Inhibitory effect of esculentoside A on prostaglandin E(2) production from murine peritoneal macrophages and rabbit synovial cells in vitro

Esculentoside A (EsA) is a saponin isolated from the roots of Phytolacca esculenta. Previous experiments have shown that it has strong antiinflammatory effects. To investigate the mechanism of anti-inflammatory effects of esculentoside A (EsA),[(3)H] arachidonic acid (AA) prelabelled murine macrophage and radioimmunoassay were used to test the effect of EsA on the total release of AA and prostaglandin E(2) in culture supernatants. The results showed that EsA had no significant effect on the total release of AA from murine macrophages. EsA (2.5-10 mumol/l), from unstimulated murine peritoneal macrophages and rabbit synovial cells, could decrease the production of prostaglandin E(2). In A(23187) and LPS-treated macrophages and synovial cells, EsA (10 mumol/l) could significantly decrease the prostaglandin E(2) production. These results confirmed that EsA exerted an inhibitory effect on prostaglandin E(2) production from murine macrophages and rabbit synovial cells.     

Interleukin-1 production by mononuclear cells from rheumatoid synovial effusions

The polypeptide interleukin-1 (IL-1) is a cytokine that may mediate inflammation and connective tissue damage in rheumatoid arthritis (RA). We examined cytokine production by normal blood and by rheumatoid synovial mononuclear cells with sensitive (picomolar) assays. The assays were immunolabeling and immunoblotting with rabbit anti-IL-1 beta sera, and proliferation of the murine D10 cell line to IL-1. Little or no cytokine was detected in rheumatoid joint fluid or in exudate mononuclear cells from patients with acute rheumatoid flares. The mononuclear cells could be induced to make IL-1 upon stimulation with lipopolysaccharide (LPS). The responsive cells were monocytes, since all could be double-labeled with anti-IL-1 and the monocyte-specific CD14 antibody. More than 80% of the synovial fluid monocytes made IL-1 beta after 24 hr in 2 ng/ml LPS. Other agents failed to induce IL-1 from enriched populations of monocytes including interferon gamma (IFN-gamma), poly (I/C), phorbol myristate acetate (PMA), concanavalin A (Con A), phytohemagglutinin (PHA), and anti-CD3 antibodies. Relatively high levels of dendritic cells (DC) were present in RA effusions, but these did not produce IL-1 in response to any of the above stimuli. Blood dendritic cells also did not make IL-1, whereas blood monocytes responded comparably to synovial exudate cells. The data indicate that rheumatoid exudate monocytes make very little IL-1 during acute flares of arthritis and that this cytokine is primarily a macrophage rather than a dendritic cell product.     

IL-6/IFN-beta 2 in synovial effusions of patients with rheumatoid arthritis and other arthritides. Identification of several isoforms and studies of cellular sources

We have looked for IL-6, a cytokine that has immunomodulating and inflammation-associated activities, in joint exudates (fluid and mononuclear cells) from patients with rheumatoid arthritis and other arthritides using both biologic and biochemical assays. IL-6 was assessed by its ability to stimulate alpha 1-antichymotrypsin secretion from the human hepatoma cell line Hep3B clone 2, an activity which is blocked by an antiserum to Escherichia coli derived IL-6, and by the growth of the IL-6-dependent murine hybridoma 7TD1 cell line. IL-6 isoforms in synovial fluid were characterized by immunoaffinity chromatography followed by Western blotting. The presence of IL-1 in synovial fluids and its production by synovial fluid mononuclear cells was monitored by Western blotting and indirect immunofluorescence with polyclonal anti-IL-1 beta antisera. In an analysis of 30 effusions from 27 rheumatoid patients with acutely inflamed joints, abundant quantities of IL-6 (greater than 2 ng/ml) were detected in 23 by the alpha 1-antichymotrypsin bioassay. Several rheumatoid synovial fluids also had elevated IL-6 levels in the 7TD1 bioassay. Seven of nine nonrheumatoid effusions also contained high levels of IL-6 (greater than 2 ng/ml). No IL-1 (less than 0.25 ng/ml) could be detected by Western blotting in 10 rheumatoid effusions even though eight of these contained high levels of IL-6. The IL-6 activity could be neutralized with a rabbit antiserum to rIL-6. Multiple IL-6 isoforms (25, 30, 45 kDa) were present in two rheumatoid and one traumatic effusion studied. Fresh mononuclear cells isolated from various synovial effusions did not appear to make IL-6 constitutively, as no IL-6 could be detected in the media of cells cultured for 12 to 18 h after isolation. Similarly, there was no constitutive production of IL-1 by these cells. However, synovial fluid mononuclear cells could be induced to secrete both IL-6 and IL-1 after stimulation with LPS. The LPS-responsive cells were monocytes and not lymphocytes or dendritic cells. These findings suggest that IL-6 is involved in inflammatory joint disease. However, the primary cells synthesizing it may be located in the synovial lining instead of the joint exudate.     

IL-27-producing CD14(+) cells infiltrate inflamed joints of rheumatoid arthritis and regulate inflammation and chemotactic migration

Interleukin (IL)-27, a heterodimeric cytokine, has been reported to be involved in the pathogenesis of autoimmune diseases through mediating differentiation of Th1 or Th17 cells and immune cell activity or survival. However, the origin and effects of IL-27 in joints of rheumatoid arthritis (RA) remain unclear. In this study, we investigated the distribution and anti-inflammatory roles of IL-27 in RA synovium. The IL-27 levels in plasma of RA patients, osteoarthritis (OA) patients, or healthy volunteers (n=15 per group) were equivalent and were at most 1 ng/ml, but the IL-27 level in synovial fluid of RA patients (n=15, mean 0.13 ng/ml; range 0.017-0.37 ng/ml) was significantly higher than that in synovial fluid of OA patients (n=15, mean 0.003 ng/ml; range 0-0.033 ng/ml) and potentially lower than in plasma. We analyzed the protein level of IL-27 produced by RA fibroblast-like synoviocytes (FLSs) or mononuclear cells (MNCs) from RA or OA synovial fluid or peripheral blood and showed that IL-27 in RA joints was derived from MNCs but not from FLSs. We also found by flow cytometry that IL-27-producing MNCs were CD14(+), and that these CD14(+)IL-27(+) cells were clearly detected in RA synovium but rarely in OA synovium by immunohistochemistry. Furthermore, we demonstrated that a relatively physiological concentration of IL-27 below 10 ng/ml suppressed the production of IL-6 and CCL20 from RA FLSs induced by proinflammatory cytokines through the IL-27/IL-27R axis. In the synovial fluid of RA, the IL-27 level interestingly had positive correlation with the IFN-γ level (r=0.56, p=0.03), but weak negative correlation with the IL-17A level (r=-0.30, p=0.27), implying that IL-27 in inflammatory joints of RA induces Th1 differentiation and suppresses the development or the migration of Th17 cells. These findings indicate that circulating IL-27-producing CD14(+) cells significantly infiltrate into inflamed regions such as RA synovium and have anti-inflammatory effects in several ways: both directly through the reduction of IL-6 production, and possibly through the induction of Th1 development and the suppression of Th17 development; and indirectly by regulation of recruitment of CCR6(+) cells, such as Th17 cells, through the suppression of CCL20 production. Our results suggest that such a serial negative feedback system could be applied to RA therapy.     

Immunostimulating effects of muramyl dipeptide, glucosaminyl muramyl dipeptide and their synthetic derivatives in vitro

The effect of muramyldipeptide (MDP), glucosaminylmuramyldipeptide (GMDP) and their six synthetic derivatives on production of tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-2 (IL-2) by murine spleen cells in vitro was studied. MDP induced insignificant TNF production and did not stimulate production of IL-1 by the murine splenocytes within a 24-hour cultivation period whereas in combination with lipopolysaccharide (LPS) it induced significant production of both the cytokins. GMDP induced marked production of TNF (54 per cent cytotoxic index) and IL-1 (stimulation index 8). Addition of LPS in an amount of 10 ng/ml increased production of TNF by the murine splenocytes under the effect of GMDP but had no effect on production of IL-1. Neither MDP nor GMDP even in combination with LPS induced production of IL-2 by splenocytes of mice DVA/2 and C57B1/6 at activation for 24 hours. All the synthetic derivatives of MDP and GMDP except the MDP polymer activated TNF production by the murine spleen cells. GMDP lysine had the highest effect: 67 per cent cytotoxic index. In combination with LPS its cytotoxic index amounted to 87 per cent. The TNF activity was always higher when LPS in an amount of 10 ng/ml was added to the glycopeptides.     

Modulation of IL-1 inflammatory and immunomodulatory properties by IL-6.

Among the major cytokines present in inflammatory lesions interleukin-1 (IL-1), tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6) share many biological activities. Since IL-1 alpha, IL-1 beta and TNF alpha have been previously demonstrated to play an important role in connective tissue destruction by stimulating the production of prostaglandin E2 (PGE2) and collagenase, these functions were investigated in the presence or absence of natural human IL-6 (nhIL-6) or recombinant human IL-6 (rhIL-6). IL-6 was found 1 degree to stimulate immunoglobulin A production by the CESS B cell line up to 19 fold without being affected by the presence of IL-1 beta and 2 degrees to stimulate murine thymocytes proliferation up to 2-4 fold, with an increase up to 60-fold in costimulation with either IL-1 alpha or beta. IL-6 alone, even at very high concentrations (up to 200 U/ml and 50 ng/ml), did not induce PGE2 production by fibroblasts and synovial cells. However, IL-1 alpha or beta induced PGE2 production by human dermal fibroblasts and by human synovial cells was inhibited (in 5/8 experiments) up to 62% by addition of IL-6. On the contrary in 2/4 experiments TNF alpha-induced PGE2 production was increased (approximately 2 fold) by the addition of IL-6. IL-1 and TNF alpha-induced collagenase production in synovial cells remained unchanged in the presence of IL-6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual effects of pertussis toxin on lymphoid cells in culture

Pertussis toxin (Ptx), a component of Bordetella pertussis, is responsible for many of the biological activities of this bacterium, including its potent adjuvant capacity. In attempt to better understand the Ptx activity on the immune response in vivo, we have examined the effect of Ptx on certain lymphoid cell responses in vitro which could be targets for the adjuvant activity of this molecule. Ptx was found to stimulate a variety of cell responses which include (a) increased production and release of interleukin-1 (IL-1) by human monocytes and murine macrophages; (b) co-mitogenesis, in combination with IL-1, in cultures of murine thymocytes; (c) mitogenesis in cultures of various peripheral lymphocytes; (d) increased production of IL-2 in cultures of human blood lymphocytes and rodent splenocytes; and (e) elevated release of IL-3 in cultures of murine spleen cells. In addition to its stimulatory effects, however, Ptx was found to inhibit responses of both mononuclear phagocytes and lymphocytes to other stimuli. Most activities of Ptx in vitro were achieved at the optimal concentration range of 1-10 micrograms/ml, which is 100-1000 times higher than that showing adjuvant effects in vivo. Possible explanations for the dual effect of Ptx and for the discrepancy in doses optimal for the effects in vivo and in vitro are discussed.     

Blockade of Toll-like receptor 2 prevents spontaneous cytokine release from rheumatoid arthritis ex vivo synovial explant cultures

Introduction

The aim of this study was to examine the effect of blocking Toll-like receptor 2 (TLR2) in rheumatoid arthritis (RA) synovial cells.

Methods

RA synovial tissue biopsies, obtained under direct visualization at arthroscopy, were established as synovial explant cultures ex vivo or snap frozen for immunohistology. Mononuclear cell cultures were isolated from peripheral blood and synovial fluid of RA patients. Cultures were incubated with the TLR1/2 ligand, Pam3CSK4 (200 ng, 1 and 10 μg/ml), an anti-TLR2 antibody (OPN301, 1 μg/ml) or an immunoglobulin G (IgG) (1 μg/ml) matched control. The comparative effect of OPN301 and adalimumab (anti-tumour necrosis factor alpha) on spontaneous release of proinflammatory cytokines from RA synovial explants was determined using quantitative cytokine MSD multiplex assays or ELISA. OPN301 penetration into RA synovial tissue explants cultures was assessed by immunohistology.

Results

Pam3CSK4 significantly upregulated interleukin (IL)-6 and IL-8 in RA peripheral blood mononuclear cells (PBMCs), RA synovial fluid mononuclear cells (SFMCs) and RA synovial explant cultures (P < 0.05). OPN301 significantly decreased Pam3CSK4-induced cytokine production of tumour necrosis factor alpha (TNF-α), IL-1β, IL-6, interferon (IFN)-γ and IL-8 compared to IgG control in RA PBMCs and SFMCs cultures (all P < 0.05). OPN301 penetration of RA synovial tissue cultures was detected in the lining layer and perivascular regions. OPN301 significantly decreased spontaneous cytokine production of TNF-α, IL-1β, IFN-γ and IL-8 from RA synovial tissue explant cultures (all P < 0.05). Importantly, the inhibitory effect of OPN on spontaneous cytokine secretion was comparable to inhibition by anti-TNFα monoclonal antibody adalimumab.

Conclusions

These findings further support targeting TLR2 as a potential therapeutic agent for the treatment of RA.     

The effect of pseudomonas exotoxin A on cytokine production in whole blood exposed to Pseudomonas aeruginosa

To determine the effect of Pseudomonas aeruginosa exotoxin A (P-ExA) on cytokine production, we studied cytokine release induced by heat-killed P. aeruginosa (HKPA) in human whole blood in the presence or absence of P-ExA. P-ExA (0.01-1 microgram ml(-1)) caused a dose-dependent decrease in HKPA-induced production of tumor necrosis factor alpha (TNF), interleukin (IL-) 10, IL-6 and IL-8 (all P<0.05). P-ExA-induced inhibition of IL-10, IL-6 and IL-8 release was not dependent on reduced TNF concentrations, since the relative attenuation of the production of these cytokines was similar in the presence or absence of a neutralizing anti-TNF antibody. The effect of P-ExA on cytokine production may offer a disadvantage to the host with respect to clearance of the infection.     

Macrolide antibiotics modulate ERK phosphorylation and IL-8 and GM-CSF production by human bronchial epithelial cells

Macrolide antibiotics decrease proinflammatory cytokine production in airway cells from subjects with chronic airway inflammation. However, in subjects with chronic obstructive pulmonary disease, short-term azithromycin (AZM) therapy causes a transient early increase in the blood neutrophil oxidative burst followed by a decrease in inflammatory markers with longer administration. We studied the effects of clarithromycin (CAM) and AZM on proinflammatory cytokine production from normal human bronchial epithelial (NHBE) cells. CAM decreased IL-8 over the first 6 h and then significantly increased interleukin (IL)-8 at 12-72 h after exposure (P < 0.0001). AZM also increased IL-8 at 24 and 48 h, and CAM increased granulocyte-macrophage colony-stimulating factor at 48 h. In the presence of LPS, both CAM and AZM dose-dependently increased IL-8 secretion over 24 h, but after 5 days of exposure to 10 microg/ml CAM there is suppression of IL-8 (P < 0.001). PD-98059, an inhibitor of MAP kinase/ERK kinase, inhibited CAM-induced IL-8 (P < 0.0001) and GM-CSF (P < 0.01) release. The p38 MAP kinase inhibitor SB-203580 increased CAM-induced IL-8 release (P < 0.001), and the c-jun NH2-terminal kinase inhibitor SP-600125 had no effect on IL-8. At 120 min and 6 h, CAM increased phospho-ERK1/2 (pERK) but not phospho-p38 or phospho-JNK. Over the first 90 min, CAM at 10 microg/ml inhibited pERK and then increased pERK in parallel with measured IL-8 secretion. After daily CAM exposure for 5 days, both IL-8 and pERK returned to baseline. The p38 MAP kinase inhibitor, SB-203580 increased ERK phosphorylation and IL-8 secretion. These results suggest that macrolide antibiotics can differentially modulate proinflammatory cytokine secretion in NHBE cells, in part through ERK.     

A murine interleukin-4-Ig fusion protein regulates the expression of Th1- and Th2-specific cytokines in the pancreas of NOD mice.

Interleukin (IL)-4 is a key cytokine in T-helper type 2 (Th2) immune response. We have constructed a dimeric IL-4 molecule consisting of the murine IL-4 and the murine Fc part of IgG2a. We first tested the biological activity of the chimeric protein by in vitro studies using isolated murine spleen cells. IL-4-Ig was found to downregulate LPS-induced IFN-gamma and TNF-alpha production. The immunomodulatory potential of the fusion protein was also analyzed in non-obese diabetic (NOD) mice, an animal model for human type 1 diabetes. Female NOD mice aged 10 weeks were treated once with cyclophosphamide to accelerate and synchronize the progression of insulitis. Treatment with IL-4-Ig induced strong modulation of the pancreatic cytokine balance. Expression was downregulated for both Th1-specific cytokine IFN-gamma and the Th2-specific cytokine IL-10. IL-12p40 expression was only slightly affected. Interestingly, infiltration increased in the islets of IL-4-Ig-treated animals, and therefore did not correlate with the decreased IFN-gamma expression. Hence, IL-4-Ig did not prevent the progression from peri- to intra-insulitis, but suppressed inflammatory cytokine production. In most experiments, the biological activity of IL-4-Ig and IL-4 was comparable. We conclude that treatment with the chimeric protein IL-4-Ig effectively downregulates Th1 responses but simultaneously augments intra-islet infiltration.     

Stimulation of interleukin-6 release by interleukin-1beta from isolated human adipocytes

The secretion of interleukin-6 (IL-6) is modulated by immune, hormonal and metabolic stimuli in a cell-specific manner. We investigated the effect of cytokines, TNFalpha and IL-1beta, and insulin on IL-6 release from human adipocytes and peripheral blood cells (PBC). Adipocytes released IL-6 constitutively (after 5 h: 5.64 [1.61-15.30]pg ml(-1), after 10 h: 15.95 [2.34-45.59]pg ml(-1), p = 0.007), while PBC secretion did not change significantly over this period. LPS stimulated IL-6 secretion in PBC after 5 h but was without effect on adipocytes. TNFalpha and insulin induced IL-6 production from PBC, but had no effect on adipocytes. IL-1beta, however, induced a substantial increase in IL-6 release in adipocytes and PBC (all p < 0.05). Adipose tissue production of IL-1beta was assessed in vivo by measuring arterio-venous differences across the subcutaneous abdominal adipose bed. Net release of IL-1beta was not observed, suggesting that under basal conditions there is no detectable release of this cytokine into the circulation from this depot. In conclusion (1) PBC demonstrate regulated IL-6 release, while the adipocyte release has a large constitutive component; (2) immune modulators, such as LPS, TNFalpha and IL-1beta, all induce PBC IL-6 release, but only IL-1beta stimulates adipocyte release. Though IL-1beta is not an endocrine signal from adipose tissue, it is an autocrine/paracrine stimulator of IL-6 release from human adipocytes.     

Effects of interleukin-6 on proliferation and proteoglycan metabolism in articular chondrocyte cultures.

Interleukin-6 (IL-6) levels are markedly increased in the synovial fluid of patients with rheumatoid arthritis or osteoarthritis. However, the effects of IL-6 on proliferation and proteoglycan metabolism in articular cartilage are not known. We demonstrated here the effects of human recombinant (hr) IL-6 on proliferation and proteoglycan metabolism in rabbit articular chondrocyte cultures. In vitro, these cells proliferated and produced abundant extracellular matrices. We found that 1-10 ng/ml of hrIL-6 inhibited proliferation to approximately 65% of control levels and suppressed colony formation induced by bFGF in soft agarose. The same concentration of hrIL-6 depressed proteoglycan synthesis to approximately 60% of control levels. Moreover, hrIL-6 significantly enhanced proteoglycan degradation induced by hrIL-1beta, although hrIL-6 alone did not affect proteoglycan degradation. These findings suggest that IL-6 is a negative regulator for chondrocyte proliferation and articular cartilage metabolism.     

Evaluation of sage phenolics for their antileishmanial activity and modulatory effects on interleukin-6, interferon and tumour necrosis factor-alpha-release in RAW 264.7 cells

A series of sage phenolics was tested for activity against a panel of Leishmania parasites and for immunomodulatory effects on macrophage functions including release of tumour necrosis factor (TNF), interleukin-6 (IL-6), and interferon (IFN)-like activities. For this, functional bioassays were employed including an in vitro model for leishmaniasis in which macrophage-like RAW 264.7 cells were infected with Leishmania parasites, an extracellular Leishmania growth-inhibition assay, a fibroblast-lysis assay for TNF-activity, a cell proliferation assay using IL-6 sensitive murine B9 hybridoma cells, and a virus protection assay for IFN-like activity. Whereas none of the test samples exhibited marked activities against extracellular Leishmania promastigotes (IC50 > 700 to > 2800 nM; > 500 microg/ml), caffeic acid, salvianolic acids K and L as well as the methyl ester of salvianolic acid I showed pronounced antileishmanial activities against intracellular amastigote stages within RAW cells (IC50 3-23 nM vs. 10-11 nM for the reference Pentostam). Noteworthy, the phenolic samples showed no cytotoxicity against the host cells (IC50 > 600 to > 2200 nM; > 400 microg/ml). Tested sage phenolics activated Leishmania-infected RAW 264.7 for release of TNF ranging 22-117 U/ml and IL-6 ranging 3-42 U/ml. In contrast, their TNF- or IL-6-inducing potential in experiments with non-infected host cells was negligible. Furthermore, caffeic acid and salvianolic acid K induced a modest release of IFN-like activity (5-9 and 2-4 U/ml, respectively) as reflected by inhibition of the cytopathic effect of encephalomyocarditis virus on L929 cells. The results support the emerging picture that plant polyphenols may be credited for the profound health-beneficial properties of various herbal medicines and agricultural products.     

Regulation of interleukin-2 and interleukin-6 production from T-cells: involvement of interleukin-1 beta and transforming growth factor-beta

The effect of recombinant (r) interleukin-1 beta (rIL-1 beta) and transforming growth factor-beta (TGF-beta) on the production of interleukin-2 (IL-2) and interleukin-6 (IL-6) from an antigen-specific (LBRM-33-1A5) and an antigen-nonspecific (EL-4-NOB-1) T-cell line was investigated. rIL-1 beta induced the production of IL-2 and IL-6 from EL-4-NOB-1 cells in a dose-related manner. The LBRM-33-1A5 cells required phytohemagglutinin (PHA) in addition to rIL-1 beta in order to produce IL-2 and IL-6. IL-2 production was found to precede IL-6 production in both cell lines. No IL-2 or IL-6 production was observed by adding r murine tumor necrosis factor-alpha or r murine interferon gamma to the cells. The presence of 1 ng/ml TGF-beta reduced IL-2 and IL-6 production from both T-cell lines by more than 80%. The inhibition of IL-2 and IL-6 production was still evident by a concentration as low as 10 pg/ml of TGF-beta. rIL-1 beta and PHA also stimulated murine thymocytes to produce IL-6 which was inhibited up to 85% in the presence of 1 ng/ml TGF-beta. Taken together these results suggest that TGF-beta may suppress immune responses by inhibiting the endogenous production of IL-2 and IL-6.     

The effect of adenosine receptor agonists on cytokine release by human mononuclear cells depends on the specific Toll-like receptor subtype used for stimulation

In the present study, we determined whether the immunomodulatory effect of adenosine receptor stimulation depends on the Toll-like Receptor (TLR) used for stimulation of cytokine release. Therefore, human mononuclear cells were stimulated by different TLR agonists in the absence and presence of A1 (CPA), A2a (CGS21680), and A3 (Cl-IB-MECA) adenosine receptor agonists. Effects of these agonists on Il-6, Il-10, IFN-gamma, TNF-alpha, and Il-1beta production were expressed as percentage inhibition/stimulation after TLR stimulation. CGS21680 inhibited TLR4-mediated TNF-alpha release and potentiated TLR3- and TLR5-mediated IL-6 release. Cl-IB-MECA inhibited TLR4-agonist-induced IFN-gamma release. Interestingly, CPA en Cl-IB-MECA tended to inhibit cytokine release only after TLR4 stimulation. In more detail, CPA potentiated TLR5-mediated IL-6 production, TLR3-mediated IFN-gamma production and TLR3-mediated Il-1beta-production compared to TLR4-mediated stimulation. Cl-IB-MECA potentiated TLR5-mediated IL-6 and Il-1beta formation as compared to TLR4-mediated stimulation. Finally, CGS21680 potentiated TLR5-mediated IL-6 production compared to TLR1-2 stimulation, and potentiated TLR3- and TLR5-mediated IL-10 production compared to TLR1-2-mediated stimulation. In conclusion, the effect of adenosine agonists on cytokine production depends on the specific TLR agonist used for stimulation. These findings suggest that well-known anti-inflammatory effects of adenosine agonists on LPS-induced inflammation cannot be extrapolated to situations in which stimulation of other TLR subtypes is involved.