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1.
J Biol Chem ; 290(4): 2368-78, 2015 Jan 23.
Article in English | MEDLINE | ID: mdl-25451941

ABSTRACT

ITF2357 (generic givinostat) is an orally active, hydroxamic-containing histone deacetylase (HDAC) inhibitor with broad anti-inflammatory properties, which has been used to treat children with systemic juvenile idiopathic arthritis. ITF2357 inhibits both Class I and II HDACs and reduces caspase-1 activity in human peripheral blood mononuclear cells and the secretion of IL-1ß and other cytokines at 25-100 nm; at concentrations >200 nm, ITF2357 is toxic in vitro. ITF3056, an analog of ITF2357, inhibits only HDAC8 (IC50 of 285 nm). Here we compared the production of IL-1ß, IL-1α, TNFα, and IL-6 by ITF2357 with that of ITF3056 in peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS), heat-killed Candida albicans, or anti-CD3/anti-CD28 antibodies. ITF3056 reduced LPS-induced cytokines from 100 to 1000 nm; at 1000 nm, the secretion of IL-1ß was reduced by 76%, secretion of TNFα was reduced by 88%, and secretion of IL-6 was reduced by 61%. The intracellular levels of IL-1α were 30% lower. There was no evidence of cell toxicity at ITF3056 concentrations of 100-1000 nm. Gene expression of TNFα was markedly reduced (80%), whereas IL-6 gene expression was 40% lower. Although anti-CD3/28 and Candida stimulation of IL-1ß and TNFα was modestly reduced, IFNγ production was 75% lower. Mechanistically, ITF3056 reduced the secretion of processed IL-1ß independent of inhibition of caspase-1 activity; however, synthesis of the IL-1ß precursor was reduced by 40% without significant decrease in IL-1ß mRNA levels. In mice, ITF3056 reduced LPS-induced serum TNFα by 85% and reduced IL-1ß by 88%. These data suggest that specific inhibition of HDAC8 results in reduced inflammation without cell toxicity.


Subject(s)
Cytokines/metabolism , Gene Expression Regulation, Enzymologic , Histone Deacetylase Inhibitors/chemistry , Repressor Proteins/antagonists & inhibitors , Animals , Apoptosis , Candida/metabolism , Caspase 1/metabolism , Caspase 3/metabolism , Caspase 7/metabolism , Cells, Cultured , Histone Deacetylases/metabolism , Humans , Inflammation , Inhibitory Concentration 50 , Interleukin-1alpha/metabolism , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides/chemistry , Mice , Mice, Inbred C57BL , Monocytes/cytology , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Repressor Proteins/metabolism , Tumor Necrosis Factor-alpha/metabolism
3.
Ann Rheum Dis ; 71(4): 563-6, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22084392

ABSTRACT

OBJECTIVE: Uveitis and retinal vasculitis are sight-threatening manifestations of Behçet's disease with limited treatment options. This pilot study aimed to evaluate the safety, pharmacokinetics and clinical activity of XOMA 052 (gevokizumab), a recombinant humanised anti-interleukin 1ß antibody, in Behçet's disease patients with uveitis. METHODS: Patients with acute posterior or panuveitis, and/or retinal vasculitis, resistant to azathioprine and/or ciclosporin, and receiving 10 mg/day or less of prednisolone, were enrolled into the 98-day study. Immunosuppressive agents were discontinued at baseline. Patients received a single infusion of XOMA 052 (0.3 mg/kg). The safety and uveitis status and pharmacokinetics of XOMA 052 were evaluated. RESULTS: Seven patients enrolled and completed the study. No treatment-related adverse event was observed. XOMA 052 treatment was associated with rapid and durable clinical response in all patients. Complete resolution of intraocular inflammation was achieved in 4-21 days (median 14 days), with a median duration of response of 49 days (range 21-97 days); one patient remained exacerbation free throughout the study. CONCLUSIONS: Well tolerated, XOMA 052 resulted in a rapid onset and sustained reduction in intraocular inflammation in patients with resistant uveitis and retinal vasculitis. Moreover, the effect was observed despite discontinuation of immunosuppressive agents and without the need to increase corticosteroid dosages.


Subject(s)
Anti-Inflammatory Agents/adverse effects , Antibodies, Monoclonal, Humanized/adverse effects , Behcet Syndrome/drug therapy , Immunosuppressive Agents/adverse effects , Acute Disease , Adult , Anti-Inflammatory Agents/blood , Anti-Inflammatory Agents/therapeutic use , Antibodies, Monoclonal, Humanized/blood , Antibodies, Monoclonal, Humanized/therapeutic use , Behcet Syndrome/blood , Behcet Syndrome/physiopathology , Female , Humans , Immunosuppressive Agents/blood , Immunosuppressive Agents/therapeutic use , Interleukin-1beta/antagonists & inhibitors , Male , Pilot Projects , Retinal Vasculitis/blood , Retinal Vasculitis/drug therapy , Retinal Vasculitis/physiopathology , Treatment Outcome , Uveitis/blood , Uveitis/drug therapy , Uveitis/physiopathology , Visual Acuity/drug effects
4.
Mol Med ; 17(5-6): 353-62, 2011.
Article in English | MEDLINE | ID: mdl-21365126

ABSTRACT

ITF2357 (givinostat) is a histone deacetylase inhibitor with antiinflammatory properties at low nanomolar concentrations. We report here a phase I safety and pharmacokinetics trial in healthy males administered 50, 100, 200, 400 or 600 mg orally. After 50 mg, mean maximal plasma concentrations reached 104 nmol/L 2 h after dosing, with a half-life of 6.9 h. After 100 mg, maximal concentration reached 199 nmol/L at 2.1 h with a half-life of 6.0 h. Repeat doses for 7 consecutive days of 50, 100 or 200 mg resulted in nearly the same kinetics. There were no serious adverse effects (AEs) and no organ toxicities. However, there was a dose-dependent but transient fall in platelets. After 7 daily doses of 50 or 100 mg, the mean decrease in platelets of 17 and 25% was not statistically significant and returned to baseline within 14 d. Blood removed from the subjects after oral dosing was cultured ex vivo with endotoxin, and the release of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-1Ra, interferon (IFN)-γ and IL-10 was determined. Maximal reduction in IL-1ß, TNFα, IL-6 and IFNγ was observed 4 h after dosing but returned to baseline at 12 h. There was no significant reduction in IL-1Ra or IL-10. With daily dosing, the fall in cytokine production in blood cultures observed on day 7 was nearly the same as that of the first day. We conclude that dosing of 50 or 100 mg ITF2357 is safe in healthy humans and transiently but repeatedly reduces the production of proinflammatory cytokines without affecting production of antiinflammatory cytokines.


Subject(s)
Anti-Inflammatory Agents/adverse effects , Anti-Inflammatory Agents/pharmacokinetics , Cytokines/blood , Hydroxamic Acids/adverse effects , Hydroxamic Acids/pharmacokinetics , Adult , Anti-Inflammatory Agents/administration & dosage , Female , Humans , Hydroxamic Acids/administration & dosage , Interferon-gamma/blood , Interleukin 1 Receptor Antagonist Protein/blood , Interleukin-10/blood , Interleukin-1beta/blood , Interleukin-6/blood , Male , Tumor Necrosis Factor-alpha/blood , Young Adult
5.
J Infect Dis ; 198(9): 1407-14, 2008 Nov 01.
Article in English | MEDLINE | ID: mdl-18793104

ABSTRACT

BACKGROUND: Many novel therapeutics have failed to reduce all-cause mortality associated with severe sepsis. Eukaryotic translation initiation factor 5A (eIF5A) is a regulator of apoptosis as well as inflammatory cell activation, making it a potential target for sepsis therapy. METHODS: In a murine model of severe sepsis, mice were intraperitoneally challenged with lipopolysaccharide (LPS). Mice were treated both before and after LPS challenge with liposome complexes containing either an eIF5A-specific or control small interference RNA (siRNA), and both survival and serum concentrations of inflammatory cytokines were monitored. The ability of eIF5A siRNA to reduce inflammatory cytokines was also tested in a model of acute lung injury established by intranasal administration of LPS to mice. RESULTS: There was a statistically significant increase in the rate of survival for mice intraperitoneally challenged with LPS that received eIF5A siRNA, compared with that noted for mice that received control siRNA (71% vs. 5%; P< .001), as well as a reduction in cytokine expression in serum. Concentrations of proinflammatory cytokines were also reduced in the lung homogenates and serum of mice that were intranasally challenged with LPS and received eIF5A siRNA (P< or = .05). CONCLUSIONS: eIF5A siRNA-liposome complexes reduced inflammation and contributed to increased survival in a model of severe sepsis, decreased inflammation in a model of acute lung injury, and should be considered for clinical use.


Subject(s)
Inflammation/therapy , Liposomes/pharmacology , Lung Diseases/therapy , Peptide Initiation Factors/metabolism , RNA, Small Interfering/pharmacology , RNA-Binding Proteins/metabolism , Sepsis , Acute Disease , Animals , Female , Inflammation/chemically induced , Lipopolysaccharides/toxicity , Liposomes/chemistry , Lung Diseases/chemically induced , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Peptide Initiation Factors/genetics , RNA Interference , RNA, Small Interfering/chemistry , RNA-Binding Proteins/genetics , Eukaryotic Translation Initiation Factor 5A
6.
J Pharmacol Exp Ther ; 322(1): 8-15, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17400889

ABSTRACT

We have demonstrated that caspase-1-deficient (caspase-1(-/-)) mice are functionally and histologically protected against cisplatin-induced acute renal failure (ARF). Caspase-1 exerts proinflammatory effects via the cytokines interleukin (IL)-1beta, IL-18, IL-6, and neutrophil recruitment. We sought to determine the role of the cytokines IL-1beta, IL-18, and IL-6 and neutrophil recruitment in cisplatin-induced ARF. We first examined IL-1beta; renal IL-1beta increased nearly 2-fold in cisplatin-induced ARF and was reduced in the caspase-1(-/-) mice. However, inhibition with IL-1 receptor antagonist (IL-1Ra) did not attenuate cisplatin-induced ARF. Renal IL-18 increased 2.5-fold; however, methods to inhibit IL-18 using IL-18 antiserum and transgenic mice that overproduce IL-18-binding protein (a natural inhibitor of IL-18) did not protect. Renal IL-6 increased 3-fold; however, IL-6-deficient (IL-6(-/-)) mice still developed cisplatin-induced ARF. We next examined neutrophils; blood neutrophils increased dramatically after cisplatin injection; however, prevention of peripheral neutrophilia and renal neutrophil infiltration with the neutrophil-depleting antibody RB6-8C5 did not protect against cisplatin-induced ARF. In summary, our data demonstrated that cisplatin-induced ARF is associated with increases in the cytokines IL-1beta, IL-18, and IL-6 and neutrophil infiltration in the kidney. However, inhibition of IL-1beta, IL-18, and IL-6 or neutrophil infiltration in the kidney is not sufficient to prevent cisplatin-induced ARF.


Subject(s)
Acute Kidney Injury/chemically induced , Antineoplastic Agents/toxicity , Cisplatin/toxicity , Interleukin-18/physiology , Interleukin-1beta/physiology , Interleukin-6/physiology , Kidney/drug effects , Neutrophil Infiltration/drug effects , Animals , Caspase 1/physiology , Kidney/immunology , Kidney/pathology , Male , Mice , Mice, Inbred C57BL , Neutrophils/physiology
7.
Arthritis Rheum ; 54(9): 2872-85, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16947419

ABSTRACT

OBJECTIVE: The roles of the transmembrane and secreted forms of tumor necrosis factor alpha (TNFalpha) in rheumatoid arthritis (RA) remain unclear. Agents used to inhibit TNFalpha have shown varying efficacy in RA patients, suggesting that anti-TNFalpha agents possess dissimilar mechanisms of action, including the ability to neutralize transmembrane (tmTNFalpha) and secreted TNFalpha. In this study, TNFalpha-knockout (TNFalpha-KO) mice that were genetically altered to express elevated levels of tmTNFalpha were constructed to further understand the roles of the 17-kd secreted, trimeric, and 26-kd transmembrane forms of TNFalpha. METHODS: A speed-congenic mating scheme was used to generate 3 unique strains of mice: 1) transgenic tmTgA86 mice overexpressing 26-kd tmTNFalpha and also secreting 17-kd trimeric TNFalpha (tmTNFalpha-transgenic), 2) TNFalpha-/- mice (TNFalpha-KO), and 3) transgenic mice overexpressing tmTNFalpha backcrossed to TNFalpha-KO mice (tmTNFalpha-transgenic/TNFalpha-KO). Mice were treated with phosphate buffered saline (as vehicle control), dexamethasone (as positive control), or modified recombinant human soluble TNF receptor (sTNFR) p55 or p75, and were assessed clinically and histopathologically for signs of inflammation and development of arthritis. RESULTS: The tmTNFalpha-transgenic/TNFalpha-KO mice were born with crinkled tails and spinal deformities similar to those in ankylosing spondylitis. By 2-4 weeks, these mice developed symmetric inflammatory arthritis, characterized by tissue swelling, pannus formation, and bone deformities. The tmTNFalpha-transgenic mice also developed spontaneous-onset arthritis, but at a slower rate (100% incidence by 10-12 weeks). Clinical and histologic progression of arthritis in the tmTNFalpha-transgenic/TNFalpha-KO mice was reduced by treatment with dexamethasone or with the p55 or p75 sTNFR (69% and 63% reduction in total histologic score, respectively). CONCLUSION: These data show that arthritis is sufficiently initiated and maintained in tmTNFalpha-transgenic/TNFalpha-KO mice, and that it can be neutralized by recombinant human p55 or p75 sTNFR, resulting in amelioration of the biologic and subsequent histologic destructive effects of tmTNFalpha.


Subject(s)
Arthritis, Experimental/therapy , Receptor, Nerve Growth Factor/therapeutic use , Receptors, Tumor Necrosis Factor, Type II/therapeutic use , Tumor Necrosis Factor-alpha/deficiency , Tumor Necrosis Factor-alpha/genetics , Animals , Crosses, Genetic , Disease Models, Animal , Female , Humans , Male , Mice , Mice, Knockout , Mice, Transgenic , Remission, Spontaneous
8.
Am J Physiol Renal Physiol ; 291(5): F1090-5, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16985210

ABSTRACT

Acute renal failure (ARF) in septic patients drastically increases the mortality to 50-80%. Sepsis induces several proinflammatory cytokines including tumor necrosis factor-alpha (TNF-alpha), a major pathogenetic factor in septic ARF. Pentoxifylline has several functions including downregulation of TNF-alpha and endothelia-dependent vascular relaxation. We hypothesized that pentoxifylline may afford renal protection during endotoxemia either by downregulating TNF-alpha and/or by improving endothelial function. In wild-type mice, pentoxifylline protected against the fall in glomerular filtration rate (GFR; 105.2 +/- 6.6 vs. 50.2 +/- 6.6 microl/min, P < 0.01) at 16 h of LPS administration (2.5 mg/kg ip). This renal protective effect of pentoxifylline was associated with an inhibition of the rise in serum TNF-alpha (1.00 +/- 0.55 vs. 7.02 +/- 2.40 pg/ml, P < 0.05) and serum IL-1beta (31.3 +/- 3.6 vs. 53.3 +/- 5.9 pg/ml, P < 0.01) induced by LPS. Pentoxifylline also reversed the LPS-related increase in renal iNOS and ICAM-1 and rise in serum nitric oxide (NO). Enhanced red blood cell deformability by pentoxifylline may have increased shear rate and upregulated eNOS. Studies were therefore performed in eNOS knockout mice. The renal protection against endotoxemia with pentoxifylline was again observed as assessed by GFR (119.8 +/- 18.0 vs. 44.5 +/- 16.2 microl/min, P < 0.05) and renal blood flow (0.86 +/- 0.08 vs. 0.59 +/- 0.05 ml/min, P < 0.05). Renal vascular resistance significantly decreased with the pentoxifylline (91.0 +/- 5.8 vs. 178.0 +/- 7.6 mmHg.ml(-1).min(-1), P < 0.01). Thus pentoxifylline, an FDA-approved drug, protects against endotoxemia-related ARF and involves a decrease in serum TNF-alpha, IL-1beta, and NO as well as a decrease in renal iNOS and ICAM-1.


Subject(s)
Acute Kidney Injury/drug therapy , Endotoxemia/complications , Enzyme Inhibitors/pharmacology , Pentoxifylline/pharmacology , Acute Kidney Injury/etiology , Alprostadil/analogs & derivatives , Alprostadil/urine , Animals , Cyclic AMP/metabolism , Cyclic GMP/metabolism , Disease Models, Animal , Down-Regulation/drug effects , Glomerular Filtration Rate/drug effects , Intercellular Adhesion Molecule-1/metabolism , Interleukin-1/blood , Kidney Cortex/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Nitric Oxide/blood , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Nitric Oxide Synthase Type III , Tumor Necrosis Factor-alpha/metabolism
9.
J Am Coll Surg ; 201(3): 418-25, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16125076

ABSTRACT

BACKGROUND: Activated macrophages defend against tumors by secreting cytokines to recruit secondary immune cells, presenting antigen to T cells, and by direct tumor cytotoxicity. Peritoneal macrophages harvested from melanoma-bearing mice are less cytotoxic to melanoma cells, and produce less superoxide, nitric oxide, and tumor necrosis factor-alpha (TNF-alpha) than those from nontumor-bearing mice. Similar impairment of macrophage activation occurs in vitro using media harvested from cultured melanoma cells. Stimulation of Toll-like receptor 4 (TLR-4) activates macrophages and results in the release of TNF-alpha. We hypothesized that melanoma inhibits macrophage activation by suppressing TLR-4 signaling. STUDY DESIGN: Melanoma conditioned media (MCM) was generated from B16 melanoma cells. Peritoneal macrophages from TLR-4 competent or TLR-4 incompetent mice were exposed to control or MCM for 24 hours; then stimulated with lipopolysaccharide. TNF-alpha secretion, TNF-alpha mRNA production, nuclear factor-kappaB (NF-kappaB) activation, and TLR-4 surface expression were measured. RESULTS: Peritoneal macrophages exposed to MCM produced considerably less TNF-alpha in response to stimulus than controls (691 pg/mL versus 2,066 pg/mL, p < 0.001). TNF-alpha production by TLR-4 incompetent macrophages was not affected by MCM (454 pg/mL versus 480 pg/mL). Stimulated TNF-alpha mRNA and activated NF-kappaB were decreased in MCM treated C57BL/6 macrophages (by 38% and 33%, respectively). TLR-4 surface expression, however, was not decreased by exposure to MCM. CONCLUSIONS: Melanoma inhibits macrophage activation by suppressing TLR-4 signaling downstream of the TLR-4 receptor.


Subject(s)
Macrophage Activation , Melanoma, Experimental/immunology , Membrane Glycoproteins/immunology , Receptors, Cell Surface/immunology , Signal Transduction , Animals , Culture Media , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , NF-kappa B/pharmacology , RNA, Messenger/metabolism , Signal Transduction/physiology , Toll-Like Receptor 4 , Toll-Like Receptors , Tumor Necrosis Factor-alpha/biosynthesis
10.
Am J Physiol Renal Physiol ; 288(5): F997-1004, 2005 May.
Article in English | MEDLINE | ID: mdl-15644489

ABSTRACT

Caspase-1-deficient (-/-) mice are protected against sepsis-induced hypotension and mortality. We investigated the role of caspase-1 and its associated cytokines in a nonhypotensive model of endotoxemic acute renal failure (ARF). Mice were injected intraperitoneally with 2.5 mg of LPS that induces endotoxemic ARF. On immunoblot analysis of whole kidney, there was an increase in caspase-1 protein in LPS-treated mice compared with vehicle-treated controls. In LPS-treated mice, the glomerular filtration rate (GFR) was significantly higher in caspase-1 -/- vs. wild-type mice at 16 and 36 h after LPS. To determine the mechanism of this protection, the caspase-1-activated cytokines IL-1beta and IL-18 were investigated. IL-1beta and IL-18 protein were significantly increased in the kidneys of LPS- vs. vehicle-treated mice. To determine the role of these cytokines, mice were treated with recombinant IL-1 receptor antagonist (IL-1Ra) or IL-18-neutralizing antiserum. In LPS-treated mice, GFR was not different in IL-1Ra-treated or IL-18-neutralizing antiserum-treated or combination therapy (IL-1Ra plus IL-18-neutralizing antiserum-treated) compared with control mice. In addition, tubular cell apoptosis, neutrophil infiltration, myeloperoxidase activity, caspase-3 activity, and calpain activity were not different between wild-type and caspase-1 -/- mice with endotoxemic ARF. In LPS- vs. vehicle-treated wild-type mice, renal IL-1alpha was significantly increased. In both LPS- and vehicle-treated caspase-1 -/- mice, renal IL-1alpha was very low. In summary, caspase-1 -/- mice are functionally protected against endotoxemic ARF. Neutralization of IL-1beta and IL-18 is not functionally protective. The role of the intracellular proinflammatory cytokine IL-1alpha in endotoxemic ARF merits further study.


Subject(s)
Acute Kidney Injury/physiopathology , Caspase 1/genetics , Endotoxemia/physiopathology , Acute Kidney Injury/immunology , Acute Kidney Injury/pathology , Animals , Antibodies/pharmacology , Calpain/metabolism , Caspase 1/metabolism , Caspase 3 , Caspases/metabolism , Endotoxemia/immunology , Endotoxemia/pathology , Glomerular Filtration Rate , Interleukin 1 Receptor Antagonist Protein , Interleukin-1/metabolism , Interleukin-18/immunology , Interleukin-18/metabolism , Kidney/immunology , Kidney/pathology , Kidney/physiopathology , Male , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Peroxidase/metabolism , Sialoglycoproteins/immunology , Sialoglycoproteins/metabolism
11.
Mol Med ; 11(1-12): 1-15, 2005.
Article in English | MEDLINE | ID: mdl-16557334

ABSTRACT

We studied inhibition of histone deacetylases (HDACs), which results in the unraveling of chromatin, facilitating increased gene expression. ITF2357, an orally active, synthetic inhibitor of HDACs, was evaluated as an anti-inflammatory agent. In lipopolysaccharide (LPS)-stimulated cultured human peripheral blood mononuclear cells (PBMCs), ITF2357 reduced by 50% the release of tumor necrosis factor-alpha (TNFalpha) at 10 to 22 nM, the release of intracellular interleukin (IL)-1alpha at 12 nM, the secretion of IL-1beta at 12.5 to 25 nM, and the production of interferon-gamma (IFNgamma) at 25 nM. There was no reduction in IL-8 in these same cultures. Using the combination of IL-12 plus IL-18, IFNgamma and IL-6 production was reduced by 50% at 12.5 to 25 nM, independent of decreased IL-1 or TNFalpha. There was no evidence of cell death in LPS-stimulated PBMCs at 100 nM ITF2357, using assays for DNA degradation, annexin V, and caspase-3/7. By Northern blotting of PBMCs, there was a 50% to 90% reduction in LPS-induced steady-state levels of TNFalpha and IFNgamma mRNA but no effect on IL-1beta or IL-8 levels. Real-time PCR confirmed the reduction in TNFalpha RNA by ITF2357. Oral administration of 1.0 to 10 mg/kg ITF2357 to mice reduced LPS-induced serum TNFalpha and IFNgamma by more than 50%. Anti-CD3-induced cytokines were not suppressed by ITF2357 in PBMCs either in vitro or in the circulation in mice. In concanavalin-A-induced hepatitis, 1 or 5 mg/kg of oral ITF2357 significantly reduced liver damage. Thus, low, nonapoptotic concentrations of the HDAC inhibitor ITF2357 reduce pro-inflammatory cytokine production in primary cells in vitro and exhibit anti-inflammatory effects in vivo.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cytokines/antagonists & inhibitors , Cytokines/biosynthesis , Histone Deacetylase Inhibitors , Hydroxamic Acids/pharmacology , Inflammation Mediators/pharmacology , Amino Acid Sequence , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Humans , Inflammation/pathology , Inflammation/prevention & control , Inflammation Mediators/administration & dosage , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Zea mays/enzymology
12.
Kidney Int ; 66(6): 2202-13, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15569309

ABSTRACT

BACKGROUND: Cisplatin is a commonly used chemotherapeutic agent which causes apoptosis or necrosis of renal tubular epithelial cells in vitro. Caspases are a family of cysteine proteases that mediate apoptosis (caspase-3) and inflammation (caspase-1). Although well studied in vitro, caspases have not been previously studied in cisplatin-induced acute renal failure (ARF) in vivo. METHODS: Cisplatin (30 mg/kg) was injected intraperitoneally into wild-type and caspase-1-deficient (-/-) C57BL/6 mice. Serum creatinine and blood urea nitrogen (BUN), and renal caspase-1, -3, -8 and -9 activity were measured on days 1, 2, and 3 after cisplatin injection. Kidneys were examined for acute tubular necrosis (ATN), neutrophils, and apoptosis on days 1, 2, and 3. RESULTS: After cisplatin injection, serum creatinine and BUN were normal on day 1, began to increase on day 2, and peaked on day 3. Similarly, ATN scores and neutrophil counts peaked on day 3. In contrast, renal apoptosis significantly increased on day 2. Renal dysfunction, apoptosis, ATN scores and neutrophil infiltration were all reduced in the caspase-1(-/-) mice. In wild-type mice, caspase-1 and -3 activity increased on days 2 and 3. Caspase-3 activity was reduced by approximately 50% in caspase-1(-/-) mice; active caspase-3 detected by immunoblot was also reduced in caspase-1(-/-) mice. In vitro, addition of recombinant caspases to kidney cytosolic extracts determined that caspase-1 activates caspase-3 in renal tissue. CONCLUSION: These results indicate that caspase-1 contributes to cisplatin-induced ARF and ATN (day 3). Furthermore, caspase-1 affects caspase-3 activation and apoptosis in cisplatin-induced ARF (day 2).


Subject(s)
Antineoplastic Agents/toxicity , Caspase 1/genetics , Caspase 1/metabolism , Cisplatin/toxicity , Kidney Tubular Necrosis, Acute/metabolism , Animals , Apoptosis/physiology , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/metabolism , Cytosol/enzymology , Immunoblotting , Kidney/enzymology , Kidney/pathology , Kidney/physiopathology , Kidney Tubular Necrosis, Acute/chemically induced , Kidney Tubular Necrosis, Acute/pathology , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Necrosis , Neutrophils/pathology
13.
J Surg Res ; 121(2): 206-13, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15501460

ABSTRACT

BACKGROUND: The inflammatory response to vascular injury is characterized by expression of cytokines, growth factors, and chemokines that conspire to promote vessel remodeling and intimal hyperplasia (IH). Interleukin-10 (IL-10) is a multifunctional cytokine that has several anti-inflammatory properties in vitro. Few studies have evaluated the effects of IL-10 in experimental atherosclerosis. The purpose of the present study was to determine the influence of IL-10 on vascular inflammation and IH following mechanical injury. METHODS: Wire carotid injury was performed in wild-type (WT) mice with and without IL-10 treatment. Immunohistochemistry, PCR, and ELISA assays were used to examine vessel production of basic fibroblast growth factor (bFGF), monocyte chemotactic protein-1 (MCP-1), and nuclear factor kappa B (NFkappaB). Vessels were morphometrically analyzed for IH. RESULTS: Carotid injury induced early expression of MCP-1 and bFGF that was abrogated in mice treated with IL-10. Similarly, injury-induced expression of NFkappaB message and protein was attenuated in mice receiving exogenous IL-10. Compared to untreated mice, IL-10 markedly decreased levels of IH. Interestingly, carotid injury in IL-10-deficient mice resulted in an augmented IH response compared to injured WT mice. CONCLUSIONS: In an in vivo model of direct vascular injury, IL-10 decreased expression of the pro-inflammatory transcription factor, NFkappaB, and the mitogenic chemokine and growth factor, MCP-1 and bFGF, respectively. These observations were associated with IL-10-induced attenuation of IH. Furthermore, endogenous IL-10 appeared to suppress the injury response. In conclusion, exogenously delivered IL-10 may represent a clinically relevant anti-inflammatory strategy for post-injury intimal hyperplasia.


Subject(s)
Carotid Arteries/pathology , Carotid Artery Injuries/complications , Carotid Artery Injuries/pathology , Interleukin-10/pharmacology , Tunica Intima/pathology , Vasculitis/etiology , Animals , Carotid Arteries/drug effects , Carotid Arteries/metabolism , Chemokines/metabolism , Growth Substances/metabolism , Hyperplasia , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-kappa B/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tunica Intima/drug effects , Wounds, Nonpenetrating/complications , Wounds, Nonpenetrating/pathology
14.
Proc Natl Acad Sci U S A ; 101(23): 8815-20, 2004 Jun 08.
Article in English | MEDLINE | ID: mdl-15161979

ABSTRACT

IL-1 and IL-18 are members of the IL-1 family of ligands, and their receptors are members of the IL-1 receptor family. Although several biological properties overlap for these cytokines, differences exist. IL-18 uniquely induces IFN-gamma from T lymphocytes and natural killer cells but does not cause fever, whereas fever is a prominent characteristic of IL-1 in humans and animals. In the present study, human epithelial cells were stably transfected with the IL-18 receptor beta chain and responded to IL-18 with increased production of IL-1alpha, IL-6, and IL-8. Five minutes after exposure to either cytokine, phosphorylation of mitogen activated protein kinase (MAPK) p38 was present; specific inhibition of p38 MAPK reduced IL-18 activity to background levels. Whereas IL-1beta induced the expression of the NF-kappaB-reporter gene and was suppressed by competitive inhibition of NF-kappaB binding, IL-18 responses were weak or absent. In contrast to IL-1beta, IL-18 also did not activate degradation of the NF-kappaB inhibitor. After 4 h, both cytokines induced comparable levels of mRNA for the chemokine IL-8 but, in the same cells, steady-state levels of cyclooxygenase (COX)-2 mRNA were high after IL-1beta but low or absent after IL-18. After 30 h, IL-18-induced COX-2 appeared in part to be IL-1 dependent. Similarly, low levels of prostaglandin E2 were measured in IL-18-stimulated A549 cells and freshly obtained primary human monocytes and mouse macrophages. We conclude that in epithelial cells, IL-18 signal transduction is primarily via the MAPK p38 pathway rather than NF-kappaB, which may explain the absence of COX-2 and the failure of IL-18 to cause fever.


Subject(s)
Interleukin-18/pharmacology , Interleukin-1/pharmacology , MAP Kinase Signaling System/drug effects , Animals , Base Sequence , Cell Line , Cyclooxygenase 2 , DNA, Complementary/genetics , Dinoprostone/metabolism , Fever/etiology , Humans , In Vitro Techniques , Interleukin-1/biosynthesis , Interleukin-6/biosynthesis , Interleukin-8/biosynthesis , Isoenzymes/genetics , Isoenzymes/metabolism , Macrophages/drug effects , Macrophages/physiology , Membrane Proteins , Mice , Mitogen-Activated Protein Kinases/metabolism , Monocytes/drug effects , Monocytes/physiology , NF-kappa B/metabolism , Phosphorylation , Prostaglandin-Endoperoxide Synthases/genetics , Prostaglandin-Endoperoxide Synthases/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins/pharmacology , Transcription Factor AP-1/metabolism , p38 Mitogen-Activated Protein Kinases
15.
Am J Physiol Regul Integr Comp Physiol ; 287(3): R592-9, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15072965

ABSTRACT

Toll-like receptor 4 (TLR-4), initially identified as an LPS receptor, is critical to the signaling of a variety of danger signals, including heat shock protein 60, fibrinogen, and fibronectin. Recent data also suggest that TLR-4 plays a role in determining survival in both endotoxemia and hemorrhagic shock. We hypothesized that a functional TLR-4 would be required for hemorrhage and endotoxin-induced acute lung injury. Hemorrhage- and endotoxin-induced lung TNF-alpha mRNA and protein production, neutrophil accumulation, and protein permeability were dependent on a functional TLR-4. Hemorrhage-induced nuclear factor (NF)-kappaB activation was independent of functional TLR-4, whereas endotoxin-induced activation of NF-kappaB requires a functional TLR-4 for full response. Therefore, we conclude that 1) hemorrhage-induced acute lung injury is TLR-4 dependent and 2) hemorrhage has a different and distinct TLR-4-dependent intracellular activation mechanism compared with endotoxemia.


Subject(s)
Hemorrhage/complications , Lung Diseases/etiology , Membrane Glycoproteins/metabolism , Receptors, Cell Surface/metabolism , Acute Disease , Animals , Endotoxemia/metabolism , Hemorrhage/metabolism , Lung/metabolism , Lung/physiopathology , Male , Mice , Mice, Inbred C3H , NF-kappa B/metabolism , Neutrophil Infiltration , Permeability , Toll-Like Receptor 4 , Toll-Like Receptors , Tumor Necrosis Factor-alpha/metabolism
16.
Proc Natl Acad Sci U S A ; 101(11): 3921-6, 2004 Mar 16.
Article in English | MEDLINE | ID: mdl-15001702

ABSTRACT

Acute graft-versus-host disease (GVHD) and leukemic relapse are the two major obstacles to successful outcomes after allogeneic bone marrow transplantation (BMT), an effective therapy for hematological malignancies. Several studies have demonstrated that the dysregulation of proinflammatory cytokines and the loss of gastrointestinal tract integrity contribute to GVHD, whereas the donor cytotoxic responses are critical for graft-versus-leukemia (GVL) preservation. Suberoylanilide hydroxamic acid (SAHA) is currently in clinical trials as an antitumor agent; it inhibits the activity of histone deacetylases and at low doses exhibits antiinflammatory effects by reducing the production of proinflammatory cytokines. Using two well characterized mouse models of BMT, we have studied the effects of SAHA on GVHD severity and GVL activity. Administration of SAHA from day +3 to day +7 after BMT reduced serum levels of the proinflammatory cytokines and decreased intestinal histopathology, clinical severity, and mortality from acute GVHD compared with vehicle-treated animals. However, SAHA had no effect on donor T cell proliferative and cytotoxic responses to host antigens in vivo or in vitro. When mice received lethal doses of tumor cells at the time of BMT, administration of SAHA did not impair GVL activity and resulted in significantly improved leukemia-free survival by using two different tumor and donor/recipient combinations. These findings reveal a critical role for histone deacetylase inhibition in the proinflammatory events contributing to GVHD and suggest that this class of pharmacologic agents may provide a strategy to reduce GVHD while preserving cytotoxic T cell responses to host antigens and maintaining beneficial GVL effects.


Subject(s)
Graft vs Host Disease/drug therapy , Histone Deacetylase Inhibitors , Hydroxamic Acids/pharmacology , Immune System/drug effects , Animals , Bone Marrow Transplantation/immunology , Cytokines/drug effects , Female , Immune System/immunology , Leukemia/immunology , Mice , Transplantation, Homologous , Vorinostat
17.
J Thorac Cardiovasc Surg ; 126(3): 688-93, 2003 Sep.
Article in English | MEDLINE | ID: mdl-14502140

ABSTRACT

OBJECTIVE: Serologic evidence of Chlamydia pneumoniae infection and atherosclerosis was first demonstrated in patients with ischemic heart disease in 1988. Subsequently, the organism has been detected in several cardiovascular lesions. Outside of observational reports, few studies mechanistically link vascular infection with C. pneumoniae and atherogenesis. To better define its pathophysiologic role, we examined the influence of C. pneumoniae infection of human vascular smooth muscle cells on vascular smooth muscle cell proliferation, cell-cycle protein expression, and inflammatory cytokine release. METHODS: Human aortic vascular smooth muscle cells were inoculated with C. pneumoniae in culture. Proliferation was assessed by mitochondrial activity, direct cell counting, and immunohistochemical staining for proliferating cell nuclear antigen. Electromobility gel shift assays probed for the antiproliferative cell-cycle protein p53. Supernatants were assayed for the mitogens interleukin-6 and interleukin-8 by enzyme-linked immunosorbent assay. RESULTS: After C. pneumoniae inoculation, vascular smooth muscle cell proliferation increased 2-fold by mitochondrial activity and more than 3-fold by cell numbers. C. pneumoniae infection promoted a 3-fold increase in proliferating cell nuclear antigen expression, which was associated with decreased nuclear binding of p53. Compared with control, C. pneumoniae inoculation resulted in a 2.5-fold increase in released interleukin-6 and interleukin-8. In each experiment, the influence of C. pneumoniae was abrogated by concomitant treatment with the macrolide antibiotic azithromycin. CONCLUSIONS: C. pneumoniae induced human vascular smooth muscle cell proliferation and proliferating cell nuclear antigen expression, down-regulated p53, and promoted release of prototypical atherogenic cytokines. These in vitro findings indicate that C. pneumoniae is more than an innocent bystander, rather it is a pathophysiologic participant in atherogenesis warranting elimination.


Subject(s)
Arteriosclerosis/microbiology , Chlamydophila Infections/complications , Chlamydophila pneumoniae , Cells, Cultured , Chlamydophila pneumoniae/isolation & purification , Humans , Muscle, Smooth, Vascular/cytology
18.
Cytokine ; 21(2): 65-73, 2003 Jan 21.
Article in English | MEDLINE | ID: mdl-12670445

ABSTRACT

Interleukin 12 (IL-12) and IL-18 act synergistically to stimulate interferon gamma (IFN-gamma) production; moreover, IL-1 and tumor necrosis factor (TNF) may also augment IFN-gamma synthesis. We have investigated the relative contributions of these cytokines in the production of IFN-gamma and TNF by the Gram-positive bacterium Staphylococcus epidermidis, using the specific cytokine inhibitors IL-18 binding protein (IL-18BP), IL-1 receptor antagonist (IL-1Ra), anti-IL-12 antibodies (anti-IL-12 Ab), and TNF binding protein. Inhibition of caspase-1 reduced IFN-gamma and IL-1beta levels (by 80 and 67%, respectively) when heat-killed S. epidermidis was added to whole human blood cultures. IL-18BP reduced S. epidermidis-induced IFN-gamma (77% maximal suppression). In contrast, blocking IL-1 receptors by IL-1Ra had no effect on IFN-gamma production. Blocking endogenous IL-12 and TNF reduced IFN-gamma production by 69 and 36%. S. epidermidis-induced TNF-alpha was inhibited by IL-18BP and IL-1Ra, but not anti-IL-12 Ab, whereas IL-8 production was unaffected by any of the specific cytokine blocking agents. In conclusion, S. epidermidis stimulates IFN-gamma which is IL-18, IL-12 and TNF-dependent, but IL-1 independent.


Subject(s)
Gene Expression Regulation, Bacterial , Interferon-gamma/metabolism , Interleukin-12/physiology , Interleukin-18/physiology , Interleukin-1/physiology , Staphylococcus epidermidis/metabolism , Tumor Necrosis Factor-alpha/physiology , Adult , Antibodies, Monoclonal/metabolism , Caspase 1/metabolism , Dose-Response Relationship, Drug , Humans , Middle Aged
19.
Am J Physiol Regul Integr Comp Physiol ; 284(5): R1213-8, 2003 May.
Article in English | MEDLINE | ID: mdl-12531783

ABSTRACT

Tumor necrosis factor-alpha (TNF-alpha) is an important mediator in the inflammatory response to vascular injury. The present study sought to determine the relative contribution of each TNF-alpha receptor subtype (p55 and p75) to intimal hyperplasia (IH) and characterize the mechanisms of transcriptional regulation after vascular injury. A murine model of wire carotid arterial injury was employed to induce IH in wild-type (WT), p55-deficient (p55-/-), and p75-deficient (p75-/-) mice. Compared with injured WT and p75-/- animals, p55-/- mice demonstrated a twofold reduction in IH. Additionally, p55-/- mice demonstrated a decrease in expression of nuclear factor-kappaB mRNA and protein. These observations suggest an important role for the p55 receptor in IH after mechanical endoluminal injury. Suppression of the transcriptional activator nuclear factor-kappaB may provide a mechanism by which p55-mediated IH is attenuated.


Subject(s)
Antigens, CD/metabolism , Hyperplasia/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Tunica Intima/metabolism , Tunica Intima/pathology , Animals , Antigens, CD/genetics , Carotid Artery Injuries/metabolism , Carotid Artery Injuries/pathology , Gene Expression Regulation , Hyperplasia/pathology , Male , Mice , Mice, Knockout , NF-kappa B/metabolism , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor, Type I , Receptors, Tumor Necrosis Factor, Type II , Tumor Necrosis Factor-alpha/metabolism , Tunica Intima/injuries
20.
Proc Natl Acad Sci U S A ; 99(21): 13723-8, 2002 Oct 15.
Article in English | MEDLINE | ID: mdl-12381835

ABSTRACT

IL-1F7 was discovered in expressed sequence tag databases as a member of the increasing family of proteins sharing sequence homology to IL-1alpha/beta, IL-1Ra, and IL-18. In the present study using immunohistochemical staining, IL-1F7 was localized in human peripheral monocytic cells, suggesting its role in immune regulation. Recombinant human IL-1F7b was shown to bind to the IL-18Ralpha but without IL-18 agonistic or antagonistic function. Using chemical cross-linking, we observed that, unlike IL-18, IL-1F7b fails to recruit the IL-18Rbeta chain to form a functionally active, ternary complex with the IL-18Ralpha chain. IL-1F7b shares two conserved amino acids with IL-18 (Glu-35 and Lys-124), which participate in the interaction of IL-18 with the IL-18Ralpha chain as well as the IL-18-binding protein (IL-18BP), a secreted protein that neutralizes IL-18 activity. In testing whether IL-1F7b interacts with IL-18BP, we unexpectedly observed that IL-1F7b enhanced the ability of IL-18BP to inhibit IL-18-induced IFNgamma by 25-30% in a human natural killer cell line. This effect was observed primarily at limiting concentrations of IL-18BP (3.12-12.5 ng/ml) and at a 50- to 100-fold molar excess of IL-1F7b. Similar results were obtained by using isolated human peripheral blood mononuclear cells. To study the molecular basis of this effect we performed binding studies of IL-1F7b and IL-18BP. After cross-linking, a high molecular weight complex consisting of IL-1F7b and IL-18BP was observed on SDS/PAGE. We propose that after binding to IL-18BP, IL-1F7b forms a complex with IL-18Rbeta, depriving the beta-chain of forming a functional receptor complex with IL-18Ralpha and thus inhibiting IL-18 activity.


Subject(s)
Glycoproteins/chemistry , Glycoproteins/metabolism , Interleukin-18/antagonists & inhibitors , Interleukin-1/chemistry , Interleukin-1/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cell Line , Gene Expression , Humans , In Vitro Techniques , Intercellular Signaling Peptides and Proteins , Interferon-gamma/biosynthesis , Interleukin-1/genetics , Interleukin-18/genetics , Interleukin-18 Receptor alpha Subunit , Killer Cells, Natural/immunology , Macromolecular Substances , Mice , Molecular Sequence Data , Monocytes/immunology , Protein Structure, Tertiary , Receptors, Interleukin/chemistry , Receptors, Interleukin/metabolism , Receptors, Interleukin-18 , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Transfection
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