Lymphotoxin-beta receptor blockade reduces CXCL13 in lacrimal glands and improves corneal integrity in the NOD model of Sjögren's syndrome.

INTRODUCTION: In Sjögren's syndrome, keratoconjunctivitis sicca (dry eye) is associated with infiltration of lacrimal glands by leukocytes and consequent losses of tear-fluid production and the integrity of the ocular surface. We investigated the effect of blockade of the lymphotoxin-beta receptor (LTBR) pathway on lacrimal-gland pathology in the NOD mouse model of Sjögren's syndrome. METHODS: Male NOD mice were treated for up to ten weeks with an antagonist, LTBR-Ig, or control mouse antibody MOPC-21. Extra-orbital lacrimal glands were analyzed by immunohistochemistry for high endothelial venules (HEV), by Affymetrix gene-array analysis and real-time PCR for differential gene expression, and by ELISA for CXCL13 protein. Leukocytes from lacrimal glands were analyzed by flow-cytometry. Tear-fluid secretion-rates were measured and the integrity of the ocular surface was scored using slit-lamp microscopy and fluorescein isothiocyanate (FITC) staining. The chemokine CXCL13 was measured by ELISA in sera from Sjögren's syndrome patients (n = 27) and healthy controls (n = 30). Statistical analysis was by the two-tailed, unpaired T-test, or the Mann-Whitney-test for ocular integrity scores. RESULTS: LTBR blockade for eight weeks reduced B-cell accumulation (approximately 5-fold), eliminated HEV in lacrimal glands, and reduced the entry rate of lymphocytes into lacrimal glands. Affymetrix-chip analysis revealed numerous changes in mRNA expression due to LTBR blockade, including reduction of homeostatic chemokine expression. The reduction of CXCL13, CCL21, CCL19 mRNA and the HEV-associated gene GLYCAM-1 was confirmed by PCR analysis. CXCL13 protein increased with disease progression in lacrimal-gland homogenates, but after LTBR blockade for 8 weeks, CXCL13 was reduced approximately 6-fold to 8.4 pg/mg (+/- 2.7) from 51 pg/mg (+/-5.3) in lacrimal glands of 16 week old control mice. Mice given LTBR blockade exhibited an approximately two-fold greater tear-fluid secretion than control mice (P = 0.001), and had a significantly improved ocular surface integrity score (P = 0.005). The mean CXCL13 concentration in sera from Sjögren's patients (n = 27) was 170 pg/ml, compared to 92.0 pg/ml for sera from (n = 30) healthy controls (P = 0.01). CONCLUSIONS: Blockade of LTBR pathways may have therapeutic potential for treatment of Sjögren's syndrome.

Inhibition of arterial lesion progression in CD16-deficient mice: evidence for altered immunity and the role of IL-10.

AIMS: Given the importance of IgG Fc receptors in immune regulation, we hypothesized that Fcg receptor type III (FcgRIII, CD16) plays an important role in atherogenesis. We therefore analysed the formation of arterial lesions in LDL receptor-deficient (LDLR(-/-)) and FcgRIII(-/-)xLDLR(-/-) double knockout mice at three different points up to 24 weeks of exposure to a high-fat diet. METHODS AND RESULTS: Analysis of Oil Red-O-stained sections revealed no difference in lesion formation between strains after 6 weeks of a high-fat diet, and a modest decrease after 14 weeks in double knockouts relative to LDLR(-/-) controls. After 24 weeks, lesion formation was decreased in the aortic root (30%) and innominate artery (50%) in FcgRIII double knockouts relative to LDLR(-/-) controls. Analysis of peripheral CD4+ T-cells by intracellular flow cytometry from double knockouts after 24 weeks of a high-fat diet revealed statistically significant increases in the percentages of cells producing interferon-gamma, interleukin (IL)-10, and IL-4 relative to controls, differences that were also observed by analyses of whole aortas for cytokine mRNA levels. As determined by flow cytometry, FcgRIII deficiency resulted in an expansion of CD4+ cells and an increase in the CD4 to CD8 ratio. Analysis of plasma anti-oxidized LDL (OxLDL) antibodies by chemiluminescent assay revealed that IgG1 and IgG2c titers to OxLDL were increased in FcgRIII (-/-)xLDLR(-/-) double knockouts relative to LDLR(-/-) controls, while total IgG levels were similar. CONCLUSION: These results reveal altered immunity in FcgRIII(-/-)xLDLR(-/-) mice and a reduction in lesion formation associated with increased production of IL-10 by an expansion of CD4+ T-cells. The reduction in lesion formation was manifest well after evidence of an immune response to OxLDL, suggesting that FcgRIII contributes to lesion progression in murine atherosclerosis.

Role of CYP3A and CYP2E1 in alcohol-mediated increases in acetaminophen hepatotoxicity: comparison of wild-type and Cyp2e1(-/-) mice.

CYP2E1 is widely accepted as the sole form of cytochrome P450 responsible for alcohol-mediated increases in acetaminophen (APAP) hepatotoxicity. However, we previously found that alcohol [ethanol and isopentanol (EIP)] causes increases in APAP hepatotoxicity in Cyp2e1(-/-) mice, indicating that CYP2E1 is not essential. Here, using wild-type and Cyp2e1(-/-) mice, we investigated the relative roles of CYP2E1 and CYP3A in EIP-mediated increases in APAP hepatotoxicity. We found that EIP-mediated increases in APAP hepatotoxicity occurred at lower APAP doses in wild-type mice (300 mg/kg) than in Cyp2e1(-/-) mice (600 mg/kg). Although this result suggests that CYP2E1 has a role in the different susceptibilities of these mouse lines, our findings that EIP-mediated increases in CYP3A activities were greater in wild-type mice compared with Cyp2e1(-/-) mice raises the possibility that differential increases in CYP3A may also contribute to the greater APAP sensitivity in EIP-pretreated wild-type mice. At the time of APAP administration, which followed an 11 h withdrawal from the alcohols, alcohol-induced levels of CYP3A were sustained in both mouse lines, whereas CYP2E1 was decreased to constitutive levels in wild-type mice. The CYP3A inhibitor triacetyloleandomycin (TAO) decreased APAP hepatotoxicity in EIP-pretreated wild-type and Cyp2e1(-/-) mice. TAO treatment in vivo resulted in inhibition of microsomal CYP3A-catalyzed activity, measured in vitro, with no inhibition of CYP1A2 and CYP2E1 activities. In conclusion, these findings suggest that both CYP3A and CYP2E1 contribute to APAP hepatotoxicity in alcohol-treated mice.

Involvement of Toll-like receptor 4 in acetaminophen hepatotoxicity.

The objective of this study was to determine whether Toll-like receptor 4 (TLR4) has a role in alcohol-mediated acetaminophen (APAP) hepatotoxicity. TLR4 is involved in the inflammatory response to endotoxin. Others have found that ethanol-mediated liver disease is decreased in C3H/HeJ mice, which have a mutated TLR4 resulting in a decreased response to endotoxin compared with endotoxin-responsive mice. In the present study, short-term (1 wk) pretreatment with ethanol plus isopentanol, the predominant alcohols in alcoholic beverages, caused no histologically observed liver damage in either C3H/HeJ mice or endotoxin-responsive C3H/HeN mice, despite an increase in nitrotyrosine levels in the livers of C3H/HeN mice. In C3H/HeN mice pretreated with the alcohols, subsequent exposure to APAP caused a transient decrease in liver nitrotyrosine formation, possibly due to competitive interaction of peroxynitrite with APAP producing 3-nitroacetaminophen. Treatment with APAP alone resulted in steatosis in addition to congestion and necrosis in both C3H/HeN and C3H/HeJ mice, but the effects were more severe in endotoxin-responsive C3H/HeN mice. In alcohol-pretreated endotoxin-responsive C3H/HeN mice, subsequent exposure to APAP resulted in further increases in liver damage, including severe steatosis, associated with elevated plasma levels of TNF-alpha. In contrast, alcohol pretreatment of C3H/HeJ mice caused little to no increase in APAP hepatotoxicity and no increase in plasma TNF-alpha. Portal blood endotoxin levels were very low and were not detectably elevated by any of the treatments. In conclusion, this study implicates a role of TLR4 in APAP-mediated hepatotoxicity.

Role of the nuclear receptor pregnane X receptor in acetaminophen hepatotoxicity.

The pregnane X receptor (PXR) is a transcriptional regulator of xenobiotic metabolizing enzymes, including cytochrome P450 3A (CYP3A), and transporters. Pretreatment of mice and rats with inducers of CYP3A increases acetaminophen (APAP) hepatotoxicity. In untreated mice, the amount of hepatic CYP3A11 mRNA is 4-fold greater in PXR(-/-) mice compared to wild-type mice (Guo et al., 2003), a finding anticipated to increase APAP hepatotoxicity in PXR(-/-) mice. We investigated APAP hepatotoxicity in wild-type and PXR(-/-) mice in a C57BL/6 background, with APAP administered by gavage. Despite a 2.5-fold higher level of total hepatic CYP3A protein and a 3.6-fold higher level of CYP3A activity compared to wild-type mice, PXR(-/-) mice were less sensitive to APAP hepatotoxicity. Hepatic levels of CYP2E1 were identical in the two mouse lines, but hepatic CYP1A2 levels were 3-fold greater in wild-type mice compared to PXR(-/-) mice. Caffeine, an inhibitor of CYP1A2 activity and an enhancer of CYP3A activity, decreased APAP hepatotoxicity in wild-type mice. APAP uptake was 1.5-fold greater in wild-type mice compared to PXR(-/-) mice. No significant differences in the formation of APAP glucuronide and sulfate-conjugated metabolites were observed between wild-type and PXR(-/-) mice. Glutathione levels were similar in the two mouse lines and were transiently decreased to similar amounts after APAP administration. Our finding that APAP hepatotoxicity was decreased in PXR(-/-) mice indicates that PXR is an important modulator of APAP hepatotoxicity, through positive modulation of constitutive CYP1A2 expression and possibly through increased APAP absorption.

Phenobarbital and phenytoin increased acetaminophen hepatotoxicity due to inhibition of UDP-glucuronosyltransferases in cultured human hepatocytes.

Here we present a preclinical model to assess drug-drug interactions due to inhibition of glucuronidation. Treatment with the antiepileptics phenobarbital (PB) or phenytoin (PH) has been associated with increased incidence of acetaminophen (APAP) hepatotoxicity in patients. In human hepatocytes, we found that the toxicity of APAP (5 mM) was increased by simultaneous treatment with phenobarbital (2 mM) or phenytoin (0.2 mM). In contrast, pretreatment with PB for 48 h prior to APAP treatment did not increase APAP toxicity unless both drugs were present simultaneously. Cells treated with APAP in combination with PB or PH experienced decreases in protein synthesis as early as 1 h, ultrastructural changes by 24 h, and release of liver enzymes by 48 h. Toxicity correlated with inhibition of APAP glucuronidation. PB or PH also inhibited APAP glucuronidation in rat and human liver microsomes and expressed human UGT1A6, 1A9, and 2B15. As with intact hepatocytes, PB and PH were neither hydroxylated nor glucuronidated, suggesting the direct inhibition of UGTs. Our findings suggest that, in multiple drug therapy, an inhibitory complex between UGT and one of the drugs can lead to decreased glucuronidation and increased systemic exposure and toxicity of a coadministered drug.

Arsenite decreases CYP3A4 and RXRalpha in primary human hepatocytes.

Arsenic is a naturally occurring, worldwide contaminant implicated in numerous pathological conditions in humans, including cancer and several forms of liver disease. One of the contributing factors to these disorders may be the alteration of cytochrome P450 (P450) levels by arsenic. P450s are involved in the oxidative metabolism and elimination of numerous toxic chemicals. CYP3A4, a major P450 in humans, is involved in the metabolism of half of all currently used drugs. Acute exposure to arsenite decreases the induction of CYP1A1/2 proteins and activities in cultured human hepatocytes, as well as CYP3A23 in cultured rat hepatocytes. Here, in primary cultures of human hepatocytes, we assessed the effects of acute arsenite exposure on CYP3A4 and several transcription factors involved in CYP3A4 expression. The concentrations of arsenite used in these studies were nontoxic to the hepatocytes and failed to elicit an oxidative response. Treatment with arsenite in the presence of CYP3A4 inducers, rifampicin (Rif) or phenobarbital, caused major decreases in CYP3A4 mRNA, protein, and activity. In addition, the levels of CYP3A4 in untreated cells were decreased following arsenite treatment. Transcription of the CYP3A4 gene is primarily regulated by heterodimers of the retinoid X receptor alpha (RXRalpha) and the pregnane X receptor (PXR). We found that arsenite failed to affect expression of PXR or the transcription factor Sp1, yet caused a significant decrease in PXR responsiveness to Rif. Arsenite caused a large decrease in nuclear RXRalpha protein and, to a lesser extent, RXRalpha mRNA. These results suggest that arsenite inhibits both untreated and induced CYP3A4 transcription in primary human hepatocytes by decreasing the activity of PXR, as well as expression of the nuclear receptor RXRalpha.

Role of mouse CYP2E1 in the O-hydroxylation of p-nitrophenol: comparison of activities in hepatic microsomes from Cyp2e1(-/-) and wild-type mice.

Enzymatic activities are routinely used to identify the contribution of individual forms of cytochrome P450 in a particular biotransformation. p-Nitrophenol O-hydroxylation (PNPH) has been widely used as a measure of CYP2E1 catalytic activity. However, rat and human forms of CYP3A have also been shown to catalyze this activity. In mice, the contributions of CYP3A and CYP2E1 to PNPH activity are not known. Here we used hepatic microsomes from Cyp2e1(-/-) and wild-type mice to investigate the contributions of constitutively expressed and alcohol-induced murine CYP2E1 and CYP3A to PNPH activity. In liver microsomes from untreated mice, PNPH activity was much greater in wild-type mice compared with Cyp2e1(-/-) mice, suggesting a major role for CYP2E1 in catalyzing PNPH activity. Hepatic PNPH activities were not significantly different in microsomes from male and female mice, although the microsomes from females have dramatically higher levels of CYP3A. Treatment with a combination of ethanol and isopentanol resulted in induction of CYP3A proteins in wild-type and Cyp2e1(-/-) mice, as well as CYP2E1 protein in wild-type mice. The alcohol treatment increased PNPH activities in hepatic microsomes from wild-type mice but not from Cyp2e1(-/-) mice. Our findings suggest that in untreated and alcohol-treated mice, PNPH activity may be used as a specific probe for CYP2E1 and that constitutively expressed and alcohol-induced forms of mouse CYP3A have little to no role in catalyzing PNPH activity.

Effect of caffeine on acetaminophen hepatotoxicity in cultured hepatocytes treated with ethanol and isopentanol.

Pretreatment of cultured rat hepatocytes with ethanol alone or in combination with isopentanol, the major higher chain alcohol in alcoholic beverages, significantly increased CYP3A and acetaminophen (APAP) bioactivation, with no increase in APAP toxicity. Caffeine has previously been shown to activate CYP3A activity in vitro and to increase APAP hepatotoxicity in rodents pretreated with prototypic inducers of CYP3A. Here we found that caffeine enhanced APAP toxicity in cultured rat hepatocytes pretreated with the alcohols. The caffeine-mediated increase in APAP toxicity was similar in cells treated with ethanol or isopentanol alone or in combination. These findings suggest that even small increases in CYP3A are sufficient to support caffeine-enhanced APAP toxicity. Triacetyloleandomycin inhibited CYP3A activity in intact hepatocytes and protected alcohol-pretreated cells from caffeine enhancement of APAP toxicity. This protection was associated with decreased formation of the toxic metabolite of APAP. The results indicate that CYP3A is responsible for the caffeine-mediated stimulation of APAP toxicity. Our results suggest that caffeine may be an additional risk factor for developing alcohol-mediated APAP hepatotoxicity.