose LPS-induced liver injury/hepatocyte apoptosis.

FIGURE 1.

Liver injury in LFA-1−/−, LFA-1+/−, and C57BL/6 mice following challenge with d-GalN plus low dose LPS. Mice were injected i.v. with LPS (1 μg) together with d-GalN (8 mg), and livers were collected at 6 and/or 8 h. Livers were cryosectioned and stained with H&E (A), TUNEL (B), anti-Ly6G mAb (C), or F4/80 mAb (D). Bar = 50 μm.

FIGURE 1.

Liver injury in LFA-1−/−, LFA-1+/−, and C57BL/6 mice following challenge with d-GalN plus low dose LPS. Mice were injected i.v. with LPS (1 μg) together with d-GalN (8 mg), and livers were collected at 6 and/or 8 h. Livers were cryosectioned and stained with H&E (A), TUNEL (B), anti-Ly6G mAb (C), or F4/80 mAb (D). Bar = 50 μm.

Close modal

TNF-α plays a central role in low dose LPS-induced shock (6, 7, 8, 9). We therefore compared serum levels of TNF-α in LFA-1−/−, LFA-1+/−, and C57BL/6 mice following challenge with LPS together with d-GalN. Since IFN-γ is involved in low dose LPS-induced shock (10), and this cytokine is induced by IL-12 (25), serum levels of these cytokines were analyzed as well. Serum levels of TNF-α, IL-12 (p40), IL-12 (p70), and IFN-γ in C57BL/6 mice peaked at 1, 4, 4, and 6 h, respectively, after LPS challenge (data not shown). We therefore compared cytokine levels in the sera of LFA-1−/−, LFA-1+/−, and C57BL/6 mice at the respective time points. Serum levels of TNF-α and IL-12 (p40) were significantly higher in LFA-1−/− mice than in LFA-1+/− and C57BL/6 mice after challenge (Fig. 2,A). Serum levels of IL-12 (p70) were also significantly higher in LFA-1−/− mice than in LFA-1+/− and C57BL/6 mice, although the levels were markedly lower than IL-12 (p40) levels (Fig. 2 A). In contrast, serum levels of IFN-γ slightly increased in these mouse strains following challenge, and the levels were comparable among these mouse strains (data not shown). Thus, serum levels of TNF-α and IL-12 following low dose LPS challenge were elevated in LFA-1−/− mice, although these mice resisted low dose LPS-induced shock.

FIGURE 2.

Levels of TNF-α, IL-12, and IL-10 in sera, and frequencies of these cytokine-producing cells in peripheral blood, liver, and spleen of LFA-1−/−, LFA-1+/−, and/or C57BL/6 mice following challenge with d-GalN plus low dose LPS. Mice were injected i.v. with LPS (1 μg) together with d-GalN (8 mg). A, Sera were collected at 1 h (for TNF-α), 4 h (for IL-12 (p40, p70)), and 2 h (for IL-10) after challenge, and serum levels of these cytokines were determined by ELISA. Each marker represents serum levels of cytokine in an individual animal. The horizontal lines indicate mean serum levels. Before LPS challenge, the above cytokines were virtually undetectable in sera. B, Peripheral blood, liver, and spleen were collected at 20 min (for TNF-α), 2.5 h (for IL-12 (p40, p70)), and 1 h (for IL-10) after challenge, and PBL, hepatic leukocytes, and splenocytes were prepared thereafter. The frequencies of these cytokine-producing cells were determined by ELISPOT assay. Data represent the mean of five mice per group. SFC, spot-forming cells. ∗, p < 0.01, LFA-1−/− vs LFA-1+/−.

FIGURE 2.

Levels of TNF-α, IL-12, and IL-10 in sera, and frequencies of these cytokine-producing cells in peripheral blood, liver, and spleen of LFA-1−/−, LFA-1+/−, and/or C57BL/6 mice following challenge with d-GalN plus low dose LPS. Mice were injected i.v. with LPS (1 μg) together with d-GalN (8 mg). A, Sera were collected at 1 h (for TNF-α), 4 h (for IL-12 (p40, p70)), and 2 h (for IL-10) after challenge, and serum levels of these cytokines were determined by ELISA. Each marker represents serum levels of cytokine in an individual animal. The horizontal lines indicate mean serum levels. Before LPS challenge, the above cytokines were virtually undetectable in sera. B, Peripheral blood, liver, and spleen were collected at 20 min (for TNF-α), 2.5 h (for IL-12 (p40, p70)), and 1 h (for IL-10) after challenge, and PBL, hepatic leukocytes, and splenocytes were prepared thereafter. The frequencies of these cytokine-producing cells were determined by ELISPOT assay. Data represent the mean of five mice per group. SFC, spot-forming cells. ∗, p < 0.01, LFA-1−/− vs LFA-1+/−.

Close modal

We compared numbers of TNF-α- and IL-12-producing cells in blood, liver, and spleen of LFA-1−/− and LFA-1+/− mice following low dose LPS challenge. Before challenge, TNF-α and IL-12 (p40) producers were low in both mouse strains, and no significant difference was found in these mice (data not shown). The frequencies of TNF-α and IL-12 (p40) producers among hepatic leukocytes and splenocytes were markedly increased in both mouse strains following low dose LPS challenge, and they were significantly higher in LFA-1−/− mice than in LFA-1+/− mice (Fig. 2,B). Whereas high frequencies of TNF-α-producing cells were detected among peripheral blood leukocytes in both LFA-1−/− and LFA-1+/− mice following LPS challenge, those of IL-12 (p40) producers were low in both mouse strains (Fig. 2 B). The frequencies of TNF-α- and IL-12 (p40)-producing cells were virtually comparable among peripheral blood leukocytes in LFA-1−/− and LFA-1+/− mice. Thus, higher levels of TNF-α and IL-12 in LFA-1−/− mice following low dose LPS challenge correlate with higher numbers of the respective cytokine-producing cells in liver and spleen.

Vα14+NKT cells have been shown to play a crucial role in the induction of liver injury using various experimental systems (26, 27, 28, 29, 30). We and others have previously shown that the numbers of Vα14+NKT cells are markedly reduced in the liver of LFA-1−/− mice (31, 32). We therefore wondered whether increased resistance of LFA-1−/− mice to low dose LPS-induced shock was due to the reduction of Vα14+NKT cells in the liver. To clarify this issue, the susceptibilities of TCRβ−/−, β2m−/−, and CD1d−/− mice, all of which are devoid of Vα14+NKT cells (20, 33, 34, 35, 36), to low dose LPS-induced shock were compared. The susceptibilities of TCRβ−/− and CD1d−/− mice to low dose LPS-induced shock were comparable to that of C57BL/6 mice, and susceptibility was slightly increased in β2m−/− mice (Table I). Consistent with this, C57BL/6 mice were not rescued from the lethal effects of LPS by in vivo depletion of NK1+ cells (Table II). These results not only exclude the possibility that increased resistance of LFA-1−/− mice to low dose LPS-induced shock is caused by reduced numbers of Vα14+NKT cells in the liver, but also suggest that neither conventional T cells nor NK cells are required in low dose LPS-induced shock.

Table II.

Influence of in vivo depletion of NK11 cells, granulocytes, or Kupffer cells on mortality rates of C57BL/6 and/or LFA-1−/− mice following challenge with d-GalN plus low dose LPS

d-GalN (mg)LPS (μg)Mortality Rate (%)a
C57BL/6LFA-1−/−
Anti-NK1.1 mAbAnti-Ly6G mAbPBSC12MBP-LPBS-LPBSC12MBP-LPBS-L
0.001 0 (3) 0 (3) 0 (3) 0 (3) 0 (3) 0 (5) 0 (6) 0 (3) 
0.01 78 (9) 100 (5) 100 (10) 20 (5)b 100 (5) 0 (5) 50 (8)b 0 (8) 
0.1 100 (5) 100 (3) 100 (6) 100 (3) 100 (3) 0 (5) 100 (6)c 0 (6) 
100 (3) ND 100 (3) 100 (3) 100 (3) 0 (5) 100 (6)c 0 (6) 
d-GalN (mg)LPS (μg)Mortality Rate (%)a
C57BL/6LFA-1−/−
Anti-NK1.1 mAbAnti-Ly6G mAbPBSC12MBP-LPBS-LPBSC12MBP-LPBS-L
0.001 0 (3) 0 (3) 0 (3) 0 (3) 0 (3) 0 (5) 0 (6) 0 (3) 
0.01 78 (9) 100 (5) 100 (10) 20 (5)b 100 (5) 0 (5) 50 (8)b 0 (8) 
0.1 100 (5) 100 (3) 100 (6) 100 (3) 100 (3) 0 (5) 100 (6)c 0 (6) 
100 (3) ND 100 (3) 100 (3) 100 (3) 0 (5) 100 (6)c 0 (6) 
a

Mice were treated i.p. with anti-NK1.1 mAb (500 μg), anti-Ly6G mAb (150 μg), C12MBP-L (200 μl), or PBS-L (200 μl) 2, 1, 2, or 2 days, respectively, before challenge with various doses of LPS and d-GalN (8 mg), and mortality rates were scored on day 3 after challenge. Numbers in parentheses represent the numbers of mice examined. Since no significant difference was found among mouse IgG2a-treated (isotype-matched mAb for anti-NK1.1 mAb), rat IgG2a-treated (isotype-matched mAb for anti-Ly6G mAb), and PBS-treated groups in another experiment, PBS was used as a control.

b

p < 0.05, C12MBP-L-treated group vs PBS-L-treated group.

c

p < 0.001, C12MBP-L-treated group vs PBS-L-treated group.

Since granulocytes have been suggested to participate in low dose LPS-induced shock (13, 14, 15), we compared numbers of granulocytes in the liver following challenge with LPS and d-GalN between LFA-1−/− and LFA-1+/− mice. Comparable numbers of Ly6G+ cells were detected in the liver of LFA-1−/− and LFA-1+/− mice before LPS challenge (Fig. 1,C). Although numbers of Ly6G+ cells were markedly increased in the liver of both mouse strains following LPS challenge, they were slightly higher in LFA-1−/− mice than in heterozygous littermates (Fig. 1,C). Susceptibility of C57BL/6 mice to low dose LPS-induced shock was virtually unchanged by in vivo depletion of granulocytes (Table II). These results exclude a role of granulocytes in low dose LPS-induced shock and indicate a dispensable role of LFA-1 in the infiltration of granulocytes in the liver.

Because macrophages have been considered to play a major role in low dose LPS-induced shock (37, 38, 39), we compared numbers of Kupffer cells following challenge with LPS and d-GalN between LFA-1−/− and LFA-1+/− mice. Comparable numbers of F4/80+ cells were detected in the liver of homozygous and heterozygous mouse mutants before LPS challenge, which were equally diminished in both mouse strains following LPS challenge (Fig. 1,D). The susceptibility of C57BL/6 mice to low dose LPS-induced shock was slightly, although significantly, reduced by in vivo depletion of tissue macrophages (Table II). These results argue against a critical role of Kupffer cells in low dose LPS-induced shock, at least in the presence of LFA-1.

Blood coagulation is a major event at the terminal stage of endotoxemia (40, 41). Because LFA-1 is expressed on platelets (42), we compared numbers of platelets in the liver following challenge with LPS and d-GalN between LFA-1−/− and LFA-1+/− mice. Immunohistochemical analysis of liver sections revealed an early prominent accumulation of platelets in the liver of LFA-1+/− mice following LPS challenge (Fig. 3). In contrast, the numbers of platelets were only marginally increased in LFA-1−/− mice following LPS challenge (Fig. 3). Thus, in the absence of LFA-1, the numbers of platelets were markedly reduced in livers following low dose LPS challenge.

FIGURE 3.

Accumulation of platelets in the liver of LFA-1−/− and LFA-1+/− mice following challenge with d-GalN plus low dose LPS and the influence of endogenous IL-10 neutralization. Mice were left untreated or were treated i.p. with anti-IL-10 mAb (500 μg) and 2 h later injected i.v. with LPS (1 μg) together with d-GalN (8 mg). Livers were collected at 8 h after LPS challenge, fixed in 4% paraformaldehyde in PBS, sectioned, and stained with anti-CD41 mAb. Bar = 50 μm.

FIGURE 3.

Accumulation of platelets in the liver of LFA-1−/− and LFA-1+/− mice following challenge with d-GalN plus low dose LPS and the influence of endogenous IL-10 neutralization. Mice were left untreated or were treated i.p. with anti-IL-10 mAb (500 μg) and 2 h later injected i.v. with LPS (1 μg) together with d-GalN (8 mg). Livers were collected at 8 h after LPS challenge, fixed in 4% paraformaldehyde in PBS, sectioned, and stained with anti-CD41 mAb. Bar = 50 μm.

Close modal

IL-10 plays a protective role against both high dose (43, 44, 45) and low dose LPS-induced shock (46, 47). To determine whether IL-10 participates in the increased resistance of LFA-1−/− mice to low dose LPS-induced shock, serum levels of IL-10 were compared in LFA-1−/−, LFA-1+/−, and C57BL/6 mice following challenge with LPS and d-GalN. Since serum levels of IL-10 peaked at 2 h after LPS challenge in C57BL/6 mice (data not shown), serum levels of IL-10 were compared at this time point. In these mouse strains, IL-10 was undetectable in sera before LPS challenge. After LPS challenge, serum levels of IL-10 were significantly higher in the absence of LFA-1 (Fig. 2 A).

We compared numbers of IL-10-producing cells in blood, liver, and spleen of LFA-1−/− and LFA-1+/− mice following low dose LPS challenge. IL-10 producers were virtually undetectable in both mouse strains before challenge (data not shown). In contrast, the frequencies of IL-10 producers among peripheral blood leukocytes, hepatic leukocytes, and splenocytes were markedly increased in both mouse strains following low dose LPS challenge, and they were significantly higher in LFA-1−/− mice than in LFA-1+/− mice (Fig. 2 B). Thus, higher levels of IL-10 in LFA-1−/− mice following low dose LPS challenge correlate with higher numbers of IL-10 producers.

The susceptibility of LFA-1+/− and C57BL/6 mice to low dose LPS-induced shock was virtually unchanged by endogenous IL-10 neutralization (Table III). In contrast, the susceptibility of LFA-1−/− mice to low dose LPS-induced shock was increased up to 1000-fold (Table III), and the numbers of platelets in the liver were elevated (Fig. 3). These results indicate a direct relationship between resistance and elevated IL-10 levels in low dose LPS-induced shock in the absence of LFA-1.

Table III.

Influence of endogenous IL-10 neutralization on mortality rates of LFA-1−/−, LFA-1+/−, and C57BL/6 mice following challenge with d-GalN plus low dose LPS

d-GalN (mg)LPS (μg)Mortality Rate (%)a
C57BL/6LFA-1−/−LFA-1+/−
PBSAnti-IL-10 mAbPBSAnti-IL-10 mAbPBSAnti-IL-10 mAb
0.001 50 
0.01 100 100 100b 100 100 
0.1 100 100 100b 100 ND 
100 100 ND 100 ND 
10 ND ND 100 ND 100 ND 
d-GalN (mg)LPS (μg)Mortality Rate (%)a
C57BL/6LFA-1−/−LFA-1+/−
PBSAnti-IL-10 mAbPBSAnti-IL-10 mAbPBSAnti-IL-10 mAb
0.001 50 
0.01 100 100 100b 100 100 
0.1 100 100 100b 100 ND 
100 100 ND 100 ND 
10 ND ND 100 ND 100 ND 
a

Groups of four mice were treated i.p. with anti-IL-10 mAb (500 μg) or PBS 2 h before challenge with various doses of LPS and d-GalN (8 mg), and mortality rates were scored on day 3 after challenge. Since no significant difference was found between rat IgG1-treated (isotype-matched mAb for anti-IL-10 mAb) and PBS-treated groups in another experiment, PBS was used as a control. ND, not determined.

b

p < 0.05, anti-IL-10 mAb-treated group vs PBS-treated group.

TNF-α-induced release of IL-10 during endotoxemia has been reported (48). Because not only IL-10, but also TNF-α, serum concentrations were elevated in LFA-1−/− mice following low dose LPS challenge, we assessed the influence of TNF-α neutralization on IL-10. Serum levels of IL-10 were virtually unaffected by neutralization of endogenous TNF-α (Fig. 4). The in vivo efficacy of anti-TNF-α mAb was verified in a parallel group of C57BL/6 mice by assessing susceptibility to LPS-induced shock. Neutralization of TNF-α resulted in a >1000-fold increase in the resistance of these mice to low dose LPS-induced shock.

FIGURE 4.

Influence of endogenous TNF-α neutralization or granulocyte depletion on serum levels of IL-10 in LFA-1−/− mice following challenge with d-GalN plus low dose LPS. Mice were treated i.p. with anti-TNF-α mAb (500 μg) or anti-Ly6G mAb (150 μg) 2 h or 1 day, respectively, before challenge with LPS (1 μg) together with d-GalN (8 mg), and sera were collected at 2 h after challenge. Serum levels of IL-10 were determined by ELISA. Each symbol represents serum levels of IL-10 in an individual animal. The horizontal lines indicate mean serum levels. Since no significant difference was found in the rat IgG1-treated group (isotype-matched mAb for anti-TNF-α mAb), the rat IgG2b-treated group (isotype-matched mAb for anti-Ly6G mAb), and the PBS-treated group in another experiment, PBS was used as a control. Before LPS challenge, the above cytokines were virtually undetectable in sera.

FIGURE 4.

Influence of endogenous TNF-α neutralization or granulocyte depletion on serum levels of IL-10 in LFA-1−/− mice following challenge with d-GalN plus low dose LPS. Mice were treated i.p. with anti-TNF-α mAb (500 μg) or anti-Ly6G mAb (150 μg) 2 h or 1 day, respectively, before challenge with LPS (1 μg) together with d-GalN (8 mg), and sera were collected at 2 h after challenge. Serum levels of IL-10 were determined by ELISA. Each symbol represents serum levels of IL-10 in an individual animal. The horizontal lines indicate mean serum levels. Since no significant difference was found in the rat IgG1-treated group (isotype-matched mAb for anti-TNF-α mAb), the rat IgG2b-treated group (isotype-matched mAb for anti-Ly6G mAb), and the PBS-treated group in another experiment, PBS was used as a control. Before LPS challenge, the above cytokines were virtually undetectable in sera.

Close modal

Recently, LPS was found to induce IL-10 secretion by monocytes in the presence of apoptotic granulocytes (49). Because the numbers of granulocytes infiltrating the liver were higher in LFA-1−/− mice than in LFA-1+/− mice, we examined the influence of granulocyte depletion on serum levels of IL-10 following challenge with LPS and d-GalN. In vivo depletion of granulocytes did not induce IL-10 production by itself (data not shown), and serum levels of IL-10 in LFA-1−/− mice following LPS challenge were virtually unaffected by in vivo depletion of granulocytes (Fig. 4). These results argue against participation of TNF-α and granulocytes in the control of IL-10 secretion in LFA-1−/− mice.

Because macrophages secrete IL-10 in response to LPS (50, 51), we assessed the influence of tissue macrophage depletion on serum levels of IL-10 following low dose LPS challenge. Tissue macrophage depletion did not result in measurable alterations in serum levels of IL-10 in LFA-1+/− mice (Fig. 5). In contrast, in LFA-1−/− mice serum levels of IL-10 were significantly diminished after tissue macrophage depletion (Fig. 5). Note that serum levels of IL-10 in tissue macrophage-depleted LFA-1−/− mice were comparable to those in LFA-1+/− and C57BL/6 mice (see Fig. 2 A). These results suggest that tissue macrophages are responsible for higher levels of IL-10 in LFA-1−/− mice.

FIGURE 5.

Influence of tissue macrophage depletion on serum levels of IL-10, TNF-α, and IL-12 in LFA-1−/− and/or LFA-1+/− mice following challenge with d-GalN plus low dose LPS. Mice were treated i.p. with 200 μl of CL2 MBP-L or PBS 2 days before challenge with LPS (1 μg) together with d-GalN (8 mg), and sera were collected at 2 h (for IL-10), 1 h (for TNF-α), or 4 h (for IL-12 (p70)) after challenge. Serum levels of these cytokines were determined by ELISA. Each symbol represents serum levels of each cytokine in an individual animal. The horizontal lines indicate mean serum levels. Before LPS challenge, the above cytokines were virtually undetectable in sera.

FIGURE 5.

Influence of tissue macrophage depletion on serum levels of IL-10, TNF-α, and IL-12 in LFA-1−/− and/or LFA-1+/− mice following challenge with d-GalN plus low dose LPS. Mice were treated i.p. with 200 μl of CL2 MBP-L or PBS 2 days before challenge with LPS (1 μg) together with d-GalN (8 mg), and sera were collected at 2 h (for IL-10), 1 h (for TNF-α), or 4 h (for IL-12 (p70)) after challenge. Serum levels of these cytokines were determined by ELISA. Each symbol represents serum levels of each cytokine in an individual animal. The horizontal lines indicate mean serum levels. Before LPS challenge, the above cytokines were virtually undetectable in sera.

Close modal

We examined the influence of tissue macrophage depletion on the susceptibility of LFA-1−/− mice to low dose LPS-induced shock. In contrast to C57BL/6 mice, the susceptibility of LFA-1−/− mice was >100-fold increased by tissue macrophage depletion (Table II). These results suggest that tissue macrophages have a protective role in LFA-1−/− mice to low dose LPS-induced shock. Tissue macrophage depletion diminished serum levels of TNF-α following LPS challenge (Fig. 5). In contrast, serum levels of IL-12 (p70) were increased by tissue macrophage depletion. Note that the levels of TNF-α in tissue macrophage-depleted LFA-1−/− mice were comparable to those in LFA-1+/− and C57BL/6 mice (see Fig. 2 A). Thus, the susceptibility of LFA-1−/− mice to low dose LPS-induced shock was markedly increased by tissue macrophage depletion despite reduced TNF-α levels.

This paper describes participation of LFA-1 in low dose LPS-induced shock/liver injury and points to IL-10 as critical mediator of resistance to shock/liver injury in its absence. Although TNF-α is critical for low dose LPS-induced shock (6, 7, 8, 9), and LPS is considered a ligand for LFA-1 (12), serum levels of TNF-α were significantly higher in the resistant LFA-1−/− mice compared with controls. These results imply that a factor(s) downstream of TNF-α signaling participates in the increased resistance of LFA-1−/− mice to low dose LPS and suggest that this increased resistance is not caused by the absent interactions between LPS and LFA-1. Moreover, our data suggest dominant effects of the anti-inflammatory cytokine IL-10 over the proinflammatory cytokines TNF-α and IL-12.

It could be speculated that increased resistance of LFA-1−/− mice to low dose LPS was due to reduced numbers of Vα14+NKT cells in the liver. However, mice deficient in Vα14+NKT cells were susceptible to low dose LPS, and the susceptibility of C57BL/6 mice was only slightly affected by NK1+ cell depletion. The susceptibility of β2m−/− mice to low dose LPS was slightly higher than that of other mouse strains. Following low dose LPS challenge, serum levels of IFN-γ in β2m−/− mice were significantly higher than those in other mouse strains (M. Emoto and Y. Emoto, unpublished observations). Hence, we consider it likely that slightly increased susceptibility of β2m−/− mice to low dose LPS-induced shock is due to higher levels of IFN-γ. In any case, our data suggest that a numerical reduction of Vα14+NKT cells in the liver is not responsible for the increased resistance of LFA-1−/− mice to low dose LPS-induced shock/liver injury.

Granulocytes infiltrate the liver in response to low dose LPS challenge and play a critical role in low dose LPS-induced shock/liver injury (13, 14, 15, 16, 17). Although by 8 h after low dose LPS challenge, granulocytes infiltrated the liver, the number of liver granulocytes was slightly higher in LFA-1−/− mice than in heterozygous littermates. Moreover, susceptibility of C57BL/6 mice to low dose LPS was virtually unchanged by in vivo depletion of granulocytes. Hence, we assume that granulocytes are not responsible for increased resistance to low dose LPS in the absence of LFA-1.

Our study does not formally exclude that increased resistance of LFA-1−/− mice to low dose LPS was caused by a lack of interactions between LFA-1 and its physiological ligands. It has been reported that anti-LFA-1 mAb treatment does not rescue mice from low dose LPS-induced lethal shock (17). This finding raises the possibility that increased resistance of LFA-1−/− mice to low dose LPS occurred independently from interactions between LFA-1 and its physiological ligands. We cannot exclude that the mAb treatment failed to block LFA-1 interactions with a ligand(s) hidden within tissue or involving additional, unknown ligands and binding epitopes. Consecutive administration of anti-LFA-1 mAb increases the resistance of Propionibacterium acnes-primed mice to low dose LPS challenge (52). However, LFA-1 is expressed on virtually all leukocytes, which could be depleted by anti-LFA-1 mAb treatment, resulting in increased resistance to LPS-induced shock. Although in the mouse, ICAM-1 is one of the physiological ligands for LFA-1 (11), we found that the susceptibility of ICAM-1−/− mice was comparable to that of controls. We assume that increased resistance of LFA-1−/− mice to low dose LPS is not a direct consequence of cognate interactions between LFA-1 and ICAM-1 in situ. In addition to ICAM-1, ICAM-2 is a ligand for LFA-1 (11). Hence, we cannot exclude that interactions between LFA-1 and ICAM-2 are involved in this mechanism.

Mac-1 can compensate for the lack of LFA-1 (53). Moreover, Mac-1 expression is up-regulated on leukocytes following LPS challenge, and this molecule participates in low dose LPS-induced shock (16, 17). It is also possible that Mac-1 participates in increased resistance of LFA-1−/− mice to low dose LPS. However, we found that Mac-1 expression on various cell populations was comparable in LFA-1−/− and LFA-1+/− mice even after LPS challenge (M. Emoto and Y. Emoto, unpublished observations). We therefore consider it unlikely that increased resistance of LFA-1−/− mice to low dose LPS occurred independently of Mac-1 expression.

IL-10 plays a protective role in low dose LPS-induced shock/liver injury (46, 47). After low dose LPS challenge, serum levels of IL-10 were significantly, although modestly, higher in LFA-1−/− mice than in LFA-1+/− and C57BL/6 mice. Whereas endogenous IL-10 neutralization only marginally increased the susceptibility of LFA-1+/− mice to low dose LPS, IL-10 neutralization diminished the resistance of LFA-1−/− mice by several orders of magnitude. Differential efficacy of IL-10 neutralization in these mouse strains was probably due to different serum levels of TNF-α. IL-10 has been found to limit TNF-α secretion after low dose LPS challenge (47). Yet serum levels of both TNF-α and IL-10 were higher in LFA-1−/− mice than in heterozygous littermates. We therefore consider it unlikely that increased resistance of LFA-1−/− mice to low dose LPS is a direct consequence of impaired TNF-α secretion by IL-10. The production of various proinflammatory cytokines and chemokines is regulated by IL-10 (54, 55), and IL-10 increases the secretion of soluble TNF-α receptor p55 (56). It is thus possible that LFA-1 participates in these mechanisms by controlling IL-10 secretion.

IFN-γ participates in low dose LPS-induced shock/liver injury (10). Because serum levels of IFN-γ were comparable in LFA-1−/−, LFA-1+/−, and C57BL/6 mice, we assume that IFN-γ is not responsible for the increased resistance of LFA-1−/− mice to low dose LPS-induced shock/liver injury. At present, we cannot provide a conclusive answer for why serum levels of IFN-γ were comparable in LFA-1−/−, LFA-1+/−, and C57BL/6 mice after low dose LPS challenge, although IL-12 levels were higher in LFA-1−/− mice than in other mouse strains. However, IL-10 prevents IFN-γ production (57, 58), and it is possible that IFN-γ production in LFA-1−/− mice was prevented by higher levels of IL-10.

Blood coagulation is a major event at the terminal stage of endotoxemia (40, 41). 1) Platelets play a pivotal role in the blood coagulation cascade; 2) a critical role of platelets in low dose LPS-induced shock has been suggested (59); 3) LFA-1 is expressed on platelets (42); and 4) high numbers of platelets were detected in the liver of LFA-1+/− mice following low dose LPS challenge, whereas platelet accumulation in LFA-1−/− mice was marginal. Hence, it is possible that LFA-1 expressed on platelets directly participates in blood coagulation during endotoxemia. However, endogenous IL-10 neutralization markedly increased the numbers of platelets in the liver of LFA-1−/− mice following low dose LPS challenge, and an inhibitory role of IL-10 in fibrin formation has been reported (60, 61, 62, 63, 64). Therefore, we consider it more likely that higher levels of IL-10 increased the resistance of LFA-1−/− mice to low dose LPS by impairing the blood coagulation cascade.

Serum levels of IL-12 in LFA-1−/− mice following LPS challenge were further increased by tissue macrophage depletion. This raises the question of whether increased levels of IL-12 are responsible for the increased susceptibility of tissue macrophage-depleted LFA-1−/− mice to low dose LPS-induced shock. Yet the susceptibility of tissue macrophage-depleted LFA-1−/− mice to low dose LPS-induced shock/liver injury was unchanged by endogenous IL-12 neutralization (M. Emoto and Y. Emoto, unpublished observation). Hence, the role and cellular source of IL-12 in low dose LPS-induced shock/liver injury of LFA-1−/− mice remain elusive.

Depletion of tissue macrophages did not significantly reduce the serum levels of IL-10 in LFA-1+/− mice, arguing against tissue macrophages as a major source of IL-10 during endotoxemia in the presence of LFA-1. In contrast, in LFA-1−/− mice serum levels of IL-10 were markedly reduced by tissue macrophage depletion. Serum levels of IL-10 were comparable in tissue macrophage-depleted LFA-1−/− mice and nondepleted LFA-1+/− and C57BL/6 mice following low dose LPS challenge, and higher frequencies of IL-10 producers were detected in LFA-1−/− mice compared with LFA-1+/− mice. We conclude that tissue macrophages are responsible for higher levels of IL-10 in the absence of LFA-1.

The susceptibility of C57BL/6 mice to low dose LPS was only slightly reduced by in vivo depletion of tissue macrophages. Moreover, considerable levels of TNF-α were detected in the sera of tissue macrophage-depleted C57BL/6 mice following low dose LPS challenge (M. Emoto and Y. Emoto, unpublished observations). Hence, we assume that in wild-type mice cells other than tissue macrophages play a central role in low dose LPS-induced shock/liver injury by producing TNF-α. In contrast, in LFA-1−/− mice tissue macrophage depletion reduced serum levels of TNF-α. Serum levels of TNF-α were comparable in tissue macrophage-depleted LFA-1−/− mice and nondepleted LFA-1+/− and C57BL/6 mice following low dose LPS challenge, and high frequencies of TNF-α producers were detected in LFA-1−/− mice compared with LFA-1+/− mice. We conclude that tissue macrophages are a major source not only of the anti-inflammatory cytokine IL-10 but also of the proinflammatory cytokine TNF-α in the absence of LFA-1.

Because the susceptibility of LFA-1−/− mice to low dose LPS-induced shock was markedly increased by tissue macrophage depletion despite the fact that not only IL-10, but also TNF-α, were diminished, we consider it likely that the host is rescued from LPS-induced lethal shock/liver injury when IL-10 levels exceed a certain threshold level even in the presence of elevated levels of TNF-α. Consistent with this idea, our additional experiments revealed that serum levels of IL-10 in the susceptible mouse strains were lower than those in LFA-1−/− mice (M. Emoto and Y. Emoto, unpublished observations; see also Fig. 2 A).

The present study does not conclusively answer the question of why IL-10 is elevated in the absence of LFA-1 following low dose LPS challenge. TNF-α is a major mediator of the cytokine cascade that leads to endotoxic shock, and this cytokine has been suggested to participate in the release of IL-10 during endotoxemia (48). However, in our hands serum levels of IL-10 were virtually unaffected by endogenous TNF-α neutralization, which is consistent with previous findings by others (65). It is therefore possible that a factor(s) independent from TNF-α signaling participates in IL-10 production in LFA-1−/− mice. LPS has recently been shown to induce the prompt release of IL-10 from monocytes in the presence of apoptotic granulocytes (49). However, granulocyte depletion did not affect serum levels of IL-10 in LFA-1−/− mice. We therefore consider it unlikely that elevated levels of IL-10 in LFA-1−/− mice were caused by increased numbers of granulocytes.

Mice deficient in LFA-1, CD18, P-selectin, L-selectin, and/or E-selectin display leukocytosis (66, 67, 68, 69, 70, 71, 72), and serum levels of G-CSF and IL-17 are elevated in mice deficient in CD18, P-selectin, and/or E-selectin mice (72). We have recently shown that numbers of leukocytes in peripheral blood and serum levels of G-CSF and IL-17 are markedly increased in LFA-1−/− mice (73). It is therefore possible that the higher levels of pro- and anti-inflammatory cytokines in LFA-1−/− mice are a consequence of leukocytosis and altered regulatory interactions as observed in other mouse strains deficient in cell adhesion molecules. It is tempting to assume that leukocytosis at least in part is responsible for the elevated levels of IL-10 in LFA-1−/− mice following low dose LPS challenge.

Our results show that LFA-1 deficiency confers resistance to low dose LPS-induced shock/liver injury, and that LFA-1 deficiency results in higher IL-10 production in response to low dose LPS. Elevated serum levels of the anti-inflammatory cytokine IL-10 and the proinflammatory cytokines TNF-α and IL-12 in LFA-1−/− mice suggest a dominant role for the inhibitory cytokine IL-10 over the proinflammatory cytokines TNF-α and IL-12. Hence, IL-10 is the critical mediator of resistance to low dose LPS-induced shock/liver injury as a corollary of LFA-1 deficiency. In summary, therefore, our findings define a novel role of the cell adhesion molecule LFA-1 in the regulation of the proinflammatory/anti-inflammatory cytokine balance.

We thank Drs. Marina A. Freudenberg, Rudolf Schmits, and Albert Bendelac for S. abortus equi-derived LPS and breeding pairs of LFA-1−/− and CD1d−/− mice, respectively. We are grateful to Daniela Groine-Triebkorn for screening of mice, and to Beatrix Fauler and Ulrike Reichard for help with histological procedures.

1

This work was supported by a grant from the German Science Foundation (SFB421).

3

Abbreviations used in this paper: d-GalN, d-galactosamine; β2m, β2-microglobulin; Cl2 MBP-L, liposome-encapsulated dichloromethylene bisphosphonate; PBS-L, liposome-encapsulated PBS.

1
Glauser, M. P., G. Zanetti, J. D. Baumgartner, J. Cohen.
1991
. Septic shock: pathogenesis.
Lancet
338
:
732
.
2
Beutler, B., A. Cerami.
1989
. The biology of cachectin/TNF-α primary mediator of the host response.
Annu. Rev. Immunol.
7
:
625
.
3
Gutierrez-Ramos, J. C., H. Bluethmann.
1997
. Molecules and mechanisms operating in septic shock: lessons from knockout mice.
Immunol. Today
18
:
329
.
4
Hack, C. E., L. A. Aarden, L. G. Thijs.
1997
. Role of cytokines in sepsis.
Adv. Immunol.
66
:
101
.
5
Galanos, C., M. A. Freudenberg, W. Reutter.
1979
. Galactosamine-induced sensitization to the lethal effects of endotoxin.
Proc. Natl. Acad. Sci. USA
76
:
5939
.
6
Lehmann, V., M. A. Freudenburg, C. Galanos.
1987
. Lethal toxicity of lipopolysaccharide and tumor necrosis factor in normal and d-galactosamine treated mice.
J. Exp. Med.
165
:
657
.
7
Tiegs, G., M. Wolter, A. Wendel.
1989
. Tumor necrosis factor is a terminal mediator in d-galactosamine/endotoxin-induced hepatitis in mice.
Biochem. Pharmacol.
38
:
627
.
8
Pfeffer, K., T. Matsuyama, T. M. Kündig, A. Wakeham, K. Kishihara, A. Shahinian, K. Wiegmann, P. S. Ohashi, M. Krönke, T. W. Mak.
1993
. Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxin shock, yet succumb to L. monocytogenes infection.
Cell
73
:
457
.
9
Rothe, J., W. Lesslauer, H. Lötscher, Y. Lang, P. Koebel, F. Kötgen, A. Althage, R. Zinkernagel, M. Steinmetz, H. Bluethmann.
1993
. Mice lacking the tumor necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly susceptible to infection by Listeria monocytogenes.
Nature
364
:
798
.
10
Car, B. D., V. M. Eng, B. Schnyder, L. Ozmen, S. Huang, P. Gallay, D. Heumann, M. Aguet, B. Ryffel.
1994
. Interferon γ receptor deficient mice are resistant to endotoxic shock.
J. Exp. Med.
179
:
1437
.
11
Springer, T. A..
1995
. Trafic signals on endothelium for lymphocyte recirculation and leukocyte emigration.
Annu. Rev. Physiol.
57
:
827
.
12
Wright, S. D., M. T. C. Jong.
1986
. Adhesion-promoting receptors on human macrophages recognize Escherichia coli by binding to lipopolysaccharide.
J. Exp. Med.
164
:
1876
.
13
Hewett, J. A., P. A. Jean, S. L. Kunkel, R. A. Roth.
1993
. Relationship between tumor necrosis factor and neutrophils in endotoxin-induced liver injury.
Am. J. Physiol.
265
:
G1011
.
14
Komatsu, Y., Y. Shiratori, T. Kawase, N. Hashimoto, K. Han, S. Shiina, M. Matsumura, Y. Niwa, M. Tada, Y. Ikeda, et al
1994
. Role of polymorphonuclear leukocytes in galactosamine hepatitis: mechanism of adherence to hepatic endothelial cells.
Hepatology
20
:
1548
.
15
Jaeschke, H., C. W. Smith.
1997
. Mechanisms of neutrophil-induced parenchymal cell injury.
J. Leukocyte Biol.
61
:
647
.
16
Jaeschke, H., A. Farhood, C. W. Smith.
1991
. Neutrophil-induced liver cell injury in endotoxin shock is a CD11b/CD18-dependent mechanism.
Am. J. Physiol.
261
:
G1051
.
17
Chang, H. R., C. Vesin, G. E. Grau, P. Pointaire, D. Arsenijevic, M. Strath, J.-C. Pechere, P.-F. Piguet.
1993
. Respective role of polymorphonuclear leukocytes and their integrins (CD11/18) in the local or systemic toxicity of lipopolysaccharide.
J. Leukocyte Biol.
53
:
636
.
18
Schmits, R., T. M. Kündig, D. M. Baker, G. Shumaker, J. J. L. Simard, G. Duncan, A. Wakeham, A. Shahinian, A. van der Heiden, M. F. Bachmann, et al
1996
. LFA-1-deficient mice show normal CTL responses to virus but fail to reject immunogenic tumor.
J. Exp. Med.
183
:
1415
.
19
Emoto, M., M. Miyamoto, I. Yoshizawa, Y. Emoto, U. E. Schaible, E. Kita, S. H. E. Kaufmann.
2002
. Critical role of NK cells rather than Vα14+NKT cells in lipopolysaccharide-induced lethal shock in mice.
J. Immunol.
169
:
1426
.
20
Emoto, M., Y. Emoto, S. H. E. Kaufmann.
1995
. IL-4 producing CD4+ TCR αβint liver lymphocytes: influence of thymus, β2-microglobulin and NK1.1 expression.
Int. Immunol.
7
:
1729
.
21
Emoto, Y., M. Emoto, S. H. E. Kaufmann.
1997
. Transient control of interleukin-4-producing natural killer T cells in the livers of Listeria monocytogenes-infected mice by interleukin-12.
Infect. Immun.
65
:
5003
.
22
van Rooijen, N., A. Sanders.
1994
. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications.
J. Immunol. Methods
174
:
83
.
23
Seiler, P., P. Aichele, B. Raupach, B. Odermatt, U. Steinhoff, S. H. E. Kaufmann.
2000
. Rapid neutrophil response controls fast-replicating intracellular bacteria but not slow-replicating Mycobacterium tuberculosis.
J. Infect. Dis.
181
:
671
.
24
Hirschfeld, M., Y. Ma, J. H. Weis, S. N. Vogel, J. J. Weis.
2000
. Repurification of lipopolysaccharide eliminates signaling through both human and murine Toll-like receptor 2.
J. Immunol.
165
:
618
.
25
Trinchieri, G..
1995
. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity.
Annu. Rev. Immunol.
13
:
251
.
26
Toyabe, S., S. Seki, T. Iiai, K. Takeda, K. Shirai, H. Watanabe, H. Hiraide, M. Uchiyama, T. Abo.
1997
. Requirement of IL-4 and liver NK1+ T cells for concanavalin A-induced hepatic injury in mice.
J. Immunol.
159
:
1537
.
27
Takeda, K., Y. Hayakawa, L. van Kaer, H. Matsuda, H. Yagita, K. Okumura.
2000
. Critical contribution of liver natural killer T cells to a murine model of hepatitis.
Proc. Natl. Acad. Sci. USA
97
:
5498
.
28
Kaneko, Y., M. Harada, T. Kawano, M. Yamashita, Y. Shibata, F. Gejyo, T. Nakayama, M. Taniguchi.
2000
. Augmentation of Vα14 NKT cell-mediated cytotoxicity by interleukin 4 in an autocrine mechanism resulting in the development of concanavalin A-induced hepatitis.
J. Exp. Med.
191
:
105
.
29
Ogasawara, K., K. Takeda, W. Hashimoto, M. Satoh, R. Okuyama, N. Yanai, M. Obinata, K. Kumagai, H. Takada, H. Hiraide, et al
1998
. Involvement of NK1+ T cells and their IFN-γ production in the generalized Shwartzman reaction.
J. Immunol.
160
:
3522
.
30
Dieli, F., G. Sireci, D. Russo, M. Taniguchi, J. Ivanyi, C. Fernandez, M. Troye-Blomberg, G. de Leo, A. Salerno.
2000
. Resistance of natural killer T cell-deficient mice to systemic Shwartzman reaction.
J. Exp. Med.
192
:
1645
.
31
Ohteki, T., C. Maki, S. Koyasu, T. W. Mak, P. S. Ohashi.
1999
. LFA-1 is required for liver NK1.1+ TCRα/β+ cell development: evidence that liver NK1.1+ TCRα/β+ cells originate from multiple pathways.
J. Immunol.
162
:
3753
.
32
Emoto, M., H.-W. Mittrücker, R. Schmits, T. W. Mak, S. H. E. Kaufmann.
1999
. Critical role of leukocyte function-associated antigen-1 in liver accumulation of CD4+NKT cells.
J. Immunol.
162
:
5094
.
33
Bendelac, A., M. N. Rivera, S. Park, J. H. Roark.
1997
. Mouse CD1-specific NK1 T cells: development, specificity, and function.
Annu. Rev. Immunol.
15
:
535
.
34
Mendiratta, S. K., W. D. Martin, S. Hong, A. Boesteanu, S. Joyce, L. van Kaer.
1997
. CD1d1 mutant mice are deficient in natural T cells that promptly produce IL-4.
Immunity
6
:
469
.
35
Smiley, S. T., M. H. Kaplan, M. J. Grusby.
1997
. Immunoglobulin E production in the absence of interleukin-4-secreting CD1-dependent cells.
Science
275
:
977
.
36
Chen, Y., N. M. Chiu, M. Mandal, N. Wang, C. Wang.
1997
. Impaired NK1+ T cell development and early IL-4 production in CD1-deficient mice.
Immunity
6
:
459
.
37
Chojkier, M., J. Fierer.
1985
. d-Galactosamine hepatotoxicity is associated with endotoxin sensitivity and mediated by lymphoreticular cells in mice.
Gastroenterology
88
:
115
.
38
Freudenberg, M. A., D. Keppler, C. Galanos.
1986
. Requirement for lipopolysaccharide-responsive macrophages in galactosamine-induced sensitization to endotoxin.
Infect. Immun.
51
:
891
.
39
Shiratori, Y., T. Kawase, S. Shiina, K. Okano, T. Sugimoto, H. Teraoka, S. Matano, K. Matsumoto, K. Kamii.
1988
. Modulation of hepatotoxicity by macrophages in the liver.
Hepatology
8
:
815
.
40
McKay, D. G., W. Margaretten, I. Csavossy.
1966
. An electron microscope study of the effects of bacterial endotoxin on the blood-vascular system.
Lab. Invest.
15
:
1815
.
41
Dhainaut, J. F., N. Marin, A. Mignon, C. Vinsonneau.
2001
. Hepatic response to sepsis: interaction between coagulation and inflammatory processes.
Crit. Care Med.
29
:
S42
.
42
McCaffery, P. J., M. V. Berridge.
1986
. Expression of the leukocyte functional molecule (LFA-1) on mouse platelets.
Blood
67
:
1757
.
43
Gerard, C., C. Bruyns, A. Marchant, D. Abramowicz, P. Vandenabeele, A. Delvaux, W. Fiers, M. Goldman, T. Velu.
1993
. Interleukin-10 reduces the release of tumor necrosis factor and prevents lethality in experimental endotoxemia.
J. Exp. Med.
177
:
547
.
44
Howard, M., T. Muchamuel, S. Andrade, S. Menon.
1993
. Interleukin-10 protects mice from lethal endotoxemia.
J. Exp. Med.
177
:
1205
.
45
Marchant, A., C. Bruyns, P. Vandenabeele, M. Ducarme, C. Gerard, D. Delvaux, D. de Groote, D. Abramowicz, T. Velu, M. Goldman.
1994
. Interleukin-10 controls interferon-gamma and tumor necrosis factor production during experimental endotoxemia.
Eur. J. Immunol.
24
:
1167
.
46
Santucci, L., S. Fiorucci, M. Chiorean, P. M. Brunori, F. M. Di Matteo, A. Sidoni, G. Migliorati, A. Morelli.
1996
. Interleukin 10 reduces lethality and hepatic injury induced by lipopolysaccharide in galactosamine-sensitized mice.
Gastroenterology
111
:
736
.
47
Louis, H., O. Le Moine, M.-O. Peny, B. Gulbis, F. Nisol, M. Goldman, J. Deviere.
1997
. Hepatoprotective role of interleukin 10 in galactosamine/lipopolysaccharide mouse liver injury.
Gastroenterology
112
:
935
.
48
van der Poll, T., J. Jansen, M. Levi, H. ten Cate, J. W. ten Cate, S. J. van Deventer.
1994
. Regulation of interleukin 10 release by tumor necrosis factor in humans and chimpanzees.
J. Exp. Med.
180
:
1985
.
49
Byrne, A., D. J. Reen.
2002
. Lipopolysaccharide induces rapid production of IL-10 by monocytes in the presence of apoptotic neutrophils.
J. Immunol.
168
:
1968
.
50
Moore, K. W., A. O’Garra, R. de Waal Malefyt, P. Vieira, T. R. Mosmann.
1993
. Interleukin-10.
Annu. Rev. Immunol.
11
:
165
.
51
Knolle, P., J. Schlaak, A. Uhrig, P. Kempf, K. H. Meyer zum Buschenfelde, G. Gerken.
1995
. Human Kupffer cells secrete IL-10 in response to lipopolysaccharide (LPS) challenge.
J. Hepatol.
22
:
226
.
52
Tanaka, Y., K. Kobayashi, A. Takahashi, I. Arai, S. Higuchi, S. Otomo, S. Habu, T. Nishimura.
1993
. Inhibition of inflammatory liver injury by a monoclonal antibody against lymphocyte function-associated antigen-1.
J. Immunol.
151
:
5088
.
53
Andrew, D. P., J. P. Spellberg, H. Takimoto, R. Schmits, T. W. Mak, M. M. Zukowski.
1998
. Transendothelial migration and trafficking of leukocytes in LFA-1-deficient mice.
Eur. J. Immunol.
28
:
1959
.
54
de Waal Malefyt, R., J. Abrams, B. Bennett, C. G. Figdor, J. E. de Vries.
1991
. Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes.
J. Exp. Med.
174
:
1209
.
55
de Waal Malefyt, R., C. G. Figdor, R. Huijbens, S. Mohan-Peterson, B. Bennett, J. Culpepper, W. Dang, G. Zurawski, J. E. de Vries.
1993
. Effects of IL-13 on phenotype, cytokine production, and cytotoxic function of human monocytes: comparison with IL-4 and modulation by IFN-γ or IL-10.
J. Immunol.
151
:
6370
.
56
Leeuwenberg, J. F., T. M. Jeunhomme, W. A. Buurman.
1994
. Slow release of soluble TNF receptors by monocytes in vitro.
J. Immunol.
152
:
4036
.
57
Nagaki, M., M. Tanaka, A. Sugiyama, H. Ohnishi, H. Moriwaki.
1999
. Interleukin-10 inhibits hepatic injury and tumor necrosis factor-α and interferon-γ mRNA expression induced by staphylococcal enterotoxin B or lipopolysaccharide in galactosamine-sensitized mice.
J. Hepatol.
31
:
815
.
58
Moore, K. W., R. de Waal Malefyt, R. L. Coffman, A. O’Garra.
2001
. Interleukin-10 and the interleukin-10 receptor.
Annu. Rev. Immunol.
19
:
683
.
59
Piguet, P. F., C. Vesin, J. E. Ryser, G. Senaldi, G. E. Grau, F. Tacchini-Cottier.
1993
. An effector role for platelets in systemic and local lipopolysaccharide-induced toxicity in mice, mediated by a CD11a- and CD54-dependent interaction with endothelium.
Infect. Immun.
61
:
4182
.
60
Ramani, M., V. Ollivier, F. Khechai, T. Vu, C. Ternisien, F. Bridey, D. de Prost.
1993
. Interleukin-10 inhibits endotoxin-induced tissue factor mRNA production by human monocytes.
FEBS Lett.
334
:
114
.
61
Praidier, O., C. Gerard, A. Delvaux, M. Lybin, D. Abramowicz, P. Capel, T. Velu, M. Goldman.
1993
. Interleukin-10 inhibits the induction of monocyte procoagulant activity by bacterial lipopolysaccharide.
Eur. J. Immunol.
23
:
2700
.
62
Ramani, M., F. Khechai, V. Ollivier, C. Ternisien, F. Bridey, J. Hakim, D. de Prost.
1994
. Interleukin-10 and pentoxifylline inhibit C-reactive protein-induced tissue factor gene expression in peripheral human blood monocytes.
FEBS Lett.
356
:
86
.
63
Vasse, M., J. Paysant, J. Soria, J. P. Collet, J. P. Vannier, C. Soria.
1996
. Regulation of fibrinogen biosynthesis by cytokines: consequences on the vascular risk.
Haemostasis
26
:(Suppl. 4):
331
.
64
Okada, K., T. Fujita, K. Minamoto, H. Liao, Y. Naka, D. J. Pinsky.
2000
. Potentiation of endogenous fibrinolysis and rescue from lung ischemia/reperfusion injury in interleukin (IL)-10-reconstituted IL-10 null mice.
J. Biol. Chem.
275
:
21468
.
65
Junger, W. G., D. B. Hoyt, H. Redl, F. C. Liu, W. H. Loomis, J. Davies, G. Schlag.
1995
. Tumor necrosis factor antibody treatment of septic baboons reduces the production of sustained T-cell suppressive factors.
Shock
3
:
173
.
66
Scharffetter-Kochanek, K., H. Lu, K. Norman, N. van Nood, F. Munoz, S. Grabbe, M. McArthur, I. Lorenzo, S. Kaplan, K. Ley, et al
1998
. Spontaneous skin ulceration and defective T cell function in CD18 null mice.
J. Exp. Med.
188
:
119
.
67
Ding, Z. M., J. E. Babensee, S. I. Simon, H. Lu, J. L. Perrard, D. C. Bullard, X. Y. Dai, S. K. Bromley, M. L. Dustin, M. L. Entman, et al
1999
. Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration.
J. Immunol.
163
:
5029
.
68
Mayadas, T. N., R. C. Johnson, H. Rayburn, R. O. Hynes, D. D. Wagner.
1993
. Leukocyte rolling and extravasation are severely compromised in P selectin-deficient mice.
Cell
74
:
541
.
69
Forlow, S. B., E. J. White, S. C. Barlow, S. H. Feldman, H. Lu, G. J. Bagby, A. L. Beaudet, D. C. Bullard, K. Ley.
2000
. Severe inflammatory defect and reduced viability in CD18 and E-selectin double mutant mice.
J. Clin. Invest.
106
:
1457
.
70
Wilson, R. W., C. M. Ballantyne, C. W. Smith, C. Montogomery, A. Bradley, W. E. O’Brien, A. L. Beaudet.
1993
. Gene targeting yields a CD18-mutant mouse for study of inflammation.
J. Immunol.
151
:
1571
.
71
Frenette, P. S., T. N. Mayadas, H. Rayburn, R. O. Hynes, D. D. Wagner.
1996
. Susceptibility to infection and altered hematopoiesis in mice deficient in both P- and E-selectins.
Cell
84
:
563
.
72
Forlow, S. B., J. R. Schurr, J. K. Kolls, G. J. Bagby, P. O. Schwarzenberger, K. Ley.
2001
. Increased granulopoiesis through interleukin-17 and granulocyte colony-stimulating factor in leukocyte adhesion molecule-deficient mice.
Blood
98
:
3309
.
73
Miyamoto, M., M. Emoto, Y. Emoto, V. Brinkmann, I. Yoshizawa, P. Seiler, P. Aichele, E. Kita, S. H. E. Kaufmann.
2003
. Neutrophilia in LFA-1-deficient mice confers resistance to listeriosis: possible contribution of granulocyte-colony-stimulating factor and IL-17.
J. Immunol.
170
:
5228
.