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Journal of Leukocyte Biology logoLink to Journal of Leukocyte Biology
. 2008 Oct 9;85(1):146–153. doi: 10.1189/jlb.0308161

Mice with heterozygous deficiency of lipoic acid synthase have an increased sensitivity to lipopolysaccharide-induced tissue injury

Xianwen Yi *, Kuikwon Kim †, Weiping Yuan ‡, Longquan Xu *, Hyung-Suk Kim *, Jonathon W Homeister *, Nigel S Key §, Nobuyo Maeda *,1
PMCID: PMC2626770  PMID: 18845616

Abstract

α-Lipoic acid (1, 2-dithiolane-3-pentanoic acid; LA), synthesized in mitochondria by LA synthase (Lias), is a potent antioxidant and a cofactor for metabolic enzyme complexes. In this study, we examined the effect of genetic reduction of LA synthesis on its antioxidant and anti-inflammatory properties using a model of LPS-induced inflammation in Lias+/– mice. The increase of plasma proinflammatory cytokine, TNF-α, and NF-κB at an early phase following LPS injection was greater in Lias+/– mice compared with Lias+/+ mice. The circulating blood white blood cell (WBC) and platelet counts dropped continuously during the initial 4 h. The counts subsequently recovered partially in Lias+/+ mice, but the recovery was impaired totally in Lias+/– mice. Administration of exogenous LA normalized the recovery of WBC counts in Lias+/– mice but not platelets. Enhanced neutrophil sequestration in the livers of Lias+/– mice was associated with increased hepatocyte injury and increased gene expression of growth-related oncogene, E-selectin, and VCAM-1 in the liver and/or lung. Lias gene expression in tissues was 50% of normal expression in Lias+/– mice and reduced further by LPS treatment. Decreased Lias expression was associated with diminished hepatic LA and tissue oxidative stress. Finally, Lias+/– mice displayed enhanced mortality when exposed to LPS-induced sepsis. These data demonstrate the importance of endogenously produced LA for preventing leukocyte accumulation and tissue injury that result from LPS-induced inflammation.

Keywords: antioxidant, neutrophil, inflammation, sepsis, knockout mice

INTRODUCTION

LPS, or bacterial endotoxin, is known to contribute to the pathologic manifestations of sepsis as a consequence of dysregulation and amplification of the host inflammatory response [1]. Experimental and clinical studies indicate that sepsis is associated with increased oxidative and nitrosative stresses [2,3,4,5,6]. The concept that reduced endogenous antioxidant capacity renders the host susceptible to sepsis has been proposed and is being studied [7,8,9]. Studies have shown that plasma antioxidants such as ascorbate, α-tocopherol, and glutathione are decreased in septic patients [10,11,12,13].

α-Lipoic acid (1, 2-dithiolane-3-pentanoic acid; LA) is an essential cofactor for the pyruvate dehydrogenase complex (PDC) and α-ketoglutarate dehydrogenase complex, which both take part in energy formation. LA and its reduced form, dihydrolipoic acid (DHLA), also act as potent micronutrients with diverse pharmacologic and antioxidant properties [14, 15]. LA/DHLA can quench almost all types of free radicals by chelating redox-active transition metals and regenerating other antioxidants such as reduced glutathione [16, 17]. These biological properties, coupled with the current clinical evidence for the safety and efficacy of LA treatment in these diseases [18] suggest that LA should be considered as a therapeutic agent for the treatment of sepsis.

LA is endogenously synthesized by LA synthase (Lias) in mitochondria. Lack of Lias is embryonic-lethal in mice. Lias+/– mice develop normally and are apparently healthy, but they exhibit a slightly reduced endogenous antioxidant capacity [19]. Thus, Lias+/– mice are a suitable animal model to investigate how genetic reduction of LA production may influence the manifestations of sepsis induced by LPS challenge. Our data demonstrate that Lias+/– mice with half-normal expression of the Lias gene have an increased accumulation of inflammatory cells in the lung and liver, resulting in increased tissue damage following LPS exposure.

MATERIALS AND METHODS

LPS-induced endotoxic shock

The Lias+/– mice and their wild-type littermates were F1 offspring of Lias+/+ C57BL/6J mice and Lias+/–129/SvEv mice heterozygous for the disrupted Lias gene [19]. Three-month-old female Lias+/– and Lias+/+ mice were given a single dose (20 mg/kg body weight, unless stated otherwise) of LPS (Escherichia coli 055:B5, Sigma Chemical Co., St. Louis, MO, USA) or saline i.p. The experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of North Carolina (Chapel Hill, NC, USA) and complied with National Institutes of Health (NIH) standards.

Recemic LA (Sigma Chemical Co.) was dissolved in PBS, and pH of the solution was adjusted to 7.0 with NaOH.

Biochemical assays

Blood glucose was monitored by the OneTouch Ultra glucometer (LifeScan, Milpitas, CA, USA). Body temperature was recorded using rectal temperature sensors (Physitemp Instruments Inc., Clifton, NJ, USA) just before LPS injection and every 60 min for 4 h after the injection. Blood was withdrawn from the retro-orbital sinus of anesthetized mice at 0, 2, 4, 8, and 24 h after LPS injection and anticoagulated with EDTA [20, 21]. Leukocyte and platelet counts were measured on an animal blood counter, CBC-Diff™ (Heska Inc., Loveland, CO, USA). Liver and kidney injury was assessed by measuring the activities of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) using a Vitro 250 chemical analyzer (Ortho-Clinical Diagnostics, Inc., Raritan, NJ, USA). Plasma level of IL-6, IL-10, TNF-α, and MCP-1 was measured using the Linco multiplex mouse cytokine kit (Linco Research, Inc., St. Charles, MO, USA) on a Luminex 100 system, according to the manufacturer’s protocols (Luminex Corp., Austin, TX, USA).

Thiobarbituric acid reactive substances (TBARS) in plasma and tissue in the presence of butyl hydroxytoluene were determined as described previously [22]. Reduced glutathione (GSH) in erythrocytes and tissue was measured with a commercial assay kit (Calbiochem, San Diego, CA, USA). Plasma and liver metabolites of nitric/NO (NOx) were measured using a nitrite assay kit (Cayman Chemical, Ann Arbor, MI, USA). Myeloperoxidase (MPO) activity was measured in lung and liver homogenates as described [23]. Protein concentrations were determined based on the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA). Nuclear extract of livers was freshly prepared by using a nuclear extraction kit (Active Motif, Carlsbad, CA, USA), and NF-κB activation was monitored using a Trans-AM NF-κB p65 transcription factor assay kit (Active Motif).

Gene expression

Total RNAs in liver and lung were isolated using an ABI 6700 automated nucleic acid workstation following the manufacturer’s protocol (Applied Biosystems, Foster City, CA, USA). Expression of the genes for Lias, manganese superoxide dismutase 2 (MnSOD2), ICAM-1, VCAM-1, P- and E-selectin, TNF-α, growth-related oncogene (GRO), MIP-1α, MCP-1, inducible NO synthase (iNOS), TLR-4, CD14, and type 1 plasminogen activator inhibitor (PAI-1), was determined by quantitative real-time RT-PCR with β-actin as the reference gene in each reaction [24].

Histology and immunofluorescence

Liver and lung were harvested at 8 and 24 h and fixed in 4% buffered paraformaldehyde (pH 7.4). Paraffin sections (5 μm) were prepared and stained with H&E. LA in tissue sections was visualized by immunofluorescence with a rabbit anti-LA polyclonal antibody (Calbiochem) and a goat anti-rabbit IgG antibody conjugated with Alexa Fluor 594 (Invitrogen, Carlsbad, CA, USA).

Statistical analysis

Data are shown as mean ± sem. Intergroup variations were analyzed by two-factorial ANOVA followed by Tukey-Kramer’s honestly significant different (HSD) test for post hoc pair-wise analysis using JMP software (SAS, Cary, NC, USA). A P value <0.05 was accepted as a significant difference. Kaplan-Meier analysis was used for the survival-time analysis.

RESULTS

Changes of energy metabolism after LPS challenge

The LPS-treated mice showed a typical biphasic response of hepatic carbohydrate metabolism, as in septic shock patients [25], with initial hyperglycemia followed by a profound hypoglycemic phase (Fig. 1A). No significant differences were observed in blood glucose levels at any time-points between Lias+/– and Lias+/+ mice. Marked change in body temperature is a hallmark of endotoxemia in laboratory animals [26], and hypothermia was observed in all LPS-treated mice of both genotypes. The body temperature was reduced to a mean nadir of 28°C at 4 h after LPS administration (Fig. 1B).

Fig. 1.

Fig. 1.

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Plasma glucose, body temperature, and hematological indices in Lias+/– and Lias+/+ mice. (A) Plasma glucose concentration following LPS injection (i.p). (B) Body temperature. (C) White blood cell (WBC) count in circulating blood in Lias+/– mice versus Lias+/+ mice without supplementation of exogenous LA; *, P < 0.05. (D) WBC count in circulating blood in Lias+/– mice versus Lias+/+ mice with supplementation of LA (40 mg/kg body weight). (E) Platelet count in circulating blood and Lias+/– mice without supplementation of exogenous LA; ***, P < 0.001.

Impaired recovery of circulating platelet and neutrophil numbers after LPS challenge

LPS administration resulted in a similar decrease of circulating blood platelet counts in mice of both genotypes up to the 4-h time-point. By the 8-h time-point, platelet counts partially recovered in Lias+/+ mice but remained low in Lias+/– mice (Fig. 1E). Mean platelet volume in the two groups was not altered by LPS treatment (data not shown), implying that decreased platelet numbers in Lias+/– mice were not a result of reduction of platelet generation in the bone marrow. Likewise, the circulating WBC count in Lias+/+ mice decreased markedly, reaching a nadir at 4 h postinjection, followed by a recovery to about 50% of the normal level at 8 h. Lias+/– mice showed a similar degree of reduction, but the recovery was impaired significantly at the 8-h time-point (Fig. 1C). When exogenous LA (40 mg/kg, i.p.) was given at 30 min after LPS injection, the WBC recovery at 8 h in Lias+/– mice was enhanced significantly to the same levels as in Lias+/+ mice (Fig. 1D). In contrast, injection of LA did not significantly increase platelet count at 8 h after LPS challenge in Lias+/– mice (data not shown). To investigate the early phase of inflammation, mice were treated with 4 mg/kg LPS. We observed similar degrees of an enhanced response to a lower dose of LPS (4 mg/kg) in Lias+/– mice compared with Lias+/+ mice, excepting that the WBC count dropped significantly faster at 30 min in Lias+/– mice than in wild-type mice (data not shown).

Organ injury and enhanced mortality in Lias+/– mice after LPS injection

To test whether the decreased, circulating WBC count was a result of enhanced neutrophil sequestration in liver and lung of LPS-challenged Lias+/– animals, we quantitated tissue MPO activity at 8 and 24 h after LPS challenge. MPO activity in the Lias+/– tissues was increased significantly compared with the activity in Lias+/+ tissues (Fig. 2, A and B). Serum levels of ALT, AST, and BUN in the Lias+/– and Lias+/+ mice were similar at the baseline. However, at 8 and 24 h after injection of LPS, serum ALT and AST activities were elevated significantly by LPS treatment (P<0.001) in both genotypes, but the increase was more pronounced in Lias+/– mice than in Lias+/+ mice (Fig. 2, C and D). The serum BUN concentration was increased markedly by LPS treatment in Lias+/– and Lias+/+ animals, but no significant genotype effect was observed at 8 and 24 h after LPS injection (Fig. 2E).

Fig. 2.

Fig. 2.

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Liver and kidney injury at 8 and 24 h after i.p. injection of LPS. Effect of LPS on MPO activity in the lung (A) and liver (B). Solid bars represent Lias+/– mice, and open bars represent Lias+/+ mice. Error bars represent se, and numbers in the bars or above the bars indicate animal numbers for each group. (C) Activity of serum ALT and (D) activity of AST in Lias+/– and Lias+/+ mice. (E) BUN. The ALT, AST, and BUN results represent means sem of values obtained from seven mice per group. P values are determined using Tukey-Kramer’s HSD test.

Light microscopic evaluation of the lungs at 8 and 24 h after LPS injection confirmed increased sequestration of neutrophils within the capillaries of alveolar walls in Lias+/+ and Lias+/– mice. In addition, at 8 h, platelet aggregates plugged small caliber arterioles and/or venules in both groups. Qualitative differences in these features between groups were not detected. Histologic examination of liver at 8 h showed that one of 17 Lias+/– mice had severe acute hepatic necrosis, and one of 16 Lias+/+ mice had scattered small foci of acute hepatic necrosis. Liver obtained at 24 h after LPS injection had scattered hepatocytes with markedly pyknotic nuclei and fragmented nuclei that tended to be more frequent in Lisa+/– mice than Lias+/+ mice (2.2±0.3, n=3, vs. 1.1±0.5, n=3, per high-power field, respectively).

Finally, enhanced mortality of Lias+/– mice after exposure to LPS was evidenced by the death of all eight Lias+/– mice, compared with only 50% of wild-type mice by Day 3 (P<0.05; Fig. 3).

Fig. 3.

Fig. 3.

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Endogenous LA reduces the mortality of LPS-induced sepsis. LPS was injected i.p. in Lias+/– and Lias+/+ mice (eight mice per group). Survival rate was monitored during a subsequent 5-day period. The statistical significance of mortality and survival time was determined by the Kaplan-Meyer method.

Induction of plasma cyokines and activation of NF-κB

To examine whether the increased tissue damage is associated with an enhanced inflammatory response to LPS challenge in the Lias+/– mice, serum levels of cytokines TNF-α, IL-6, MCP-1, and IL-10 were examined at 0, 2, 4, and 8 h after LPS injection. Baseline TNF-α, IL-6, IL-10, and MCP-1 in the sera of Lias+/– mice and Lias+/+ mice were not different. At 2 h after LPS administration, all plasma cytokine levels were increased significantly compared with the baseline levels (Fig. 4). The mean serum TNF-α level at 2 h of 7.4 ± 0.5 ng/ml in Lias+/– mice was significantly higher than 4.3 ± 1.0 ng/ml in Lias+/+ mice (P<0.01, Fig. 4A). TNF-α level decreased with time, and at the 8-h time-point, serum levels in Lias+/– and Lias+/+ mice were not different. IL-6, IL-10, and MCP-1 levels increased by 2 h, and IL-6 and MCP-1 remained elevated for 8 h in mice of each genotype (Fig. 4, C and D).

Fig. 4.

Fig. 4.

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Plasma levels of TNF-α (A), IL-10 (B), IL-6 (C), and MCP-1 (D) following LPS treatment. (○) Lias+/+ mice; (•) Lias+/– mice. **, P < 0.01, between Lias+/– and Lias+/+ mice at the time-point.

To investigate whether the enhanced sensitivity of Lias+/– mice to LPS treatment occurs via activation of the NF-κB signaling pathway, NF-κB activity was examined in nuclear extracts of liver and lung tissue, from mice 1 h after administration with 4 mg/kg LPS. NF-κB p65 was highly activated in the liver and lung from mice of both genotypes. Moreover, liver NF-κB activity in Lias+/– was significantly higher than Lias+/+ mice (Fig. 5).

Fig. 5.

Fig. 5.

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NF-κB activity in liver and lung nuclear extract at 1 h after LPS injection (4 mg/kg body weight) was determined by Trans-AM NF-κB ELISA kit for assay of NF-κB/p65. Bars represent mean ± sem of liver and lung of seven mice with LPS treatment and five mice with saline in each genotype. P < 0.001 for effects of LPS; P < 0.05 for genotype effects in liver by ANOVA.

Increased oxidative stress in liver and lung

The antioxidant capacities in Lias+/– mice against LPS-induced oxidative stress [27,28,29] were estimated using GSH quantitation in erythrocytes, lung, and liver and TBARS quantitation in plasma, liver, and lung at 8 h after LPS injection. Plasma TBARS were increased, and erythrocyte GSH was decreased by LPS injection, but they did not differ significantly between the two genotypes (Fig. 6, A and B). In contrast, TBARS in livers and lungs of Lias+/– mice at 8 h post-LPS injection were significantly higher, and liver GSH was significantly lower in Lias+/+ mice (Fig. 6, C and D). A single injection of LA after LPS treatment significantly attenuated the LPS-induced increase in liver TBARS but did not affect lung TBARS or liver or lung GSH levels (Fig. 6, E and F). Plasma NOx levels were increased 10 times in response to LPS injection in both genotypes (P<0.001) with nonsignificant differences between the genotypes (Fig. 6G). Similarly, liver NOx in both genotypes was increased markedly at 8 h after LPS injection compared with untreated animals, with no significant difference between genotypes (Fig. 6H). However, exogenous LA administration reduced iNOS gene expression in Lias+/– mice significantly by 41% (data not shown).

Fig. 6.

Fig. 6.

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LPS-induced oxidative stress. (A) TBARS in plasma. (B) Reduced GSH in erythrocytes. (C and D) TBARS and GSH in the liver and lung homogenates at 8 h after LPS injection. (E and F) TBARS and GSH levels in the Lias+/– liver and lung at 8 h after single-dose LA (40 mg/kg body weight) i.p. injection at 30 min following LPS administration. Twelve mice were injected with exogenous LA. (G) Plasma NOx before (0 h) and at 8 h after the LPS treatment. (H) NOx in the liver homogenates before and at 8 h after LPS treatment. Values are mean ± sem. Significance is determined using Tukey-Kramer’s HSD test. The numbers in the bars or above the bars indicate animal numbers. NS, No significance.

As expected, Lias gene expression in the Lias+/– mice is approximately half of Lias+/+ mice. At 8 h after LPS injection, the Lias gene expression was reduced markedly by about 70% in Lias+/+ and Lias+/– mice (P<0.001; Fig. 7). Immunostaining of the liver sections with anti-LA antibody showed that the number of LA-positive hepatocytes decreased noticeably more in Lias+/– mice than Lias+/+ mice after LPS challenge (Fig. 8, A–D).

Fig. 7.

Fig. 7.

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Lias gene expression. mRNA levels isolated from livers at 8 h after LPS administration for the Lias gene determined by quantitative real-time PCR. The data are expressed as the mean ± sem relative to the mean values of the levels in Lias+/+ mice without treatment as 1.0. Numbers in each bar indicate numbers of mice in each group. Solid bars indicate Lias+/– mice, and open bars represent Lias+/+ mice.

Fig. 8.

Fig. 8.

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(A–D) LA content in tissue liver sections visualized by immunofluorescence (Texas Red) with a rabbit anti-LA polyclonal antibody in Lias+/+ (A and C) and Lias+/– (B and D), without (A and B) and with (C and D) LPS treatment. Original magnification, ×200 for all panels.

Enhanced expression of genes for recruitment of inflammatory cells

To gain further insights into the mechanisms underlying the enhanced tissue sequestration of leukocytes in the Lias+/– and Lias+/+ mice, we analyzed the expression of some inflammatory genes in the liver and lung at 8 h post-LPS treatment by real-time RT-PCR (Table 1, A and B). There was no genotype-dependent difference in the expression of genes in saline-treated control mice. LPS injection induced gene expression for certain cytokines, chemokines, and leukocyte trafficking molecules. Among the cytokines and chemokines examined, TNF-α expression was increased significantly in the lung. Liver expression of the gene encoding for GRO, a major chemoattractant protein for neutrophils, was three-fold higher in Lias+/– mice than in Lias+/+ mice. Expression of the genes for MIP-1α and MCP-1, two major chemoattractant proteins for macrophages, were not significantly different between the two genotypes in the liver and lung. Of the genes related to leukocyte trafficking, significantly higher liver expression of VCAM-1 was seen in the Lias+/– mice than the Lias+/+ mice. ICAM-1 expression was also higher, although the difference was not statistically significant. Neither E- nor P-selectin gene expressions were elevated compared with control at 8 h. As selectin expression has been demonstrated in the early phase of LPS injection [30], we also measured mRNA levels in tissues from mice 1 h after LPS injection at 4 mg/kg. Expression of P-selectin and E-secletin was elevated significantly compared with control mice. E-selectin, but not P-selectin, expression was significantly higher in liver and lung of Lias+/– mice compared with wild-type mice. LPS induced iNOS gene expression up-regulation, but no difference was observed between the genotypes at 8 h LPS injection. The liver and lung expression of MnSOD, TLR-4, CD14, and PAI-1 genes did not change between LPS-treated mice and PBS-treated mice at 8 h postinjection. Exogenous LA administration 30 min after LPS injection significantly reduced ICAM-1 and VCAM-1 gene expressions in Lias+/– mice to levels similar to those in the Lias+/+ mice (data not shown). In contrast, GRO expression after LA administration remained 2.4 times higher than Lias+/+ mice.

TABLE 1A.

Relative Gene Expression in the Lung and Liver of Mice 8 h after LPS Exposure

Gene Lung
Liver
Control
With LPS
Control
With LPS
Lias+/+ Lias+/– Lias+/+ Lias+/– Lias+/+ Lias+/– Lias+/+ Lias+/–
GRO 100 ± 21 (6) 112 ± 17 (6) 212 ± 40 (5) 308 ± 36 (5) 100 ± 14 (6) 121 ± 11 (6) 203 ± 54 (6) 570 ± 5 (6)a
MIP-1α 100 ± 15 (6) 109 ± 12 (6) 145 ± 32 (5) 182 ± 44 (5) 100 ± 19 (6) 95 ± 12 (6) 130 ± 25 (6) 224 ± 96 (6)
MCP-1 100 ± 22 (6) 95 ± 21 (6) 126 ± 20 (5) 104 ± 10 (5) 100 ± 12 (6) 96 ± 21 (6) 140 ± 35 (6) 126 ± 16 (5)
TNF-α 100 ± 14 (6) 92 ± 15 (6) 187 ± 18 (5) 330 ± 39 (5)b 100 ± 14 (6) 124 ± 15 (6) 200 ± 58 (6) 302 ± 44 (5)
ICAM-1 100 ± 16 (6) 113 ± 17 (6) 168 ± 32 (5) 203 ± 24 (5) 100 ± 8 (6) 117 ± 17 (6) 141 ± 44 (12) 425 ± 64 (11)
VCAM-1 100 ± 12 (6) 90 ± 11 (6) 346 ± 34 (5) 667 ± 70 (5)b 100 ± 15 (6) 97 ± 18 (6) 315 ± 48 (12) 545 ± 75 (11)b
E-selectin 100 ± 15 (6) 89 ± 15 (6) 118 ± 25 (5) 133 ± 22 (5) 100 ± 18 (6) 89 ± 15 (6) 115 ± 35 (6) 130 ± 23 (5)
P-selectin 100 ± 20 (6) 115 ± 25 (6) 123 ± 14 (5) 154 ± 21 (5) 100 ± 23 (6) 115 ± 25 (6) 105 ± 15 (6) 131 ± 15 (5)
MnSOD 100 ± 24 (6) 107 ± 9 (6) 82 ± 9 (5) 71 ± 8 (5) 100 ± 25 (6) 107 ± 9 (6) 85 ± 9 (12) 93 ± 13 (11)
iNOS 100 ± 14 (6) 113 ± 19 (6) 212 ± 4 (5) 265 ± 12 (5) 100 ± 11 (6) 113 ± 19 (6) 200 ± 52 (9) 262 ± 70 (8)
TLR4 100 ± 12 (6) 86 ± 16 (6) 125 ± 17 (5) 145 ± 13 (5) 100 ± 19 (6) 86 ± 16 (6) 94.2 ± 16 (9) 88.4 ± 10 (8)
CD14 100 ± 19 (6) 89 ± 22 (6) 115 ± 11 (5) 178 ± 38 (5) 100 ± 21 (6) 112 ± 17 (6) 85 ± 16 (9) 74 ± 10 (8)
Pai 1 100 ± 12 (6) 127 ± 25 (6) 123 ± 13 (5) 159 ± 16 (5) 100 ± 15 (6) 109 ± 12 (6) 87 ± 12 (6) 74 ± 15 (5)
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TABLE 1B.

Relative Gene Expression in the Lung and Liver of Mice 1 h after LPS Exposure

Gene Lung
Liver
Control
With LPS
Control
With LPS
Lias+/+ Lias+/– Lias+/+ Lias+/– Lias+/+ Lias+/– Lias+/+ Lias+/–
E-selectin 100 ± 15 (6) 98 ± 16 (6) 378 ± 196 (8) 805 ± 235 (8)b 100 ± 18 (6) 121 ± 11 (6) 385 ± 135 (8) 998 ± 165 (8)b
P-selectin 100 ± 20 (6) 128 ± 18 (6) 312 ± 56 (8) 490 ± 104 (8) 100 ± 18 (6) 114 ± 23 (6) 289 ± 94 (8) 496 ± 106 (8)
TNF-α 100 ± 17 (6) 135 ± 24 (6) 1257 ± 508 (8) 1130 ± 244 (8) 100 ± 24 (6) 117 ± 25 (6) 583 ± 200 (8) 1145 ± 342 (8)
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Data are expressed as mean ± sem relative to the mean expression in the non-LPS-treated Lias+/+ tissue as 100. Numbers in parentheses indicate animal numbers for each group. P values between the two genotypes were calculated by Student’s t-test. 

a

P < 0.01; 

b

P < 0.05. 

DISCUSSION

In this study, we found that the Lias+/– mice are more sensitive to LPS-induced oxidative stress and tissues damage. LPS treatment induced a similar degree of thrombocytopenia and leukopenia in Lias+/+ and Lias+/– mice, but recovery in Lias+/– mice was impaired significantly. Higher expression of genes for GRO, VCAM-1, and E-selectin, stronger activation of NF-κB, and diminished LA in the Lias+/– liver compared with Lias+/+ liver suggest that the reduced ability to combat cellular oxidative stress led to increased neutrophil recruitment and sequestration and tissue damage in Lias+/– mice. In addition, the mortality of Lias+/– mice was increased. These findings suggest an important role for the endogenous production of LA in preventing the detrimental effects of endotoxemia associated with sepsis.

Our results showed that the WBC counts dropped during the first 4 h after LPS injection but started to recover after 4 h in Lias+/+ mice. The initial drop could be explained by destruction and margination of peripheral blood cells [31] and/or hemorrhage or sequestration within the tissues. The subsequent rise of blood leukocyte counts in these mice may be explained by cellular release from the bone marrow reservoir or a shift from the marginated pool to the circulating pool. Reduced Lias expression in the heterozygotes did not affect the initial decrease in circulating cell counts despite higher plasma concentrations of proinflammatory TNF-α during the early stage of inflammation. Instead, recovery of the circulating cell counts in the Lias+/– mice was impaired at 8 h. The tissue oxidative stress was increased in the Lias+/– mice compared with those of Lias+/+ mice, reflecting a diminished cellular LA and endogenous antioxidant reserve. Administration of exogenous LA shortly after the LPS injection significantly accelerated WBC recovery in Lias+/– mice, strongly suggesting that these LPS-induced alterations are related to oxidative stress.

In response to a variety of inflammatory stimuli, endothelial cells activate expression of the genes for adhesion molecules, including VCAM-1, ICAM-1, and E-selectin, which play an important role in regulating the movement of WBC from blood to foci of inflammation [32, 33]. We found that at 8 h after the LPS injection, the gene expression of VCAM-1 and ICAM-1 was higher in the Lias+/– livers than in Lias+/+ livers and that administration of exogenous LA completely reversed this increase in expression. Likewise, expression of E-selectin at 1 h after LPS injection was significantly higher in Lias+/– mice than in Lias+/+ mice. Marui et al. [34] have shown that LPS-mediated transcriptional activation of the human VCAM-1 promoter through NF-κB-like DNA enhancer elements is regulated through an antioxidant-sensitive mechanism in vascular endothelial cells. Similarly, Faruqi et al. [35] found that NF-κB binding to its consensus sequences in the VCAM-1 gene is redox-sensitive, therefore allowing for differential gene expression regulated by the same transcription factor. Thus, it is possible that reduced production of endogenous LA increases cellular levels of NF-κB activation, leading to an increased downstream gene expression of VCAM-1 in the Lias+/– liver. However, NF-κB in lungs of both genotypes was highly activated without a detectable genotype difference. Thus, other regulatory factors in addition to NF-κB must be involved in the significantly enhanced VCAM gene expression in the Lias+/– lung. Our in vivo findings are consistent with those of Zhang and Frei [36] and coworkers [37], who showed that exogenous LA can inhibit TNF-α-induced expression of ICAM-1 and VCAM-1 at the mRNA level. Similarly, Chaudhary et al. [38] demonstrated the ability of dietary LA to inhibit up-regulation of ICAM-1 and VCAM-1 in endothelial cells within the CNS in mice. However, the effects of exogenously given LA may not exactly mimic those caused by a subtle difference in the production of endogenous LA. For example, increased expression of the gene for GRO in the liver suggests that enhanced endothelial activation contributes to the recruitment of neutrophils to the Lias+/– tissue, but exogenous LA supplementation had no effect on the GRO expression levels. In addition, although reduction in the endogenous LA production did not alter the levels of iNOS gene expression, exogenous LA significantly reduced the iNOS expression in the Lias+/– mice. Furthermore, LA supplementation did not significantly increase platelet recovery. The reasons for these observations are not clear at present.

TNF-α is one of the first cytokines to become elevated in plasma following LPS administration and initiates a cascade of host responses [39,40,41]. It is noteworthy that the TNF-α response in mice with heterozygous deficiency of Lias was exaggerated in the early phase of LPS-induced inflammation. Enhanced activation of NF-κB at the early stage of Lias+/– tissues may contribute to the elevated TNF-α. However, this enhanced response did not appear to contribute to the course of physiological changes such as plasma glucose, body temperature, and plasma levels of cytokines in later stages. The mechanism by which LA regulates TNF-α production is largely unknown. However, Zhang and Frei [36] speculated that LA inhibits TNF-α-induced endothelial activation, probably as a result of metal chelation by LA rather than its general antioxidant effects. Recently, Lee et al. [42] reported that LA inhibits TNF-α-induced MAPK signaling in rheumatoid arthritis fibroblast-like synovial cells. In our present study, the TLR4/CD14 gene expression was not significantly different between the tissues of Lias+/– and wild-type mice after LPS treatment, suggesting that the regulation of TNF-α production by LA is not likely mediated via the TLR4 signaling pathway. Similarly, up-regulation of iNOS by LPS likely contributes to hepatocellular damage, directly or indirectly, by forming reactive nitrogen intermediates [43]. However, we observed no significant differences in the NOx levels and LPS-induced iNOS gene expression between the two genotypes, suggesting that the reduction of endogenous LA does not affect the nitrosyl stress.

Finally, the dramatic reduction of the Lias gene expression and LA in tissues after LPS challenge requires comment. LPS challenge also results in transcriptional suppression of the PDC genes in various organs [44]. This is partly because the body attempts to cope with an increased oxidative stress through reduction of glucose oxidative metabolism. In the current work, we found that LPS markedly decreased Lias gene expression. As LA is a mandatory cofactor for PDC and α-ketoglutarate dehydrogenase, decreased Lias expression is consistent with reduction of an oxidative metabolism during sepsis. This metabolic change induced by LPS is the same as the switch required for cellular adaptation to hypoxia, although it has been debated whether sepsis induces the hypoxic condition [45, 46]. An adverse consequence of this down-regulation of the Lias gene is the reduction of cellular antioxidant capacity. Further studies are necessary to elucidate whether the antioxidant and anti-inflammatory properties of endogenous LA are similar to or distinct from dietary LA.

In summary, LPS-treated Lias+/– mice show impaired recovery of the WBC and platelet counts. This is likely a result of a prolonged net accumulation of neutrophils in tissues as a result of increased expression of E-selectin, GRO, and VCAM-1, affected by activation of NF-κB and oxidative stress. These results raise the possibility that genetic variations affecting the expression levels of the Lias gene may influence the severity of endotoxemia in humans.

Acknowledgments

This work was supported by grants (HL42630 and HL87946) from the NIH. We thank Dr. Alisa Wolberg and Mr. John Hagaman for helpful discussion and valuable advice and Mr. Lance Johnson, Ms. Sylvia Hiller, and Drs. Jose Arbones-Mainar and Raymond Givens for helpful comments about the manuscript.

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