Gold sodium thiomalate (GSTM) inhibits lipopolysaccharide stimulated tumor necrosis factor-α through ceramide pathway

Cellular Immunology 219 (2002) 1–10 www.academicpress.com

Gold sodium thiomalate (GSTM) inhibits lipopolysaccharide stimulated tumor necrosis factor-a through ceramide pathway Ashutosh Kumar Mangalam, Amita Aggarwal, and Sita Naik* Department of Immunology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014, India Received 26 June 2001; accepted 2 October 2002

Abstract TNF-a has emerged as the major pro-inflammatory cytokine involved in the pathogenesis of rheumatoid arthritis (RA). LPS is a potent stimulator of TNF-a production by human monocytes. Ceramide, a structural homolog of LPS and a second messenger in the sphingomyelin signal transduction pathway has been shown to stimulate TNF-a production from murine macrophages. We have previously shown that GSTM, an anti-rheumatic drug inhibits LPS stimulated TNF-a production by normal PBMCs. We studied the ability of ceramide to stimulate TNF-a production by human PBMCs and the mechanism of action of GSTM on ceramide and LPS induced TNF-a production. LPS induced significant TNF-a production in PBMCs and THP-1. However, C2 ceramide stimulated TNF-a production in 5 of 10 PBMCs (ceramide responder); it did not do so in the other 5 PBMCs (ceramide non-responder) or the THP-1 cell line. GSTM inhibited LPS stimulated TNF-a productions in PBMCs of all 5 ceramide responders both at protein and mRNA expression level. We also found that GSTM inhibited LPS induced NF-jB level only in ceramide responder. Thus, we for the first time report that GSTM inhibits LPS stimulated TNF-a production through ceramide pathway and anti-inflammatory activity of GSTM in treatment of RA may depend on its ability to inhibit NF-jB activation and TNF-a production. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Human; Inflammation; Cytokines; Lipopolysaccharide; Ceramide; Inflammation; RT-PCR and transcription factors

1. Introduction Lipopolysaccharide (LPS) is a potent stimulator of TNF-a production by cells of monocyte/macrophage lineage [1]. This effect of LPS is mediated primarily by a serum protein, LPS-binding protein, and receptors on the surface of macrophages/monocytes, such as CD14, a GPI anchored receptor [2], and toll-like receptors [3,4]. LPS activates several intracellular signaling pathways; these include mitogen-activated protein kinases (ERK, JNK, and p38 kinases) [5–7], protein kinase C [8], srcrelated kinases (lyn and hck) [9], G proteins [10], and nuclear factor-jB (NF-jB) activating kinases ([11], reviewed in [12]). *

Corresponding author. Fax: +91-522-440017 or 440973. E-mail address: [email protected] (S. Naik).

Ceramide is a lipid messenger, generated from membrane sphingomyelin by the action of sphingomyelinase (SMase) [13] and has been shown to have structural homology with lipid A moiety of LPS [14]. It activates cytosolic ceramide-activated protein phosphatase (CAPP) [15] and membrane-associated ceramide-activated protein kinase (CAPK) [14], and has been shown to phosphorylate Raf-1 [16]. Ceramide has been reported to act as second messenger in LPS, TNF-a, and interleukin-1 (IL-1)-induced signaling [13,17]. The activation of these pathways is believed to cause nuclear translocation of transcription factors such as AP-1 and NF-jB, which control expression of various cytokines including TNF-a. Ceramide has recently been shown to mimic some functions of LPS such as expression of TNF-a and IL-1 by murine peritoneal macrophages [18,19]. However, its role in inducing production of

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inflammatory cytokines from human monocytes/macrophages is not well understood. TNF-a plays a pivotal role in synovial proliferation and joint destruction in patients with rheumatoid arthritis (RA) and is believed to be the major cytokine responsible for maintenance and progression of this disease [20–21]. Blockade of TNF pathway has been shown to ameliorate joint disease in murine collagen induced arthritis, an animal model of human RA [22–23]. Further, a TNF-blocking chimeric (human/mouse) monoclonal antibody (cA2) caused significant reduction in disease activity in patients with RA [24,25] through downregulation of several cytokines, cytokine inhibitors, acute phase proteins, leukocyte migration, and adhesion molecules expression [26]. Despite the fact that overproduction of TNF-a plays a major role in chronic inflammatory disease, little is known regarding regulation of its production. Gold sodium thiomalate (GSTM) is widely used as a disease-modifying agent for treatment of RA. The mechanism of its therapeutic action is, however, poorly understood, and has been attributed to inhibition of TNF-a and other pro-inflammatory cytokines [27–31]. In the current study, we looked at the ability of ceramide analog C2 to stimulate TNF-a production by human PBMCs and the molecular mechanisms by which GSTM suppresses TNF-a production in response to LPS and ceramide.

2. Materials and methods 2.1. Materials LPS, Escherichia coli 055B:5, n-acetyl sphingosine (C2 ceramide), RPMI-1640, Histopaque, oligo(dT)12–18 , NP40, acrylamide, bisacrylamide, and cocktail protease inhibitors were obtained from Sigma Chemicals (St. Louis, USA). GSTM was a kind gift from Dagenham Pharmaceuticals (Dagenham, UK). TNF-a ELISA kits were obtained from R&D system (Minneapolis, USA). FCS, Trizol, dithiothreitol, antibiotic mix were obtained from Life Technologies (Rockville, USA), gel shift assay system from Promega (Madison, USA), AMV-RT, Taq DNA polymerase and dNTPs from Bangalore Genei (Bangalore, India), and bovine serum albumin, sucrose and Tween 20 from SRL (Mumbai, India). LPS was dissolved in PBS, pH 7.2 and C2 ceramide in DMSO:ethanol (1:1). 2.2. Methods 2.2.1. Cell lines THP-1, a human monocytic cell line (National Center for Cell Sciences, Pune, India) was grown in complete RPMI-1640 (RPM1-1640 with 2 mM L -glutamine, 1 mM sodium pyruvate, 6.2 g/L sodium bicarbonate,

10 mM HEPES, and antibiotic–antimycotic mix) supplemented with 10% FCS. The cells were seeded at 1
105 cells/ml in 25 cm2 culture flasks (Tarson, Kolkota, India), grown at 37 °C in an atmosphere of 95% air and 5% CO2 and serially passaged every third day. 2.2.2. Cell culture Peripheral blood mononuclear cells (PBMCs) were separated from venous blood of healthy volunteers by density gradient centrifugation [32] using Histopaque, washed thrice with phosphate-buffered saline (PBS) and resuspended in complete RPMI-1640 supplemented with 10% FCS. PBMC cultures (1
106 cells/ml/well) were set up in duplicates with GSTM in different doses, along with LPS (10 ng/ml) [31] or C2 ceramide. THP-1 cells (1
106 cells/ml) were cultured in duplicate with varying doses of GSTM (10, 100, 1000, and 10000 ng/ml) and LPS (100, 500, 1000, 10,000, and 20,000 ng/ml) and C2 ceramide (5 and 20 lM). Cultures without stimulants or drug were set up as controls. All cultures were incubated at 37 °C in 5% CO2 and 95% humidity for 18 h; supernatants were collected by centrifugation and stored at )70 °C. The viability of cells at the end of the culture as estimated by trypan blue exclusion test and MTT dye reduction test was >85% in all cases. The maximal inhibition of TNF-a with GSTM was seen at the dose of 1000 ng/ml and this concentration was used in all further experiments [31]. 2.2.3. ELISA for TNF-a TNF-a was measured in culture supernatants using a commercial sandwich ELISA according to manufacturerÕs protocol. In brief, plates were coated with antihuman TNF-a in PBS (pH 7.3) and non-specific binding was blocked with 1% BSA, 5% sucrose, and 0.05% Tween 20 in PBS. Samples and standards (recombinant human TNF-a, 15–1000 pg/ml) were added to duplicate wells. Biotinylated polyclonal anti-TNF-a was used as detection antibody, followed by addition of streptavidin-HRP in dilution buffer. Color was developed using TMB as substrate and OD was taken at 450 nm. Reduction of P 20% in TNF-a production on addition of GSTM as compared to TNF-a production in the presence of either ceramide or LPS alone was defined as significant suppression [31]. 2.2.4. RT-PCR for TNF-a Total RNA was extracted from 2
106 PBMCs using Trizol reagent according to manufacturerÕs instruction. Approximately 1–5 lg of total RNA was reverse transcribed to first strand cDNA using oligo(dT) at 37 °C. The reaction mix had 2 ll of 10
reverse transcription buffer, 1 ll of oligo(dT)12–18 (0.5 mg/ml), 10 IU AMVRT, 1 ll of 10 mM dNTP, 2 ll of 100 mM dithiothretiol, DEPC treated water to make a final volume of 20 ll. PCR for TNF-a and, b-actin, was carried out using

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Table 1 Nucleotide sequence of primers for b-actin and TNF-a SN

Gene

Primer sequence

1

b-Actina

50 -CACTCTTCCAGCCTTCCTTCC-30 (forward) 50 -CGGACTCGTCATACTCCTGCTT-30 (reverse)

2

TNF-ab

50 -GAGTGACAAGCCTGTAGCCCATGTTGTAGCA-30 (forward) 50 -GCAATGATCCCAAAGTAGACCTGCCCAGACT-30 (reverse)

a b

Ref. [31]. Ref. [34].

1–5 ll of cDNA and specific primers (Table 1). cDNA was amplified by addition of 50 pmol of each primer to the PCR reaction mixture containing 5 ll of 10-fold PCR buffer (100 mM Tris–HCl, 500 mM KCl, 15 mM MgCl2 , and 0.01% gelatin), 200 lmol of dNTP mixture and 1.5 U Taq DNA polymerase. PCR cycling conditions were initial denaturation at 95 °C for 2 min, followed by 30–40 cycles of 94, 55, and 72 °C for 1 min each, with a final extension at 72 °C for 10 min. PCR products were subjected to 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining.

OCT-1 (Table 2) were end-labeled using ½32 P]dATP. Binding reaction was performed with 4 lg of the nuclear extract and radiolabelled probe in the presence of 1 lg of poly(dI:dC) in a binding buffer (25 mM Hepes, pH 7.9, 0.5 mM EDTA, 0.5 mM DTT, 1% NP-40, and 50 mM NaCl) for 20 min at 37 °C, as described previously [36]. The DNA–protein complex was separated from free oligonucleotide on a 7.5% native polyacrylamide gel in a buffer containing 50 mM Tris, 200 mM glycine, pH 8.5, and 1 mM EDTA [37]. The gel was exposed to X-ray film for 48 h and developed.

2.2.5. Electrophoretic mobility shift assay (EMSA) Cultures were set up as described earlier except for the difference in cell number. PBMCs (4
106 ) or THP1 (2
106 ) cells in complete RPMI supplemented with 10% FCS were treated for 1 h with various doses of GSTM followed by addition of LPS. PBMCs were incubated for a further 1 h and THP-1 for 2 h [33]. Control cultures were set up without LPS or GSTM. Nuclear extract was prepared according to the method of Schreiber et al. [34] with minor modifications. Briefly, the cells were washed with cold PBS, suspended in 0.4 ml of a lysis buffer (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and cocktail of protease inhibitors) for 25 min and lysed by addition of 12.5 ll of 10% NP-40. Nuclear pellet collected by brief centrifugation was resuspended in 25 ll of ice-cold extraction buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and cocktail of protease inhibitors), incubated at 4 °C for 30 min to lyse the nuclei. Supernatant containing nuclear extract was then collected by centrifugation and protein concentration was measured by the method of Bradford [35]. For electrophoretic mobility shift assay, doublestranded oligonucleotides of NF-jB, AP-1, AP-2, and

2.2.6. Statistical analysis Paired data were analyzed using WilcoxonÕs signed rank test.

3. Results 3.1. Effect of ceramide on TNF production Freshly isolated human PBMCs (n ¼ 10, mean age 30  8 years; 6 males & 4 females) were stimulated with LPS or C2 ceramide, a cell permeable analog of ceramide, for 18 h and TNF-a levels were measured in the cell free supernatant. In preliminary experiments PBMCs were stimulated with different concentration of C2 ceramide (0.1, 1, 5, and 20 lM) and maximum increase in TNF-a production was seen with 5 lM dose in some of the samples while it failed to induce TNF-a production in others at all the tested doses (data not shown). Spontaneous median TNF-a production was 875 pg/ml (range 160–4400 pg/ml); LPS at a dose of 10 ng/ml significantly stimulated TNF-a to 2000 pg/ml (580–6720 pg/ml, p < 0:01, Fig. 1). C2 ceramide (5 lM) did not significantly increased TNF-a level (median

Table 2 Oligonucleotide sequences of probes used in electrophoretic mobility shift assay (EMSA) Transcription factor

Sequence

AP-1 AP-2 Oct-1 NF-jB

50 50 50 50

CGC TTG ATG AGT CAG CCG GAA 30 GAT CGA ACT GAC CGC CCG CGG CCC GT 30 TGT CGA ATG CAA ATC ACT AGA A 30 AGT TGA GGG GAC TTT CCC AGG C 30

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Fig. 1. TNF-a production by PBMCs from healthy human volunteers ðn ¼ 10Þ stimulated with LPS or C2 ceramide. Box plots show median and range of TNF-a levels in PBMCs and bars show 25th and 75th percentile. ‘‘*’’ represents outlier. *Statistical analysis (Wilcoxon signed rank test) revealed significant differences between unstimulated and LPS stimulated values ðp < 0:01Þ; differences between unstimulated and C2 stimulated was not significant.

1300 pg/ml; range 160–8000 pg/ml; Fig. 1). Nine of the 10 individuals showed TNF-a of >1.5 times the unstimulated values in response to LPS, however among these 10 individuals, PBMCs from 5 subjects showed ceramide stimulated TNF-a levels exceeding 1.5 times the basal levels (responders) and remaining 5 subjects failed to show such a rise (non-responder). The mean (SD) TNF-a level of the 5 responders was 1526  1722 pg/m without stimulant, 3860  2598 pg/ml in response to LPS stimulation ðp < 0:05Þ and 3800  2668 pg/ml in response to C2 ceramide ðp < 0:05Þ (Fig. 2A). The mean (SD) spontaneous TNF-a level in the 5 non-responder PBMCs was 1224  572 pg/ml and LPS caused a significant stimulation in TNF-a levels of all but one subject with a mean TNF-a level of 1852  885 pg/ml. The mean TNF-a level in C2 ceramide (5 lM) treated cultures was 684  539 pg/ml, which was comparable to spontaneous TNF-a level (Fig. 2B). Vehicle control (DMSO:ethanol) did not have any effect on TNF-a production (Figs. 2A and B). 3.2. Effect of GSTM on spontaneous, ceramide, and LPS stimulated TNF-a production To study the effect of GSTM on LPS or C2 ceramide stimulated TNF-a production, PBMCs were simultaneously treated with drug and LPS or C2 ceramide for 18 h. TNF-a levels were determined in cell free supernatants. GSTM suppressed LPS induced TNF-a production in the ceramide responders but not in the non-responders (2A and B, respectively). Interestingly,

Fig. 2. Effect of GSTM on LPS and ceramide stimulated TNF-a production by human PBMCs of ceramide responders (A) and nonresponders (B) ðn ¼ 5Þ. Control cultures were set up without stimulant or drug. *Difference between LPS and LPS plus GSTM and C2 ceramide and C2 ceramide plus GSTM was significant at p < 0:05 in ceramide responder group by Wilcoxon signed rank test for paired sample. Inset picture shows effect of GSTM on LPS or ceramide stimulated TNF-a gene transcription in human PBMCs of ceramide responder (A, lanes 1–9) and ceramide non-responder (B, lanes 1–6). Lower panel represent internal control b-actin. Lane 1, unstimulated; lane 2, LPS; lane 3, LPS + GSTM (10 ng/ml); lane 4, LPS + GSTM (100 ng/ml), lane 5, LPS + GSTM (1000 ng/ml); lane 6, C2 ceramide; lane 7, C2 ceramide + GSTM (10 ng/ml); lane 8, C2 ceramide + GSTM (10 ng/ml); lane 9, C2 ceramide + GSTM (10 ng/ml); lane 10, 100 bp DNA ladder.

GSTM also suppressed ceramide induced TNF-a production in human PBMCs of ceramide responders (p < 0:05, Fig. 2A) but failed to affect TNF-a level in ceramide non-responders (Fig. 2B). These results show that GSTM inhibits LPS induced TNF-a production only in ceramide responders. 3.3. Effect of ceramide and GSTM on TNF-a gene expression level To determine whether GSTM-mediated suppression in TNF-a protein secretion is at transcriptional level, RT-PCR assay was performed. PBMCs were cultured either in presence of drug plus LPS/C2 ceramide or left untreated and after 18 h mRNA levels were estimated by RT-PCR. Results demonstrated that after normalization for the b-actin signal, C2 ceramide at 5 lM dose induced an increase in TNF-a mRNA expression

A.K. Mangalam et al. / Cellular Immunology 219 (2002) 1–10

in ceramide responder (Fig. 2A, (inset) lane 6); this increase was comparable to that seen with LPS (Fig. 2A, (inset) lane 2). In ceramide responder, both LPSstimulated and C2 ceramide-stimulated TNF-a mRNA showed a dose dependent decrease with GSTM (Fig. 2A, (inset) lanes 3–5 and 7–9, respectively). In ceramide non-responder, LPS upregulated TNF-a mRNA expression but C2 ceramide did not (Fig. 2B (inset), lanes 2 and 6, respectively). GSTM did not have any effect on LPS-stimulated TNF-a mRNA expression in PBMC of the non-responder (Fig. 2B (inset), lanes 3–5). 3.4. Effect of GSTM on LPS stimulated transcription factors To delineate role of the transcription factors in GSTM-mediated TNF-a suppression, PBMCs were preincubated for 1 h with different concentrations of GSTM and then treated with LPS (10 ng/ml) for 1 h at 37 °C. Nuclear extracts were prepared and assayed for NF-jB, AP-1, AP-2, and Oct-1 activation by EMSA. LPS stimulated cells showed a significant increase in nuclear levels of NF-jB (Fig. 3A, lane 2); there was a dose dependent decrease of NF-jB in GSTM treated cells of C2 ceramide responder (Fig. 3A, lanes 3–5). Higher doses of GSTM reduced NF-jB almost to basal level. LPS stimulated cells showed an increased nuclear level of AP-1 (Fig. 3B, lane 2); GSTM had no

Fig. 3. Effect of GSTM on LPS induced DNA binding activity of NFjB (A) and AP-1 (B) of ceramide responder and non-responder PBMC. Samples were separated on a native 1
Tris–glycine–EDTA polyacrylamide gel and exposed to an X-ray film.

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effect on AP-1 levels at any of the doses used (Fig. 3B, lanes 3–5). In non-responder PBMC, although LPS caused a significant increase in NF-jB and AP-1 levels (Fig. 3A, lane 2 and Fig. 3B, lane 2, respectively), GSTM did not have any effect on activated nuclear level of these transcription factors (Figs. 3A and B, lanes 3–5, respectively). LPS showed no effect on nuclear levels of AP-2 and OCT-1 transcription factors in PBMCs of both responder and non-responder (data not shown). 3.5. Effect of ceramide and GSTM on TNF-a production in human monocytic cell line THP-1 The effect of GSTM on LPS and C2 ceramide stimulated TNF-a production was also studied in the human monocytic cell line, THP-1. In order to optimize the dose of LPS, THP-1 was stimulated with varying concentration of LPS (100, 500, 1000, 10,000, and 20,000 ng/ml). Since the TNF-a levels plateaued at 1000 ng/ml, this dose was used for further experiments (data not shown). In contrast to LPS, C2 ceramide at doses of 5 lm and 20 lM did not show any stimulation of TNF-a (Fig. 4); GSTM also did not suppress LPS-induced TNF-a levels or TNF-a gene expression (Fig. 5, lane 6 in inset). GSTM did not have any affect on TNF-a in C2 ceramide treated cultures (Fig. 5). LPS upregulated nuclear level of NF-jB and AP-1 (Figs. 6A and B, lane 2); however GSTM failed to suppress these transcription factors (Figs. 6A and B, lanes 3–5). Neither LPS nor GSTM had any effect on AP-2 and OCT-1 levels (data not shown). These data show that THP-1 behaved like PBMCs from ceramide non-responder in that ceramide failed to induce TNFa production, with significant LPS induced TNF-a production. GSTM failed to inhibit LPS induced TNF-a production in these cells. LPS induced nuclear level of NF-jB and AP-1 was also unaffected by GSTM.

Fig. 4. Effect of C2 ceramide on TNF-a production from THP-1 cells. The data are means of three separate experiments.

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Fig. 5. Effect of GSTM on LPS stimulated TNF-a levels in THP-1 cells. The data are means of three separate experiments. Inset picture shows mRNA expression of TNF-a (lanes 4–6) and b-actin (lanes 1–3) in THP-1 cells. Lanes 1 and 4, unstimulated; lane 2 and 5, LPS (1000 ng/ml); lanes 3 and 6, LPS (1000 ng/ml) + GSTM (1000 ng/ml).

Fig. 6. Effect of GSTM on LPS induced DNA binding activity of NFjB and AP-1 in THP-1 cells. Samples were separated on a native polyacrylamide gel in 1
Tris–glycine–EDTA buffer and exposed to an X-ray film.

4. Discussion The sphingomyelin pathway is a newly described signal transduction pathway mediating the action of several extracellular responses [13,38]. Sphingomyelin is hydrolyzed by sphingomyelinase (SMase), a sphingomyelin-specifc form of PLC, to produce phosphoryl choline and ceramide. Ceramide in turn is converted to glucosyl-ceramide by glucosyl transferase and to sphingosine by ceramidase, which remove a fatty acid from ceramide [39]. Ceramide has emerged as a second messenger molecule which is considered to mimic most of the cellular effects of LPS, IL-1b, and TNF-a in proinflammatory responses, terminal differentiation, apoptosis, and cell cycle arrest and it appears that a complex

interaction between LPS and ceramide exists in the proinflammatory response. In this study we investigated the involvement of sphingomyelin pathway involved in activation of human PBMCs by LPS. We found that ceramide mimics LPS by inducing production of TNF-a in human PBMC; however, this effect was seen only in a subgroup of normal PBMCs (ceramide responders). LPS induced TNF-a levels in 9 of 10 PBMCs and the THP-1 cell line, whereas C2 ceramide could induce TNF-a only in 5 of these 10 PBMCs. When we used an anti-rheumatic gold compound GSTM to block LPS and ceramide stimulated TNF-a production the drug suppressed both ceramide and LPS induced TNF-a production only in ceramide responders which was due to inhibition of NFjB activation and resultant decrease in TNF-a gene transcript. This is the first report describing TNF-a production by human PBMCs on ceramide stimulation and is similar to reports in mouse macrophages and macrophage cell lines [18,19]. Sphingomyelinase and cell permeable analogues of ceramide have been shown to induce cellular responses that were previously reported with LPS such as the induction of interleukin-6 (IL-6) gene expression in human fibroblasts [40], accumulation of inducible nitric oxide synthase in RAW 264.7 macrophages [41], induction of IL-6 production by rat glomerular mesangial cells [42], and of TNF-a by murine macrophages [19,41]. The most convincing evidence for a link between ceramide and LPS signaling pathways was provided by the observation that macrophages derived from LPS hypo-responsive mice failed to respond to ceramide [18], whereas LPS-responsive (Lpsn ) macrophages could be stimulated by ceramide to increase expression of LPS induced genes such as TNF-a and IL-1b. It has also been shown that ceramide/sphingomyelin pathway is not responsible for all cellular function of LPS since LPS activation results in production of TNF-a and IFN-c protein, while SMase and ceramide analogs only activate TNF-a and have no effect on IFN-c [19]. In this study pre-exposure of mouse macrophages to LPS made them refractory to challenge with SMase or LPS, while pre-exposure to Smase did not result in macrophages being refractory to restimulation with LPS or SMase [19]. Together with these observations, our data suggests that ceramide does not mimic the entire LPS signaling pathway and ceramide pathway may be one of the several pathways used by LPS for its function. This does not seem surprising for a biologically important molecule such as LPS. The TNF-a production by PBMCs in response to ceramide was heterogeneous, and the population was divided into two groups based on an arbitrary cut-off of 1.5-fold increase in TNF-a levels in response to stimulation with ceramide analog C2 . Five individuals were

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ceramide responders while five showed no increase in TNF-a production and were labeled as ceramide nonresponders. Interestingly ceramide also failed to induce TNF-a production in the human monocytic cell line, THP-1 which is LPS responsive. Whereas ceramide analogs have been shown to stimulate TNF-a, IL-6, and inducible nitric oxide (NO) from human or mice cells [19,40,41,42], they have failed to stimulate TNF-a production from rabbit alveolar macrophages and rat microglia cells. C6 ceramide also failed to induce production of TNF-a from rabbit alveolar macrophages [43] while C8 ceramide failed to induce TNF-a, IL-1b, and NO from rat microglia cells [44]. In a study investigating effect of ceramide on LPS induced nitric oxide synthase induction from mouse macrophages, C2 ceramide inhibited LPS-mediated NO by murine macrophage cell line, RAW 264.7, while it failed to alter LPS-mediated NO induction in peritoneal macrophages isolated from BALB/c mice [45]. A different form of glycosphingolipid carrying zwitterionic substitutents like phosphocholine showed wide heterogeneity in its potential to induce cytokines by human cell lines (differentiated and undifferentiated) like U937, THP-1, and HL-60, as was the difference between mouse and human PBMCs ([46], G. Lochnit, personal communication). These studies suggest that the cellular response to ceramide is very variable and is influenced both by the responding cell type and its genetic background. This heterogeneity may be reflected in the varied responses of human PBMCs to ceramide. Inhibition of LPS activated pro-inflammatory cytokines by GSTM seen in our study is in agreement with previous reports [27–31]. Only Bondenson et al. [47] have failed to show any suppression of LPS activated TNF-a by GSTM which could be due to the difference in the cell type (murine macrophages vs. human PBMCs in our study) used in that study. In this study, we found that GSTM inhibited LPS stimulated TNF-a production only in 5 of 10 normal PBMCs and, surprisingly all five were ceramide responders. In contrast, there was no effect of GSTM on LPS induced TNF-a by PBMCs of five ceramide non-responders both at protein as well mRNA level. Thus it seems that GSTM inhibits TNF-a production at the transcriptional level and may affect some post-receptor signal transduction step involved in the induction of TNF-a. We and others have previously found that LPS response in the population is heterogeneous [48,31] and the present data show that only some LPS responders have C2 ceramide response; GSTM suppression appears to be related to their ability to respond to ceramide. Concordance of GSTM inhibition of LPS induced TNF-a production and C2 ceramide induced TNF-a production suggests that GSTM inhibits TNF-a production through ceramide pathway. This was further supported by the data on THP-1 cell line, where GSTM failed to inhibit LPS

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induced TNF-a production both at protein and mRNA level. TNF-a secreted by macrophages in rheumatoid synovium, is thought to be the key cytokine responsible for maintenance of disease in RA and is strongly regulated by NF-jB. Active form of NF-jB is detected in the nucleus of synovial macrophages and endothelial cells suggesting a role for NF-jB in expression of inflammatory genes in these cells [49,50]. Thus it is quite possible that GSTM downregulates TNF-a by blocking activation of NF-jB. This was indeed true, as we found that GSTM is a potent and specific inhibitor of NF-jB activation in cells where there was suppression of TNFa production. GSTM failed to inhibit LPS induced NF-jB activation in ceramide non-responder PBMC and THP-1 cell line. In addition, GSTM did not inhibit activation of AP-1, which was induced by LPS and also had no effect on AP-2 and OCT-1. The differential effect of GSTM on LPS stimulated NF-jB and AP-1 seen in this study is in agreement with data with other anti-inflammatory agents. Wahl et al. [51] reported that sulphasalazine, another DMARD, specifically suppressed activated levels of NF-jB in LPS induced SW260 colon cells. Manna et al. [52] has shown that the anti-inflammatory tridecapeptide, a-melanocyte stimulating hormone (MSH) completely abolished NFjB activation induced by LPS but activation of AP-1 by LPS was unaffected. LPS-induced activation of NFjB has been shown to be suppressed by another gold compound, auranofin in a mouse macrophage cell line RAW 264.7 [53]. Thus, the beneficial effect of GSTM in RA appears to be mainly due to inhibition of NF-jB activation while AP-1 does not seem to play a role. Previous studies have shown that blockade of NF-jB activation by adenoviral transfer of IjBa suppressed expression of TNF-a in cultured human synoviocytes [54] and intracellular injection of an oligo deoxynucleotide containing IjB binding sequence (NF-jB decoy) inhibited development of joint inflammation in an animal model of arthritis [55]. We hypothesize that the inhibition of NF-jB by GSTM is through the ceramide pathway and this is the first report on the molecular mechanism of action of GSTM in human PBMCs. LPS induces NF-jB by activating IKK, which phosphorylates IjB with release of active NF-jB that in turn translocates to nucleus and activates target gene(s). The intermediate steps in the ceramide-signaling pathway, which results in inhibition of NF-jB activation are not known but following proteins stimulated by ceramide could be the targets. The signaling molecules that are activated by ceramide are, cytosolic ceramide-activated protein phosphatase (CAPP) [15], membrane-associated ceramide-activated protein kinase (CAPK) [14], PKC-f [56], stress activated protein kinase [41] and phosphorylation of Raf-1 [57]. Ceramide-activated

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protein kinase and PKC-f have been shown to have definite role in LPS-mediated signal transduction such as the activation of MAP kinase and nuclear localization of NF-jB and AP-1, which result in increased expression of TNF-a [14,58]. It is possible that GSTM downregulates increased levels of TNF-a and NF-jB by blocking one of these pathways. It has been previously reported that hydrophilic gold compounds such as GSTM, aurothioglucose, and AuCl3 suppress in vitro IKK enzyme activity by blocking IKKa and IKKb and thus inhibiting NF-jB activation [53]. Glucocorticoids, sulphasalazine, aspirin, and sodium salicylate that are frequently used for treatment of RA have also been shown to suppress NF-jB activation [11,51,59]. In summary, our data shows that ceramide can mimic LPS function and there is heterogeneity in the ceramide response in normal population. We report for the first time that GSTM inhibits expression of pro-inflammatory cytokine TNF-a by blocking LPS-mediated induction of NF-jB through the ceramide pathway. The heterogeneity in the ceramide response of human PBMCs might explain the heterogeneity of response of patients with RA to chrysotherapy.

[6]

[7]

[8]

[9]

[10]

[11]

[12] [13]

[14]

Acknowledgments We thank S.R. Naik and R. Aggarwal for their critical review of the manuscript and Dagenham Pharmaceuticals, U.K. for providing preservative-free gold sodium thiomalate. We also thank Dr. G unter Lochnit of Justus-Liebig University, Giessen, Germany for his advice and comments on this manuscript. This work was supported in part by a research grant from Council of Scientific and Industrial Research (CSIR), New Delhi, India. The authors acknowledge to Japan International Co-operation Agency (JICA) for grant-in-aid to this institution.

[15] [16]

[17] [18]

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