Induced TNF-alpha Release Research Articles

New Lipid Metabolic Targets for Treatment of Inflammation

Abstract The human genome contains a vast number of enzymes that lack functional annotation. As a result, our basic understanding of enzymatic pathways that underlie human physiology and pathology remains largely incomplete. Molecular profiling technologies have emerged as an unbiased means to globally examine the biochemical makeup of cells and tissues and identify genes and proteins associated with pathophysiological states. In contrast, global methods for the functional characterization of enzymatic pathways in native biological systems are still lacking. I will describe our efforts to integrate activity-based protein profiling (ABPP) with functional proteomic/metabolomics methods to map novel bioactive lipid networks involved in inflammation. I will describe the development of the first selective and in vivo-active small-molecule inhibitors for the 2-arachidonoylglycerol (2-AG) biosynthetic enzyme, diacylglycerol lipase-beta (DAGLB), along with paired negative-control and tailored activity-based probes for the functional analysis of DAGLB in living systems. We utilize our newly developed chemical probes to discover and show that DAGLB inactivation lowers 2-AG production in mouse peritoneal macrophages, as well as arachidonic acid and eicosanoids in a manner that is distinct and complementary to cytosolic phospholipase-A2. A corresponding reduction in lipopolysaccharide (LPS)-induced TNF-alpha release was observed, indicating that DAGLB serves as a key metabolic hub within a lipid signaling network that regulates proinflammatory responses in macrophages. We will present recent findings that identify DAGLB as a promising anti-inflammatory target for treatment of inflammatory and neuropathic pain.

Bryostatin 1 Inhibits Phorbol Ester-Induced Apoptosis in Prostate Cancer Cells by Differentially Modulating Protein Kinase C (PKC) δ Translocation and Preventing PKCδ-Mediated Release of Tumor Necrosis Factor-α

Bryostatin 1, a macrocyclic lactone that has been widely characterized as an ultrapotent protein kinase C (PKC) activator, displays marked pharmacological differences with the typical phorbol ester tumor promoters. Bryostatin 1 impairs phorbol 12-myristate 13-acetate (PMA)-induced tumor promotion in mice and is in clinical trials as an anticancer agent for a number of hematopoietic malignancies and solid tumors. In this study, we characterized the effect of bryostatin 1 on LNCaP prostate cancer cells, a cellular model in which PKC isozymes play important roles in the control of growth and survival. Although phorbol esters promote a strong apoptotic response in LNCaP cells via PKCdelta-mediated release of TNFalpha, bryostatin 1 failed to trigger a death effect even at high concentrations, and it prevented PMA-induced apoptosis in these cells. Mechanistic analysis revealed that bryostatin 1 is unable to induce TNFalpha release, and it impairs the secretion of this cytokine from LNCaP cells in response to PMA. Unlike PMA, bryostatin 1 failed to promote the translocation of PKCdelta to the plasma membrane. Moreover, bryostatin 1 prevented PMA-induced PKCdelta peripheral translocation. Studies using a membrane-targeted PKCdelta construct revealed that the peripheral localization of the kinase is a requisite for triggering apoptosis in LNCaP cells, arguing that mislocalization of PKCdelta may explain the actions of bryostatin 1. The identification of an antiapoptotic effect of bryostatin 1 may have significant relevance in the context of its therapeutic efficacy.

Mechanical but not painful electrical stimuli trigger TNF alpha release in human skin

Pro-inflammatory cytokines–in particular tumor necrosis factor (TNF)-alpha–play an important role in pain and hyperalgesia. The stimuli inducing TNF-alpha release in humans and the time course of this release are largely unknown. We performed dermal microdialysis in healthy subjects ( n = 36) during three experimental conditions: The first condition (control) was microdialysis without stimulation, the second condition was 30 min of electrical current stimulation (1 Hz, 20 mA, moderately painful), the third condition was 30 min of repetitive mechanical stimulation via an impact stimulator (bullet 0.5 g; velocity 11 m/s, minimally painful). TNF-alpha was quantified in the samples collected at the end of the baseline perfusion (about 1 h of saline perfusion), at the end of stimulation period (exactly 30 min after stimulation commenced) and at the end of the experiment (exactly 90 min after stimulation commenced) using a commercial enzyme-linked immunosorbent assay. The C-fiber-related flare was quantified with a laser-Doppler imager. ANOVA revealed that TNF-alpha levels increased during the eluate sampling period. At 90 min TNF-alpha in the eluate of the mechanical stimulation condition was significantly increased as compared to electrical current or control condition. Flare intensity was highest in the electrical current stimulation condition and only marginally different from control in mechanical stimulation. Our results show that minimal mechanical trauma is sufficient to induce significant TNF-alpha release in the skin. These results may be relevant to the treatment of posttraumatic pain disorders.

Suppression of tumour necrosis factor production from mononuclear cells by a novel synthetic compound, CLX-090717

To evaluate the clinical efficacy of a novel synthetic peroxisome proliferator-activated receptor gamma (PPAR-gamma) agonist, CLX-090717, in several in vitro cell culture systems and murine CIA, an experimental model of RA. Peripheral blood monocytes purified by elutriation, and rheumatoid synovial cells isolated from clinical tissue were cultured with CLX-090717 and TNF-alpha release was measured. Molecular mechanism of action was analysed by western blotting and electrophoretic mobility shift assay. Thioglycollate-elicited murine peritoneal macrophages were cultured with CLX-090717 and lipopolysaccharide (LPS)-induced TNF-alpha release was assayed. Therapeutic studies were done in mice with established arthritis by evaluating clinical parameters and histology. In addition, type II collagen response of lymphocytes from mice with CIA was examined. CLX-090717 significantly inhibited spontaneous TNF-alpha release by RA synovial membrane cells, as well as LPS-induced TNF-alpha release from human and murine monocytic cells. Inhibition of TNF-alpha in monocytes was mediated partially through a nuclear factor-kappaB (NF-kappaB)-dependent pathway, as judged by sustained levels of IkappaBalpha in cytosolic extracts and a reduced level of LPS-induced NF-kappaB activity in nuclear extracts. CLX-090717 reduced clinical signs of arthritis and damage to joint architecture when administered therapeutically to arthritic mice. Mechanisms of action in CIA involved the reduction in proliferation of arthritic lymphocytes to antigen in vitro as well as reduced TNF-alpha release. Our data suggest that the synthetic compound CLX-090717 has potential as a small molecular weight anti-inflammatory therapeutic for chronic inflammatory conditions.

Iptakalim protects against MPP+-induced degeneration of dopaminergic neurons in association with astrocyte activation

Astrocyte activation observed in the MPTP mouse model and Parkinson's disease patients participates in the cascade of deleterious events that ultimately leads to death of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The present study aimed to elucidate whether inhibiting astrocyte activation was involved in the protective effects of iptakalim (Ipt), a novel ATP-sensitive potassium channel opener, on MPP+-induced degeneration of dopaminergic neurons. The results showed that Ipt could decrease MPP+-induced TNF-alpha release and p38 MAPK activation in reactive astrocytes. The effects of Ipt were reversed by the mitochondrial KATP blocker, 5-hydroxydecanoate, indicating that mitochondrial KATP channels participate in the regulation of astrocyte activation. Moreover, systematic administration of Ipt could significantly alleviate MPP+-induced behavioural symptoms in motor coordination, the loss of dopaminergic neurons, and the activation of astrocyte and microglia in the SNpc. Together, these findings suggest that Ipt may protect against MPP+-induced degeneration of dopaminergic neurons by inhibiting astrocyte activation and subsequent release of pro-inflammatory factors.

The Role of P38 MAPK and PKC in BLP Induced TNF-α Release, Apoptosis, and NFκB Activation in THP-1 Monocyte Cells

P38 mitogen activated protein kinase (p38 MAPK) is a critical mediator of the inflammatory response, which makes it a suitable candidate as a novel therapeutic strategy for inflammatory conditions. In this study, we set out to examine the precise role of both protein kinase C (PKC) and P38 MAPK signaling kinases in bacterial lipoprotein (BLP) induced nuclear factor-kappa B (NFkappaB) activation and tumor necrosis factor-alpha (TNFalpha) release in THP-1 monocytic cell line. THP-1 cells were incubated with BLP(0-1000 ng/mL), phorbol myristate acetate (PMA; 0-100 microg/mL) or a combination of both for 6 and 24 h, with or without pretreatment with SB202190, a specific inhibitor of p38 MAPK and bisindolylmaleimide I, a specific inhibitor of PKC (0-200 microm). Cell supernatants were analyzed for TNF-alpha release and apoptosis. NFkappaB activity was analyzed by electromobility supershift assay. BLP induced TNF-alpha release was significantly reduced by pretreatment with SB202190 at all concentrations (428.7 +/- 5.9 versus 51 +/- 0.8 rhog/mL, P < 0.05). Pretreatment with bis I significantly inhibited TNF-alpha release at higher concentrations (200 microM) (429.7 +/- 5.9 versus 194.9 +/- 42.68 rhog/mL, P < 0.05) but this was much less effective than SB202190. PMA induced TNF-alpha release was not inhibited at 6 h by either SB202190 or bis I, but was significantly so at 24 h (148.5 +/- 9.8 versus 24 +/- 1.7 and 25.1 +/- 4.4 rhog/mL, P < 0.05). BLP or lipopolysaccharide (LPS) did not result in apoptosis in THP-1 cells (P > 0.05) with PMA inducing apoptosis in a time- and dose-dependent manner. In combination with BLP (1000 ng/mL) but not LPS (1000 ng/mL), low dose PMA resulted in a significant increase in apoptosis, 6% +/- 0.5% (Control) versus 9.2% +/- 0.3% (P < 0.05) and 7% +/- 2.2% (Control) versus 7.7% +/- 0.3% (P > 0.05), respectively. This synergistic effect was inhibited by bisindolylmaleimide 100 nm, 8.9% +/- 0.9% (Control) versus 9.8% +/- 0.2% (P > 0.05). PMA and BLP induced rapid nuclear translocation of NFkappaB, which was inhibited by pretreatment with both SB-202190 and bis I, and SB202190 but not bis I, respectively. P38 is a critical mediator of BLP induced TNF-alpha release and NFkappaB activation, whereas PKC is only partially responsible for its response. P38 and PKC are both critical mediators of PMA induced TNF-alpha release and NFkappaB activation.

Role of MAPK signaling pathway in the activation of dendritic type cell line, THP-1, induced by DNCB and NiSO&lt;sub&gt;4&lt;/sub&gt;

The activation of dendritic cells (DC), including Langerhans cells (LC) that reside within the epidermis, is a critical event in the induction phase of allergic contact hypersensitivity. Although recently, p38 mitogen-activated protein kinase (MAPK) has been reported to play a role in the activation of DC induced by allergens, the signaling pathways involved in this process have yet to be determined. We previously found that THP-1 cells have a high capacity to induce TNF-alpha release and CD86, CD54, and CD40 expression following allergen treatment; reflecting in vitro allergen-induced DC activation during skin sensitization. In this study, we investigated the signaling pathways in THP-1 cells activated by two representative allergens, DNCB and NiSO(4). We found that DNCB and NiSO(4) induced phosphorylation of p38 MAPK and extracellular signal-regulated kinase (ERK). Inhibition of p38 MAPK activation selectively blocked DNCB-induced TNF-alpha release, but not NiSO(4)-induced release. In contrast, inhibition of ERK pathways selectively suppressed NiSO(4)-induced TNF-alpha release but not DNCB-induced release. In addition, we found that the inhibition of p38 MAPK and ERK pathways caused a selective inhibition of CD86, CD54, and/or CD40 expression following treatment with DNCB or NiSO(4). In particular, inhibition of p38 MAPK suppressed CD86, CD54, and CD40 expression induced by DNCB and CD86 expression induced by NiSO(4) while inhibition of ERK pathways suppressed CD86, CD54 and CD40 expression induced by DNCB and NiSO(4). These data indicate that both DNCB and NiSO(4) activate p38 MAPK and ERK, and thereby stimulate TNF-alpha release and phenotypic changes through the different signal transduction pathways.

IL‐7 Modulates Osteoclastogenesis in Patients Affected by Solid Tumors

High levels of interleukin-7 (IL-7) have been associated with bone loss due to its stimulatory osteoclastogenic activity. Osteolytic patients' peripheral blood mononuclear cells (PBMCs) differentiate into osteoclasts without adding stimulating factors. Now, we investigated the potential role of IL-7 in the spontaneous osteoclastogenesis occurring in these patients. We identified significant differences in serum IL-7 levels between patients with/without bone metastases, suggesting that IL-7 might be effective as a clinical marker of disease progression. In patients' PBMC cultures we demonstrated that IL-7 stimulates osteoclastogenesis by inducing TNF-alpha release by T and B cells. These findings add further details to the disclosure of the mechanisms controlling bone metastases in solid tumors.

Immunomodulatory effect of exo-polysaccharides from submerged cultured Cordyceps sinensis: enhancement of cytokine synthesis, CD11b expression, and phagocytosis

Cordyceps sinensis is widely used as a traditional medicine for treatment of a wide variety of diseases or to maintain health. The immunomodulatory activity of polysaccharides prepared from submerged cultured C. sinensis BCRC36421 was investigated in human peripheral blood. Results demonstrated that Fr. A (exo-polysaccharides, 0.025 approximately 0.1 mg/ml) induced the production of tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-6, and IL-10 dose-dependently. Fr. A, as low as 0.025 mg/ml, could significantly augment surface expression of CD11b in monocytes and polymorphonuclear neutrophils. Functional assay revealed that Fr. A (0.05 mg/ml) also elevated phagocytosis in monocytes and PMN. On the other hand, Fr. B (intracellular polysaccharides) only moderately induced TNF-alpha release, CD11b expression, and phagocytosis at the same concentrations. Our results indicate that the immunomodulatory components of submerged cultured C. sinensis mainly reside in the culture filtrate.

Effect of bisphosphonates on the stimulation of macrophages by alumina ceramic particles: A comparison with ultra-high-molecular-weight polyethylene

Wear particle-induced osteolysis and loosening is a critical process that limits the longevity of total hip arthroplasty. Despite their potential value in the management of aseptic loosening, little is known about the cellular response to bisphosphonates (BPs) in the presence of particulate debris. In the present study, we compared the effect of pamidronate and clodronate, two structurally different bisphosphonates, on the induction of TNF-alpha release by alumina ceramic (Al(2)O(3)) and ultra-high-molecular-weight-polyethylene (UHMWPE) particles. We also looked, by Trypan blue exclusion, at the viability of J774 mouse macrophages incubated with Al(2)O(3) and UHMWPE particles in combination with pamidronate or clodronate. Results showed that pamidronate and clodronate can inhibit UHMWPE particle-induced TNF-alpha release while they had no effect on Al(2)O(3)-stimulated TNF-alpha release. The co-incubation of pamidronate or clodronate and Al(2)O(3) had no effect on the induction by Al(2)O(3) of poly(ADP-ribose)polymerase (PARP) proteolysis and DNA fragmentation. On the other hand, UHMWPE particles had no effect on these apoptotic markers. However, the co-incubation of pamidronate or clodronate with UHMWPE particles led to the appearance of these markers of apoptosis. Al(2)O(3) and UHMWPE particles had no effect on macrophage cell death or the number of macrophages at the end of experiments. Co-incubation of UHMWPE particles with pamidronate and clodronate led to a significant increase in cell death. Interestingly, the number of macrophages co-incubated with particles and pamidronate or clodronate significantly decreased. In conclusion, our results suggest that the effect of BPs on particle-stimulated macrophages is, at least in part, particle composition dependent.

Haemorrhagic shock in mice – Intracellular signalling and immunomodulation of peritoneal macrophages’ LPS response

Haemorrhagic shock leads to decreased proinflammatory cytokine response which is associated with an increased susceptibility to bacterial infections. In the present study, the effect of GM-CSF on lipopolysaccharide (LPS)-induced TNF-alpha release and MAPkinase activation was analysed on the background of a possible immunostimulating activity of this substance. Male BALB/c mice were bled to a mean arterial blood pressure of 50 mm Hg for 45 min followed by resuscitation. Peritoneal macrophages were isolated 20 h after haemorrhage and incubated with 10 ng/ml GM-CSF for 6 h before LPS stimulation. TNF-alpha synthesis was studied in the culture supernatants using ELISA. Phosphorylation of ERK, p38MAPK and IκBα was detected by Western blotting. LPS-induced TNF-alpha production of peritoneal macrophages was significantly decreased 20 h after haemorrhage in comparison to the corresponding cells of sham-operated mice. In parallel the phosphorylation of IκBα was less in LPS-stimulated peritoneal macrophages from haemorrhagic mice. LPS-induced phosphorylation of ERK1/2 was also decreased in peritoneal macrophages isolated after haemorrhagic shock. In contrast, p38MAPK was phosphorylated more intensely after LPS-stimulation in macrophages collected from shocked mice. GM-CSF incubation elevated LPS-induced TNF-alpha response of macrophages from both sham-operated and shocked mice which was accompanied by an elevated IκB and ERK phosphorylation. In general, GM-CSF treatment in vitro enhanced peritoneal macrophages LPS-response both in terms of TNF-alpha synthesis and IκB and MAPK signalling, but the levels always stayed lower than those of GM-CSF-treated cells from sham-operated animals. In conclusion, GM-CSF preincubation could partly reactivate the depressed functions of peritoneal macrophages and may therefore exert immunostimulating properties after shock or trauma.

Effect of inhibitors of signal transducer and activator of transcription-1/3 on expression of tumor necrosis factor-α induced by high mobility group box-1 protein inflammatory response in rat peritoneal macrophages

To investigate the inflammatory signal transduction of high mobility group box-1 protein (HMGB1) induced inflammatory response in rat peritoneal macrophages. Peritoneal macrophages obtained from male Wistar rats were seeded in 24-well (1 x 10(6) cells/well) tissue culture plates. The cells were incubated for 3 days before they were stimulated with HMGB1, and treated with Fludarabine [the specific inhibitor of signal transducer and activator of transcription 1 (STAT1)] or Rapamycin (the specific inhibitor of STAT3). The time-dependent and dose-dependent responses between HMGB1 stimulation and tumor necrosis factor-alpha (TNF-alpha) gene expression as well as release were analyzed respectively. Moreover, the effect of Fludarabine and Rapamycin on TNF-alpha mRNA expression and protein release were also observed. (1) After HMGB1 challenge, TNF-alpha mRNA expressions were up-regulated markedly, peaked at 24 hours, and decreased at 36 hours. When HMGB1 was used at a concentration of 10 microg/ml, TNF-alpha mRNA expression increased most markedly. (2) HMGB1 could induce TNF-alpha release in rat peritoneal macrophages, with peaking at 4 hours and decreasing at 8 hours later. When the concentration of HMGB1 stimulation increased from 5 microg/ml to 25 microg/ml, TNF-alpha release was persistently enhanced. (3) It was also showed that TNF-alpha mRNA expression was significantly inhibited by treatment of either Fludarabine (100 micromol/L) or Rapamycin (25 ng/ml), while TNF-alpha release was not markedly suppressed. STAT1 and STAT3 might be involved in the regulation of TNF-alpha gene expression, but not in TNF-alpha early release (< 24 hours) induced by HMGB1 stimulation in rat peritoneal macrophages.

Questioning Current Concepts in Acute Pancreatitis: Endotoxin Contamination of Porcine Pancreatic Elastase is Responsible for Experimental Pancreatitis-Associated Distant Organ Failure

The systemic inflammatory response syndrome is responsible for pancreatitis-associated mortality. Recent in vitro and in vivo studies have suggested that pancreatic elastase is one missing link between the localized inflammatory process in the pancreas and distant organ dysfunction and failure. It has been shown that pancreatic elastase activates transcription factors, including NF-kappaB, and induces TNF-alpha secretion in myeloid cells via TLRs. In this study we demonstrate that a highly purified low endotoxin pancreatic elastase preparation (El-UP) failed both to activate NF-kappaB and to induce TNF-alpha release in RAW 264.7 cells and bone marrow-derived macrophages. In contrast, a less purified elastase preparation (El-IV) caused activation of NF-kappaB and was able to induce TNF-alpha release at very low concentrations. These effects were sensitive to pretreatment of the cells with polymyxin B and were resistant to heat inactivation. Endotoxin activity as determined by the Limulus amebocyte lysate assay was >3 orders of magnitude lower in the low endotoxin elastase preparation (El-UP) compared with less purified elastase preparations (El-IV). In contrast to contaminated elastase or LPS, elastase free of contamination (El-UP) failed to induce elevated serum TNF-alpha levels or pulmonary neutrophil infiltration after i.p. application in mice and did not induce lethality when coinjected with d-galactosamine. Failure of low endotoxin elastase (El-UP) to induce proinflammatory effects in vivo and in vitro was not due to functional inactivity of the elastase preparation, as determined by elastase activity assay. These results question current concepts of direct proinflammatory effects attributed to pancreatic elastase.

Halothane potentiates the alcohol-adduct induced TNF-alpha release in heart endothelial cells

BackgroundThe possibility exists for major complications to occur when individuals are intoxicated with alcohol prior to anesthetization. Halothane is an anesthetic that can be metabolized by the liver into a highly reactive product, trifluoroacetyl chloride, which reacts with endogenous proteins to form a trifluoroacetyl-adduct (TFA-adduct). The MAA-adduct which is formed by acetaldehyde (AA) and malondialdehyde reacting with endogenous proteins, has been found in both patients and animals chronically consuming alcohol. These TFA and MAA-adducts have been shown to cause the release of inflammatory products by various cell types. If both adducts share a similar mechanism of cell activation, receiving halothane anesthesia while intoxicated with alcohol could exacerbate the inflammatory response and lead to cardiovascular injury.MethodsWe have recently demonstrated that the MAA-adduct induces tumor necrosis factor-α (TNF-α) release by heart endothelial cells (HECs). In this study, pair and alcohol-fed rats were randomized to receive halothane pretreatments intra peritoneal. Following the pretreatments, the intact heart was removed, HECs were isolated and stimulated with unmodified bovine serum albumin (Alb), MAA-modified Alb (MAA-Alb), Hexyl-MAA, or lipopolysaccharide (LPS), and supernatant concentrations of TNF-α were measured by ELISA.ResultsHalothane pre-treated rat HECs released significantly greater TNF-α concentration following MAA-adduct and LPS stimulation than the non-halothane pre-treated in both pair and alcohol-fed rats, but was significantly greater in the alcohol-fed rats.ConclusionThese results demonstrate that halothane and MAA-adduct pre-treatment increases the inflammatory response (TNF-α release). Also, these results suggest that halothane exposure may increase the risk of alcohol-induced heart injury, since halothane pre-treatment potentiates the HEC TNF-α release measured following both MAA-Alb and LPS stimulation.

Bi‐functional effects of ATP/P2 receptor activation on tumor necrosis factor‐alpha release in lipopolysaccharide‐stimulated astrocytes

Neuroinflammation is associated with a variety of CNS pathologies. Levels of tumor necrosis factor-alpha (TNF-alpha), a major proinflammatory cytokine, as well as extracellular ATP, are increased following various CNS insults. Here we report on the relationship between ATP/P2 purinergic receptor activation and lipopolysaccharide (LPS)-induced TNF-alpha release from primary cultures of rat cortical astrocytes. Using ELISA, we confirmed that treatment with LPS stimulated the release of TNF-alpha in a concentration and time dependent manner. ATP treatment alone had no effect on TNF-alpha release. LPS-induced TNF-alpha release was attenuated by 1 mm ATP, a concentration known to activate P2X7 receptors. Consistent with this, 3'-O-(4-Benzoyl)benzoyl-ATP (BzATP), a P2X7 receptor agonist, also attenuated LPS-induced TNF-alpha release. This reduction in TNF-alpha release was not due to loss of cell viability. Adenosine and 2-chloroadenosine were ineffective, suggesting that attenuation of LPS-induced TNF-alpha release by ATP was not due to ATP breakdown and subsequent activation of adenosine/P1 receptors. Interestingly, treatment of astrocyte cultures with 10 microm or 100 microm ATP potentiated TNF-alpha release induced by a submaximal concentration of LPS. UTP and 2methylthioADP (2-MeSADP), P2Y receptor agonists, also enhanced this LPS-induced TNF-alpha release. Our observations demonstrate opposing effects of ATP/P2 receptor activation on TNF-alpha release, i.e. P2X receptor activation attenuates, whereas P2Y receptor activation potentiates TNF-alpha release in LPS-stimulated astrocytes. These observations suggest a mechanism whereby astrocytes can sense the severity of damage in the CNS via ATP release from damaged cells and can modulate the TNF-alpha mediated inflammatory response depending on the extracellular ATP concentration and corresponding type of astrocyte ATP/P2 receptor activated.

Novel p38 Inhibitors with Potent Oral Efficacy in Several Models of Rheumatoid Arthritis.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Native Versus Recombinant High-Mobility Group B1 Proteins: Functional Activity In Vitro

To compare the functional activity of native HMGB1 proteins from eukaryotic sources with HMGB1 from prokaryotic sources the cDNAs of human and murine HMGB1 were cloned and the proteins expressed in bacteria. Tissue-derived HMGB1 from calf thymus and HMGB1 secreted from Chinese hamster ovary (CHO) cells were purified. Human whole blood, THP-1 cells, and NIH/3T3 cells were exposed to HMGB1 proteins and the induction of tumor necrosis factor-alpha (TNF-alpha) release in whole blood and monocytic THP-1 cells and a proliferation assay in NIH/3T3 cells were used to study functional activity of HMGB1s in vitro. Native and recombinant HMGB1s induced TNF-alpha release in human blood and in THP-1 cells dose-dependently, but recombinant HMGB1s were more effective. Cell proliferation was induced by native and recombinant HMGB1s. The native HMGB1 proteins from eukaryotic sources exert the same (though less pronounced) biological activity in vitro as recombinant HMGB1 proteins from prokaryotic sources.

Pentoxyphylline and propentophylline are inhibitors of TNF-alpha release in monocytes activated by advanced glycation endproducts.

Non-enzymatic glycation of proteins with reducing sugars and subsequent transition metal-catalyzed oxidation leads to the formation of protein-bound "advanced glycation endproducts" (AGEs). They accumulate on long-lived protein deposits inducing senile plaques in Alzheimer's disease. AGE-modified proteins are able to activate microglia and astroglia and can cause chronic inflammation. The aim of the present study was to confirm the stimulatory effect of different AGEs on TNF-alpha release in human monocytes. Furthermore, the effects of four xanthine derivatives on AGE-induced TNF-alpha release were investigated. We show that chicken egg albumin-AGEs prepared with glucose and chicken egg albumin-AGEs prepared with methylglyoxal dose-dependently induce TNF-alpha release. The xanthine derivatives pentoxyphylline and propentophylline attenuate AGE-induced TNF-alpha release in a dose-dependent manner. Theophylline at low concentrations slightly stimulated TNF-alpha release whereas caffeine had no effect. The inhibition of the AGE-induced TNF-alpha release by pentoxyphylline and propentophylline provides interesting pharmacological strategies for diseases with local neuroinflammation such as Alzheimer's disease.

Recombinant Human Heat Shock Protein 60 Does Not Induce the Release of Tumor Necrosis Factor α from Murine Macrophages

Recent studies have shown that commercially available recombinant human heat shock protein 60 (rhHSP60) could induce tumor necrosis factor alpha (TNF-alpha) release from macrophages and monocytes in a manner similar to that of lipopolysaccharide (LPS), e.g. via CD14 and Toll-like receptor 4 complex-mediated signal transduction pathway. In this study, we demonstrated that a highly purified rhHSP60 preparation with low endotoxin activity (designated rhHSP60-1) was unable to induce TNF-alpha release from murine macrophages at concentrations of up to 10 microg/ml. In contrast, a less purified rhHSP60 preparation (designated rhHSP60-2) was able to induce a marked TNF-alpha release at concentrations as low as 1 microg/ml. Failure of rhHSP60-1 to induce TNF-alpha release was not due to defective physical properties because rhHSP60-1 and rhHSP60-2 contained a similar amount of HSP60 as determined by SDS gels stained with Coomassie Blue and Western blots probed with an anti-rhHSP60 antibody. Both rhHSP60 preparations also had similar enzymatic activities as judged by their ability to hydrolyze ATP. Polymyxin B added in the incubation media abolished the endotoxin activity but inhibited only about 50% of the TNF-alpha-inducing activity of rhHSP60-2. However, both the endotoxin activity and the TNF-alpha-inducing activity of rhHSP60-2 were essentially eliminated after passing through a polymyxin B-agarose column that removes LPS and LPS-associated molecules from the rhHSP60 preparation. The TNF-alpha-inducing activities of both rhHSP60-2 and LPS with equivalent endotoxin activity present in rhHSP60-2 were equally sensitive to heat inactivation. These results suggest that rhHSP60 does not induce TNF-alpha release from macrophages. Approximately 50% of the observed TNF-alpha-inducing activity in the rhHSP60-2 preparation is due to LPS contamination, whereas the rest of the activity was due to the contamination of LPS-associated molecule(s).

(E)-2(R)-[1(S)-(Hydroxycarbamoyl)-4-phenyl-3-butenyl]-2'-isobutyl-2'-(methanesulfonyl)-4-methylvalerohydrazide (Ro 32-7315), a selective and orally active inhibitor of tumor necrosis factor-alpha convertase.

Tumor necrosis factor-alpha (TNF-alpha), a cytokine secreted by inflammatory cells, has been implicated in several inflammatory disease states. (E)-2(R)-[1(S)-(Hydroxycarbamoyl)-4-phenyl-3-butenyl]-2'-isobutyl-2'-(methanesulfonyl)-4-methylvalerohydrazide (Ro 32-7315), is a potent, orally active inhibitor of the TNF-alpha convertase (TACE), an enzyme responsible for proteolytic cleavage of the membrane bound precursor, pro-TNF-alpha. Ro 32-7315 inhibited a recombinant form of TACE (IC(50) = 5.2 nM) with selectivity over related matrix metalloproteinases. In a cellular assay system, THP-1 cell line, and in human and rat whole blood, Ro 32-7315 significantly reduced lipopolysaccharide (LPS)-induced TNF-alpha release with IC(50) values of 350 +/- 14 nM (n = 5), 2.4 +/- 0.5 microM (n = 5), and 110 +/- 18 nM (n = 5), respectively. Oral administration of Ro 32-7315 to Wistar rats caused a dose-dependent inhibition of LPS-induced release of systemic TNF-alpha with an ED(50) of 25 mg/kg. Treatment (days 0-14) of Allen and Hamburys hooded rats with Ro 32-7315 (2.5, 5, 10, and 20 mg/kg, i.p., twice daily) significantly reduced adjuvant-induced secondary paw swelling (42, 71, 83, and 93%, respectively) as compared with the vehicle group. In the Ro 32-7315-treated group, the reduced paw swelling was associated with improved lesion score and joint mobility. Furthermore, in a placebo-controlled, single-dose study, Ro 32-7315 given orally (450 mg) significantly suppressed ex vivo, LPS-induced TNF-alpha release in the whole-blood samples taken from healthy male and female volunteers (mean inhibition of 42% over a 4-h duration, n = 6). These data collectively support the potential use of such a compound for the oral treatment of inflammatory disorders.