Erythropoietin does not affect TNF and IL-6 production directly.

The tissue-protective action of erythropoietin (EPO) in animal models is often associated with reduced inflammation. However, there are many contrasting reports of the effect of EPO on the production of inflammatory cytokines induced by lipopolysaccharide (LPS) in vitro, with different papers reporting an inhibition, an upregulation, or a lack of effect. Negative results are likely underestimated by a publication bias. As EPO has anti-inflammatory actions in models associated with tissue injury, we hypothesized that EPO could specifically inhibit the induction of inflammatory cytokines by danger signals associated with cell death, and investigated its effect on the induction of IL-6 or TNF by high-mobility group-box 1 protein (HMGB1) or by necrotic cells. We did not observe any significant effect of EPO in these models; neither EPO affected the response induced by TLR agonists different from LPS, or by extracellular ATP-mediated activation of the inflammasome. We conclude that the inhibition of inflammation by EPO is likely to be an indirect effect, secondary to its tissue-protective activity, or that it requires a prior priming induced by the injury.

Beneficial effects of PKF275-055, a novel, selective, orally bioavailable, long-acting dipeptidyl peptidase IV inhibitor in streptozotocin-induced diabetic peripheral neuropathy.

1-[(2-adamantyl)amino]acetyl-2-cyano-(S)-pyrrolidine, monohydrochloride (PKF275-055), a vildagliptin analog, is a novel, selective, potent, orally bioavailable, and long-acting dipeptidyl peptidase IV inhibitor. We studied the effect of PKF275-055 administration on the prevention, protection, and treatment of diabetic neuropathy in the streptozotocin-induced diabetic rat. PKF275-055 improved body and muscle weight. Oral glucose tolerance tests showed a marked improvement in glucose metabolism under all treatment schedules. When tested in prevention and protection experiments, PKF275-055 completely averted the decrease of Na⁺/K⁺-ATPase activity and partially counteracted the nerve conduction velocity (NCV) deficit observed in untreated diabetic rats but had no effects on abnormal mechanical and thermal sensitivity. When used in a therapeutic setting, PKF275-055 induced a significant correction in the alteration in Na⁺,K⁺-ATPase activity and NCV present in untreated diabetics. Diabetic rats developed mechanical hyperalgesia within 2 weeks after streptozotocin injection and exhibited significantly longer thermal response latencies. It is noteworthy that PKF275-055 treatment restored mechanical sensitivity thresholds by approximately 50% (p < 0.01) and progressively improved the alteration in thermal responsiveness. In conclusion, PKF275-055 showed an anabolic effect, improved oral glucose tolerance, and counteracted the alterations in Na⁺,K⁺-ATPase activity, NCV, and nociceptive thresholds in diabetic rats. The present data support a potential therapeutic effect of PKF275-055 in the treatment of rodent diabetic neuropathy.

Hemodiafiltration with endogenous reinfusion with and without acetate-free dialysis solutions: effect on ESA requirement.

BACKGROUND: Hemofiltrate reinfusion (HFR) is a form of hemodiafiltration (HDF) in which replacement fluid is constituted by ultrafiltrate from the patient 'regenerated' through a cartridge containing hydrophobic styrene resin. Bicarbonate-based dialysis solutions (DS) used in routine hemodialysis and HDF contain small quantities of acetate (3-5 mM) as a stabilizing agent, one of the major causes of intradialytic hypotension. Acetate-free (AF) DS have recently been made available, substituting acetate with hydrochloric acid. The impact of AF DS during HFR on Hb levels and erythropoietic-stimulating agent (ESA) requirement in chronic dialysis patients was assessed. PATIENTS AND METHODS: After obtaining informed consent, 30 uremic patients treated by standard bicarbonate dialysis (BHD, DS with acetate) were randomized to treatment in 3-month cycles: first AF HFR, followed by HFR with acetate, and again AF HFR. At the beginning and end of each period, Hb and ESA requirements were evaluated. RESULTS: A significant increase in the Hb level was observed throughout all periods of HFR versus BHD (from 11.1 to 11.86 g/dl; p = 0.04), with a significant decrease of ESA requirements from 29,500 to 25,033 IU/month (p = 0.04). CONCLUSION: Regardless of the presence or absence of acetate in DS, HFR per se allows a significant lowering of ESA dosage versus BHD, while at the same time increasing Hb levels. Taking for granted the clinical impact produced, HFR seems to provide a relevant decrease in end-stage renal disease patient costs.

Adrenalectomy sensitizes mice to the lethal effects of interleukin 1 and tumor necrosis factor.

To clarify the possible role of TNF and IL-1 in endotoxic shock, the lethality of rTNF (human and murine) and IL-1 in adrenalectomized mice was studied. Adrenalectomy, which has long been known to increase the susceptibility to endotoxin, rendered mice susceptible to TNF and IL-1 in terms of mortality. The lethality of endotoxin, TNF, or IL-1 was totally prevented by pretreatment with dexamethasone (minimal effective dose, 0.3 mg/Kg) but not by ibuprofen (10 mg/Kg).

Protective effect of chlorpromazine on endotoxin toxicity and TNF production in glucocorticoid-sensitive and glucocorticoid-resistant models of endotoxic shock.

The present study was designed to define the potential of chlorpromazine (CPZ) as a protective agent against lipopolysaccharide (LPS) toxicity in comparison with glucocorticoids, and to obtain initial correlations with its effects on the levels of tumor necrosis factor (TNF), a pivotal mediator of endotoxic shock. It was found that CPZ protects mice, normal or adrenalectomized, and guinea pigs against lethality of LPS, and inhibited TNF serum levels, like dexamethasone (DEX), a well-known inhibitor of TNF synthesis. CPZ protected against LPS lethality when administered 30 minutes (min) before, simultaneously, or up to 10 min after LPS and was ineffective when given 30 min after LPS, paralleling the inhibitory effect on TNF production. In another experimental model, where mice were sensitized to LPS toxicity by actinomycin D, CPZ significantly inhibited LPS lethality and hepatotoxicity, whereas under these conditions DEX was inactive. These experiments indicate that CPZ has a protective action in both glucocorticoid-sensitive and -resistant models of endotoxic shock.

Intracerebroventricular injection of interleukin 1 induces high circulating levels of interleukin 6.

IL-1 is known to have a central role in the induction of acute-phase response, and some of its activities (including induction of some acute-phase proteins) were reported to be mediated by an induction of IL-6. Administration to rats of 200 ng of human rIL-1 by intracerebroventricular injection resulted in a more marked induction of circulating IL-6 than the same dose of IL-1 administered systemically (intravenously or intraperitoneally). Induction of serum IL-6 by centrally administered IL-1 was also observed in hypophysectomized or adrenalectomized rats, suggesting that activation of the hypothalamus-pituitary-adrenal axis is not essential for this effect of IL-1. IL-6 induction was also observed after pretreatment with indomethacin, indicating that the effect was dissociated from the pyrogenic activity of IL-1. Induction of IL-6 by a central action could represent a novel pathway in IL-1-induced acute-phase response.

Defective inflammatory response in interleukin 6-deficient mice.

Systemic and localized inflammation elicit a number of host responses which include fever, cachexia, hypoglycemia, and major changes in the concentration of liver plasma proteins. Interleukin 6 (IL-6) is considered an important mediator of the inflammatory response, together with IL-1 and tumor necrosis factor alpha (TNF-alpha). The purpose of this study was to unequivocally determine the role of IL-6 in these phenomena making use of IL-6-deficient mice that we have recently generated by gene targeting. We report here that in the absence of IL-6, mice are unable to mount a normal inflammatory response to localized tissue damage generated by turpentine injection. The induction of acute phase proteins is dramatically reduced, mice do not lose body weight and only suffer from mild anorexia and hypoglycemia. In contrast, when systemic inflammation is elicited through the injection of bacterial lipopolysaccharide (LPS), these parameters are altered to the same extent both in wild-type and IL-6-deficient mice, demonstrating that under these conditions IL-6 function is dispensable. Moreover, we show that LPS-treated IL-6-deficient mice produce three times more TNF-alpha than wild-type controls, suggesting that increased TNF-alpha production might be one of the compensatory mechanisms through which a normal response to LPS is achieved in the absence of IL-6. We also show that corticosterone is normally induced in IL-6-deficient mice, demonstrating that IL-6 is not required for the activation of the hypothalamic-pituitary-adrenal axis. Our results reinforce the idea that different patterns of cytokines are involved in systemic and localized tissue damage, and identify IL-6 as an essential mediator of the inflammatory response to localized inflammation.

Thioredoxin, a redox enzyme released in infection and inflammation, is a unique chemoattractant for neutrophils, monocytes, and T cells.

Thioredoxin (Trx) is a ubiquitous intracellular protein disulfide oxidoreductase with a CXXC active site that can be released by various cell types upon activation. We show here that Trx is chemotactic for monocytes, polymorphonuclear leukocytes, and T lymphocytes, both in vitro in the standard micro Boyden chamber migration assay and in vivo in the mouse air pouch model. The potency of the chemotactic action of Trx for all leukocyte populations is in the nanomolar range, comparable with that of known chemokines. However, Trx does not increase intracellular Ca2+ and its activity is not inhibited by pertussis toxin. Thus, the chemotactic action of Trx differs from that of known chemokines in that it is G protein independent. Mutation of the active site cysteines resulted in loss of chemotactic activity, suggesting that the latter is mediated by the enzyme activity of Trx. Trx also accounted for part of the chemotactic activity released by human T lymphotropic virus (HTLV)-1-infected cells, which was inhibited by incubation with anti-Trx antibody. Since Trx production is induced by oxidants, it represents a link between oxidative stress and inflammation that is of particular interest because circulating Trx levels are elevated in inflammatory diseases and HIV infection.

Prostacyclin synthesis induced in vascular cells by interleukin-1.

Supernatants from cultures of human monocytes that had been stimulated with endotoxin or silica induced the synthesis of prostacyclin in endothelial and smooth muscle cells. The lymphokine mediating these effects on the cells of the blood vessel wall was identified as interleukin-1; interferons and interleukin-2 were inactive. Interleukin-1-induced prostacyclin synthesis represents a new aspect of the interaction between the immune system (as well as other tissues) and the vessel wall and may serve as a basis for the development of new strategies in antithrombotic therapy.

Molecular mapping and detoxification of the lipid A binding site by synthetic peptides.

Endotoxin [lipopolysaccharide (LPS)], the major antigen of the outer membrane of Gram-negative bacteria, consists of a variable-size carbohydrate chain that is covalently linked to N,O-acylated beta-1,6-D-glucosamine disaccharide 1,4'-bisphosphate (lipid A). The toxic activity of LPS resides in the lipid A structure. The structural features of synthetic peptides that bind to lipid A with high affinity, detoxify LPS in vitro, and prevent LPS-induced cytokine release and lethality in vivo were defined. The binding thermodynamics were comparable to that of an antigen-antibody reaction. Such synthetic peptides may provide a strategy for prophylaxis and treatment of LPS-mediated diseases.

Regulation of the macrophage content of neoplasms by chemoattractants.

Factor chemotactic for mononuclear phagocytes was found in supernatant fluids of cultured human and mouse tumor cells. In 11 mouse tumors there was a correlation observed between chemotactic activity and macrophage content of neoplastic tissues. Tumor-derived chemoattractants appear to participate in the regulation of tumor-associated macrophages.

Recombinant tumor necrosis factor/cachectin and interleukin 1 pretreatment decreases lung oxidized glutathione accumulation, lung injury, and mortality in rats exposed to hyperoxia.

Single, preexposure, parenteral injection with both recombinant tumor necrosis factor/cachectin (TNF/C) and interleukin-1 (IL-1) prolonged the survival of rats (144 +/- 9 h) in continuous hyperoxia (greater than 99% O2 at 1 atm) when compared with rats injected with boiled TNF/C and boiled IL-1 (61 +/- 2 h), TNF/C alone (61 +/- 2 h), IL-1 alone (62 +/- 2 h), or saline (64 +/- 3 h). After exposure to hyperoxia for 52 h, pleural effusion volume, pulmonary artery pressure, total pulmonary resistance, and lung morphologic damage were decreased in those rats given TNF/C and IL-1 as compared with saline-injected rats. In parallel, ratios of reduced (GSH) to oxidized (GSSG) glutathione were greater (P less than 0.05) in lungs of TNF/C + IL-1-injected rats (91 +/- 20) than of saline-injected rats (30 +/- 4) that had been exposed to hyperoxia for 52 h. No differences were found in superoxide dismutase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, or catalase activities in lungs of TNF/C + IL-1- or saline-treated, hyperoxia-exposed rats. Our results indicate that pretreatment with TNF/C and IL-1 favorably altered lung glutathione redox status, decreased lung injury, and enhanced survival of rats exposed to hyperoxia.

Role of reactive oxygen intermediates in the interferon-mediated depression of hepatic drug metabolism and protective effect of N-acetylcysteine in mice.

Interferon (IFN) and IFN inducers are known to depress hepatic microsomal cytochrome P-450 levels, and the liver toxicity of IFN was reported to be lethal in newborn mice. We have observed that administration to mice of IFN and IFN inducers caused a marked increase in liver xanthine oxidase activity. Because this enzyme is well known to produce reactive oxygen intermediates and cytochrome P-450 was reported to be sensitive to the oxidative damage, we have tested the hypothesis that a free radical mechanism could mediate the depression of cytochrome P-450 levels by IFN. Administration to mice of the IFN inducer polyinosinic-polycytidylic acid (2 mg/kg i.p.) caused a 29 to 52% decrease in liver cytochrome P-450. Concomitant p.o. administration of the free radical scavenger, N-acetylcysteine (as a 2.5% solution in drinking water), or the xanthine oxidase inhibitor, allopurinol (100 mg/kg), protected against the IFN-mediated depression of P-450 kg), protected against the IFN-mediated depression of P-450 levels. The results suggest that an increased endogenous generation of free radicals, possibly due to the induction of xanthine oxidase, is implicated in the IFN-mediated depression of liver drug metabolism. The relevance of these data also extends to cases in which this side effect is observed in pathological situations (e.g., viral diseases and administration of vaccines) associated with an induction of IFN.

Interleukin 1-induced augmentation of experimental metastases from a human melanoma in nude mice.

This study has examined the effect of the cytokine interleukin 1 (IL-1) on metastasis formation by the human melanoma A375M in nude mice. We have found that human recombinant IL-1 beta (a single injection greater than 0.01 micrograms per mouse i.v. given before tumor cells) induced an augmentation of experimental lung metastases from the A375M tumor cells in nude mice. This effect was rapidly induced and reversible within 24 h after IL-1 injection. A similar effect was induced by human recombinant IL-1 alpha and human recombinant tumor necrosis factor, but not by human recombinant interleukin 6. 5-[125I]odo-2'-deoxyuridine-radiolabeled A375M tumor cells injected i.v. remained at a higher level in the lungs of nude mice receiving IL-1 than in control mice. In addition, IL-1 injected 1 h, but not 24 h, after tumor cells enhanced lung colonization as well, thus suggesting an effect of IL-1 on the vascular transit of tumor cells. These findings may explain the observation of enhanced secondary localization of tumor cells at inflammatory sites and suggest that modulation of secondary spread should be carefully considered when assessing the ability of this cytokine to complement cytoreductive therapies.

Mycobacterial Cpn10 promotes recognition of the mammalian homologue by a mycobacterium-specific antiserum.

Self-tolerance, a key feature of the immune system, is still a matter of intense debate. We give here evidence for a peculiar behavior of an antiserum against Mycobacterium tuberculosis chaperonin 10 (m-Cpn10), which could have implications for the mechanism of self-recognition by antibodies against non-self. We show that this antiserum can interact in terms of both inhibition of biological activity and physical association (immunoprecipitation), with the mammalian homologue of m-Cpn10, but only if the bacterial protein is present. Several lines of evidence led us to exclude that the two proteins physically associate to form heterocomplexes: (1) the behavior of the antiserum was not shared by a monoclonal antibody against m-Cpn10; (2) a matrix selective for human Cpn10 (h-Cpn10) did not co-purify m-Cpn10; (3) the distribution pattern in non-denaturing isoelectric focusing of labeled m-Cpn10 was not altered by the presence of the unlabeled h-Cpn10. We conclude therefore that the antiserum against M. tuberculosis Cpn10 also recognizes mammalian Cpn10, with an affinity/avidity regulated by the mycobacterial protein, or by the promotion of hetero-oligomerization. This emergence of self-recognition in the presence of M. tuberculosis Cpn10 could imply a breaking of self-tolerance in situations of infection or vaccination.

PTX3, A prototypical long pentraxin, is an early indicator of acute myocardial infarction in humans.

BACKGROUND: Inflammation is an important component of ischemic heart disease. PTX3 is a long pentraxin whose expression is induced by cytokines in endothelial cells, mononuclear phagocytes, and myocardium. The possibility that PTX3 is altered in patients with acute myocardial infarction (AMI) has not yet been tested. METHODS AND RESULTS: Blood samples were collected from 37 patients admitted to the coronary care unit (CCU) with symptoms of AMI. PTX3 plasma concentrations, as measured by ELISA, higher than the mean+2 SD of age-matched controls (2.01 ng/mL) were found in 27 patients within the first 24 hours of CCU admission. PTX3 peaked at 7.5 hours after CCU admission, and mean peak concentration was 6.94+/-11.26 ng/mL. Plasma concentrations of PTX3 returned to normal in all but 3 patients at hospital discharge and were unrelated to AMI site or extent, Killip class at entry, hours from symptom onset, and thrombolysis. C-reactive protein peaked in plasma at 24 hours after CCU admission, much later than PTX3 (P<0.001). Patients >64 years old and women had significantly higher PTX3 concentrations at 24 hours (P<0.05). PTX3 was detected by immunohistochemistry in normal but not in necrotic myocytes. CONCLUSIONS: PTX3 is present in the intact myocardium, increases in the blood of patients with AMI, and disappears from damaged myocytes. We suggest that PTX3 is an early indicator of myocyte irreversible injury in ischemic cardiomyopathy.

Erythropoietin as an antiapoptotic, tissue-protective cytokine.

Erythropoietin (EPO) increases the number of circulating erythrocytes primarily by preventing apoptosis of erythroid progenitors. In addition to this proerythroid action, results of recent studies show that systemically administered EPO is protective in vivo, in several animal models of neuronal injury. In vitro, EPO prevents neuronal apoptosis induced by a variety of stimuli. This review summarizes the neuroprotective actions of EPO and discusses the underlying mechanisms in terms of signal transduction pathways involved. The understanding of these mechanisms will help differentiate the neuroprotective actions of EPO from its role in the bone marrow.

Hemodiafiltration with post-dilution reinfusion of the regenerated ultrafiltrate: a new on-line technique.

AIMS: All convective hemodiafiltration techniques require a replacement fluid, which must have an adequate electrolytic composition and must be sterile and pyrogen-free. Using an integrated adsorption cartridge, the ultrafiltrate can be "regenerated" and used as a replacement fluid (hemo-filtrate reinfusion; HFR). The aim of this study was to evaluate whether the HFR technique as suggested in its original configuration could be improved by inverting the purification sequence (post-dilution HFR; PDHFR) in order to increase the purification efficiency of the whole system. METHODS: We performed standard HFR in 6 uremic patients during 6 months and, subsequently, during further 6 months, PDHFR. The dialytic efficacy of the two techniques and the filter blood loss were evaluated. Moreover, we studied how both techniques affected cytokine levels. RESULTS: We observed a significant increase of urea extraction and of Kt/V values in PDHFR. An equally significant improvement was observed in regard to the extraction of beta2-m and the blood loss. Furthermore, IL6 and TNFalpha decreased significantly after PDHFR treatment. CONCLUSIONS: HFR has proven to be an easy-to-perform hemodiafiltration technique, capable of resolving the typical problem of the other hemodiafiltration technique, the availability and production of a sterile and ultrapure reinfusion solution. The inversion of its configuration has allowed us to improve three aspects that have characterized, in our experience, the treatments performed in the original geometry: the removal of both urea and beta2-m, and the filter. Finally, it's notable that the decrease in cytokines levels achieved with PDHFR might attenuate the uremic micro-inflammatory state.

Defective production of reactive oxygen intermediates by tumor-associated macrophages exposed to phorbol ester.

Macrophages were isolated from poorly immunogenic metastatic sarcomas (mFS6 and MN/MCA1) of C57BL/6 origin. Tumor-associated macrophages (TAM) showed little release of superoxide when exposed to phorbol myristate acetate. When exposed to a phagocytic stimulus (zymosan), TAM released appreciable amounts of superoxide. TAM had a lower number of specific binding sites for phorbol esters than resident or caseinate-elicited peritoneal macrophages, but had normal NADPH-cytochrome C reductase. The tumor environment, possibly through previously demonstrated products of neoplastic cells, may influence the functional status of in situ macrophages and, thus, impair host anti-tumor and anti-microbial defense mechanisms.

Dexamethasone modulation of in vivo effects of endotoxin, tumor necrosis factor, and interleukin-1 on liver cytochrome P-450, plasma fibrinogen, and serum iron.

Treatment of mice with endotoxin (lipopolysaccharide, LPS) and the two LPS-induced monokines, tumor necrosis factor (TNF) and interleukin-1 (IL-1), caused a depression of liver cytochrome P-450 and related drug-metabolizing enzymes, as well as other acute-phase changes including increase in plasma fibrinogen levels and hypoferremia. However, only IL-1, not TNF or LPS, depressed cytochrome P-450 in cultured hepatocytes, suggesting that the effect of TNF in vivo might be mediated by a second mediator. TNF- or LPS-stimulated monocytes released a factor capable of depressing cytochrome P-450 in cultured hepatocytes. This factor was inhibited by anti-IL-1 antiserum, and its synthesis, like that of IL-1, was inhibited by dexamethasone (DEX). Pretreatment of mice with DEX protected against the depression of liver cytochrome P-450 by LPS or TNF but not by IL-1, suggesting that IL-1 directly depresses cytochrome P-450 and that DEX acts by inhibiting IL-1 synthesis in vivo induced by LPS or TNF. However, DEX did not inhibit two other effects of LPS and TNF in vivo: increase of plasma fibrinogen levels and decrease of plasma iron, suggesting that these might not be mediated by IL-1. Therefore, the effect of DEX in vivo, although supporting the hypothesis that depression of liver cytochrome P-450 by LPS and TNF is mediated by IL-1, indicates the existence of IL-1-independent pathways in the acute-phase response.