Synthetic melanin suppresses production of proinflammatory cytokines

An overproduction of proinflammatory cytokines mediates the damaging sequelae of inflammation in pathologic conditions such as rheumatoid arthritis, graft-vs-host reaction, cachexia, and sepsis syndrome. We examined the cytokine regulatory activity of synthetic melanin, exemplified by biosynthetic l-glycine-l-tyrosine-based polymer (ME-1) and chemosynthetic dihydroxyphenylalanine-based polymer (MC-1). At nontoxic concentrations, both compounds effectively (>/=60%) and reversibly suppressed the production of tumor necrosis factor (TNF), even when applied after stimulation of human peripheral blood monocytes with lipopolysaccharide (LPS). The inhibitory activity of melanin was selective with regard to cytokine response but not inducer- or cell-type-specific. In addition to TNF, melanin inhibited production of interleukin (IL)-1beta, IL-6, and IL-10 but not granulocyte-macrophage colony-stimulating factor by the LPS-stimulated monocytes. Melanin was equally effective in inhibiting production of TNF by monocytes stimulated with the purified protein derivative of Mycobacterium tuberculosis and production of IL-6 by IL-1alpha-stimulated human fibroblasts and endothelial cells. Northern blot analysis, mRNA stability determination, immunoprecipitation studies on metabolically labeled intracellular TNF, and pulse chase experiments revealed that melanin reduced efficiency of mRNA translation. The finding that melanin arrests ongoing cytokine synthesis suggests that this compound may be useful as an adjunct therapy for conditions showing involvement of proinflammatory cytokines.     

The microglia-activating potential of thrombin: the protease is not involved in the induction of proinflammatory cytokines and chemokines

The serine protease thrombin is known as a blood coagulation factor. Through limited cleavage of proteinase-activated receptors it can also control growth and functions in various cell types, including neurons, astrocytes, and microglia (brain macrophages). A number of previous studies indicated that thrombin induces the release of proinflammatory cytokines and chemokines from microglial cells, suggesting another important role for the protease beyond hemostasis. In the present report, we provide evidence that this effect is not mediated by any proteolytic or non-proteolytic mechanism involving thrombin proper. Inhibition of the enzymatic thrombin activity did not affect the microglial release response. Instead the cyto-/chemokine-inducing activity solely resided in a high molecular weight protein fraction that could be isolated in trace amounts even from apparently homogenous alpha- and gamma-thrombin preparations. High molecular weight material contained thrombin-derived peptides as revealed by mass spectrometry but was devoid of thrombin-like enzymatic activity. Separated from the high molecular weight fraction by fast protein liquid chromatography, enzymatically intact alpha- and gamma-thrombin failed to trigger any release. Our findings may force a revision of the notion that thrombin itself is a direct proinflammatory release signal for microglia. In addition, they could be relevant for the study of other cellular activities and their assignment to this protease.     

Chronic ANG II infusion increases plasma triglyceride level by stimulating hepatic triglyceride production in rats

We recently observed that ANG II receptor blocker therapy improved the overproduction of triglyceride (TG) in fructose-fed rats and Zucker fatty rats with insulin resistance, which in turn suggests that ANG II may stimulate TG production. Accordingly, we investigated the effects of ANG II on TG production and the association with insulin resistance in normal rats. Male Wistar rats were continuously infused with ANG II (100 ng.min(-1).kg body wt(-1)) via an osmotic minipump for 14 days. ANG II infusion markedly elevated both the systolic and diastolic blood pressure. The plasma TG level increased twofold, but cholesterol was unchanged. ANG II infusion stimulated the TG secretion rate (TGSR) by twofold and increased the hepatic TG content by 31%. Lipogenesis determined by [2-(3)H]glycerol incorporation into hepatic TG was also significantly increased in ANG II-infused rats. The stimulatory effect of ANG II on TGSR was dose dependent and was not observed until 2 wk after the start of infusion. ANG II infusion significantly reduced insulin sensitivity index (SI) without affecting glucose effectiveness determined by Bergman's minimal model. The plasma TG level was positively correlated with TGSR (r = 0.88, P < 0.001) and inversely with SI (r = -0.80, P < 0.005). These results suggest that chronic ANG II infusion stimulates hepatic TG production, which is partly associated with simultaneous development of insulin resistance. Our results may suggest a new mechanism for the intimate association between hypertension and dyslipidemia.     

Royal jelly inhibits the production of proinflammatory cytokines by activated macrophages

In this study, we have examined the anti-inflammatory actions of royal jelly (RJ) at a cytokine level. When supernatants of RJ suspensions were added to a culture of mouse peritoneal macrophages stimulated with lipopolysaccharide and IFN-gamma, the production of proinflammatory cytokines, such as TNF-alpha, IL-6, and IL-1, was efficiently inhibited in a dose-dependent manner without having cytotoxic effects on macrophages. This suggests that RJ contains factor(s) responsible for the suppression of proinflammatory cytokine secretion. We named the factor for honeybees RJ-derived anti-inflammatory factor (HBRJ-AIF), and further investigated the molecular aspects of it. Size fractionation study showed that HBRJ-AIF is composed of substances of low (< 5 kDa) and high (> 30 kDa) molecular weights, with the former being a major component. Chromatographic analysis showed that MRJP3 is one candidate for the HBRJ-AIF with high molecular weights. Thus, our results suggest that RJ has anti-inflammatory actions through inhibiting proinflammatory cytokine production by activated macrophages.     

Resistin messenger-RNA expression is increased by proinflammatory cytokines in vitro

Resistin is a recently discovered polypeptide that induces insulin resistance in rodents. While in rodents resistin is predominantly expressed in adipocytes, in humans peripheral blood mononuclear cells (PBMC) seem to a be a major source of resistin. In the present study, we show that in human PBMC resistin mRNA expression-determined by fluorescence-based real-time polymerase chain reaction-is strongly increased by the proinflammatory cytokines interleukin (IL)-1, IL-6, tumor necrosis factor alpha (TNF-alpha), and also by lipopolysaccharides (LPS), respectively, while no effect was found by interferon-gamma (IFN-gamma) or leptin. Our results suggest that in humans resistin may be a link in the well-known association between inflammation and insulin resistance.     

Lethality in LPS-induced endotoxemia in C3H/HeCr mice is associated with prevalence of proinflammatory cytokines: lack of protective action of lactoferrin

C3H/HeCr mice are more susceptible to infection compared with other strains. Lactoferrin (LF), a protein involved in innate defense, was shown to protect mice against lethal endotoxemia. In this investigation we attempt to explain the cause of increased susceptibility of C3H/HeCr mice to LPS and lack of protective LF action in these mice. We found that C3H/HeCr mice produced up to 5-fold more serum TNFalpha and 66% higher IFNgamma levels in response to i.v. LPS injection than the control, CBA strain. 24 h pretreatment of C3H/HeCr mice with LF did not cause inhibition of the LPS-induced TNFalpha serum levels, whereas in CBA mice LF significantly decreased TNFalpha level. IL-6 serum levels, in turn, were lowered in C3H/HeCr mice but elevated in CBA mice. That differential regulation of cytokine production by LF in C3H/HeCr mice paralleled a decreased survival after lethal LPS injection - 10% vs. 60% in control, PBS treated mice. In addition, determination of colony forming units (CFU) in livers and spleens after administration of 10(8) Escherichia coli revealed that pretreatment of CBA mice with LF caused a marked reduction of CFU in these organs, whereas in C3H/HeCr mice the changes were insignificant. These results indicate that the altered TNFalpha/IL-6 ratio in C3H/HeCr mice, as compared to control CBA mice, as well as the increased IFNgamma level, may be responsible for the increased susceptibility to endotoxemia in that substrain. We also suggest that an association exists between the LF protective effect against endotoxic sequelae and the insult-induced systemic immune response.     

The MAPK scaffold kinase suppressor of Ras is involved in ERK activation by stress and proinflammatory cytokines and induction of arthritis

The MAPK ERK is required for LPS-induced TNF production by macrophages. Although the scaffold kinase suppressor of Ras (KSR)1 is required for efficient Erk activation by mitogenic stimuli, the role of KSR1 in ERK activation by inflammatory and stress stimuli is unknown. In this study, we examined the effects of KSR deficiency on ERK activation by stress stimuli and show that ERK activation by TNF, IL-1, and sorbitol is attenuated in the absence of KSR1. To determine the significance of this defect in vivo, we tested KSR-deficient mice using a passive transfer model of arthritis. We found that the induction of arthritis is impaired in the absence of KSR. Thus, KSR plays a role in ERK activation during inflammatory and stress responses both in vitro and in vivo.     

Tetrahydropyridine derivatives with inhibitory activity on the production of proinflammatory cytokines: Part 1

We investigated proinflammatory cytokine TNFα production inhibitors in order to develop novel anti-inflammatory agents. According to the results, we found that 17, a pyrrole derivative possessing a tetrahydropyridine group at the β-position, showed potent inhibitory activity in vitro (inhibition of lipopolysaccharide (LPS) induced TNFα production in human whole blood, IC₅₀ = 1.86 μM) and in vivo (inhibition of LPS induced TNFα production in mice, ID₅₀ = 5.98 mg/kg).     

Tetrahydropyridine derivatives with inhibitory activity on the production of proinflammatory cytokines: Part 3

In order to develop a new class of anti-rheumatic drug which inhibits production of proinflammatory cytokines such as TNFα, IL-1β, IL-6, and IL-8, a series of 3-pyridylpyrrole derivatives possessing a bicyclic tetrahydropyridine moiety at the 4-position of the pyrrole ring were synthesized and their pharmacological activities were evaluated. The derivatives were found to have potent inhibitory activities on the production of the cytokines both in vitro and in vivo. Among them, compound 4a, (S)-2-(4-fluorophenyl)-4-(1,2,3,5,6,8a-hexahydroindolizin-7-yl)-3-(pyridin-4-yl)-1H-pyrrole (R-132811), achieved the most promising results in various in vitro and in vivo tests including several rheumatoid arthritis models ((i) inhibition of p38α, p38β, p38γ, and p38δ MAP kinases: IC₅₀ = 0.034, 0.572, >10, and >10 μM, respectively; (ii) inhibition of TNFα, IL-1β, IL-6, and IL-8 production in human whole blood: IC₅₀ = 0.026, 0.020, 0.88, and 0.016 μM, respectively; (iii) inhibition of LPS induced TNFα, IL-1β and IL-6 production in mice: ID₅₀ = 0.93, 8.63, and 0.11 mg/kg, po, respectively; (iv) inhibition of anti-collagen antibody-induced arthritis in mice: ID₅₀ = 2.22 mg/kg, po; (v) inhibition of collagen-induced arthritis in mice: ID₅₀ = 2.38 mg/kg, po; (vi) prophylactic effect on adjuvant-induced arthritis in rats: ID₅₀ = 3.1 mg/kg, po; (vii) therapeutic effect on adjuvant-induced arthritis in rats: ID₅₀ = 4.9 mg/kg, po; (viii) analgesic effect on adjuvant-induced arthritic pain in rats: ID₅₀ = 2.9 mg/kg, po). As a result, compound 4a was chosen as a candidate for further pre-clinical studies.     

ANG II is required for optimal overload-induced skeletal muscle hypertrophy

ANG II mediates the hypertrophic response of overloaded cardiac muscle, likely via the ANG II type 1 (AT(1)) receptor. To examine the potential role of ANG II in overload-induced skeletal muscle hypertrophy, plantaris and/or soleus muscle overload was produced in female Sprague-Dawley rats (225-250 g) by the bilateral surgical ablation of either the synergistic gastrocnemius muscle (experiment 1) or both the gastrocnemius and plantaris muscles (experiment 2). In experiment 1 (n = 10/group), inhibiting endogenous ANG II production by oral administration of an angiotensin-converting enzyme (ACE) inhibitor during a 28-day overloading protocol attenuated plantaris and soleus muscle hypertrophy by 57 and 96%, respectively (as measured by total muscle protein content). ACE inhibition had no effect on nonoverloaded (sham-operated) muscles. With the use of new animals (experiment 2; n = 8/group), locally perfusing overloaded soleus muscles with exogenous ANG II (via osmotic pump) rescued the lost hypertrophic response in ACE-inhibited animals by 71%. Furthermore, orally administering an AT(1) receptor antagonist instead of an ACE inhibitor produced a 48% attenuation of overload-induced hypertrophy that could not be rescued by ANG II perfusion. Thus ANG II may be necessary for optimal overload-induced skeletal muscle hypertrophy, acting at least in part via an AT(1) receptor-dependent pathway.     

Tetrahydropyridine derivatives with inhibitory activity on the production of proinflammatory cytokines: Part 2

We previously reported a novel pyrrole derivative 1 which possesses a tetrahydropyridine group at the β-position with a proinflammatory cytokine TNFα production inhibitor. Herein, we report the synthesis and biological activity of N- and α-position substituted tetrahydropyridine derivatives. In this series, we found that compound 3o showed good inhibitory activity in vitro (inhibition of lipopolysaccharide (LPS)-induced TNFα production in human whole blood, IC₅₀ = 0.44 μM) and compound 3i demonstrated potent inhibitory activity in vivo (inhibition of LPS-induced TNFα production in mice, ID₅₀ = 1.42 mg/kg).     

Parthenolide attenuates LPS-induced fever, circulating cytokines and markers of brain inflammation in rats

Parthenolide, a sesquiterpene lactone, has been reported to exhibit a variety of anti-inflammatory and immunomodulatory effects. To test the effect of parthenolide on brain inflammatory responses, brain oxidative stress and fever, we treated rats with parthenolide (1 mg/kg), simultaneously or 1 h prior to a systemic (i.p.) challenge with a moderate dose (100 μg/kg) of lipopolysaccharide (LPS). The initial hypothermia was exaggerated; the second phase of the biphasic LPS-induced fever and circulating interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) were significantly attenuated only in parthenolide-pretreated animals. In the hypothalamus, markers of NFκB/NF-IL6 pathway activation (inhibitor κBα, NF-IL6 and the serin/threonin kinase-like protein mRNA expression) and markers of oxidative stress (including nuclear respiratory factor 1) and NFκB immunoreactivity were significantly reduced while NF-IL6 immunoreactivity and suppressor of cytokine signaling 3 mRNA expression remained unaltered, 8 h after LPS-stimulation with parthenolide-pretreatment. Importantly, this response was accompanied by decreased mRNA expression of the rate limiting enzyme in prostaglandin synthesis, cyclooxygenase 2 (COX2), known for its critical role in fever induction pathways. A direct action of parthenolide on brain cells was also confirmed in a primary neuro-glial cell culture of the vascular organ of the lamina terminalis a pivotal brain structure for fever manifestation with a leaky blood-brain barrier. In summary, pretreatment with parthenolide attenuates the febrile response during LPS-induced systemic inflammation by reducing circulating IL-6 and TNFα and decreasing hypothalamic NFκB/NF-IL6 activation, oxidative stress and expression of COX2. Thus parthenolide appears to have the potential to reduce brain inflammation.     

Cardiac modulations of ANG II receptor expression in rats with hypoxic pulmonary hypertension

Right ventricular myocardial hypertrophy during hypoxic pulmonary hypertension is associated with local renin-angiotensin system activation. The expression of angiotensin II type 1 (AT(1)) and type 2 (AT(2)) receptors in this setting has never been investigated. We have therefore examined the chronic hypoxia pattern of AT(1) and AT(2) expression in the right and left cardiac ventricles, using in situ binding and RT-PCR assays. Hypoxia produced right, but not left, ventricular hypertrophy after 7, 14, and 21 days, respectively. Hypoxia for 2 days was associated in each ventricle with a simultaneous and transient increase (P < 0.05) in AT(1) binding and AT(1) mRNA levels in the absence of any significant change in AT(2) expression level. Only after 14 days of hypoxia, AT(2) binding increased (P < 0.05) in the two ventricles, concomitantly with a right ventricular decrease (P < 0.05) in AT(2) mRNA. Along these data, AT(1) and AT(2) binding remained unchanged in both the left and hypertrophied right ventricles from rats treated with monocrotaline for 30 days. These results indicate that chronic hypoxia induces modulations of AT(1) and AT(2) receptors in both cardiac ventricles probably through direct and indirect mechanisms, respectively, which modulations may participate in myogenic (at the level of smooth or striated myocytes) rather than in the growth response of the heart to hypoxia.     

Triggering the immune response by proinflammatory cytokines

What determines whether or not an immune response takes place? The older view that it is only the presence or absence of T cells bearing appropriate receptors that matters has been replaced by one which lays at least equal emphasis on inflammation: whether or not antigenpresenting cells become activated and secrete proinflammatory cytokines. The evidence for this view comes partly from older work on animal models of autoimmunity and tolerance, partly signs of “immunological neglect” in transgenic mice akin to “immunological privilege”, and partly from some remarkably supportive genetics. The genetics includes genome scanning for quantitative trait loci determining disease susceptibility and case/control disease associations. Migration inhibitory factor is an interesting proinflammatory cytokine which does not yet fit into this scheme. We ask but do not answer the question whether protective T cell populations are subject to the same rules of activation.     

Phosphatidylglycerol is involved in the dimerization of photosystem II

Photosystem II core dimers (450 kDa) and monomers (230 kDa) consisting of CP47, CP43, the D1 and D2 proteins, the extrinsic 33-kDa subunit, and the low molecular weight polypeptides PsbE, PsbF, PsbH, PsbI, PsbK, PsbL, PsbTc, and PsbW were isolated by sucrose density gradient centrifugation. The photosystem II core dimers were treated with phospholipase A2 (PL-A2), which cuts phosphatidylglycerol (PG) and phosphatidylcholine molecules at the sn-2 position. The PL-A2-treated dimers dissociated into two core monomers and further, yielding a CP47-D1-D2 subcomplex and CP43. Thin layer chromatography showed that photosystem II dimers contained four times more PG than their monomeric counterparts but with similar levels of phosphatidylcholine. Consistent with this was the finding that, compared with monomers, the dimers contained a higher level of trans-hexadecanoic fatty acid (C16:1Delta3tr), which is specific to PG of the thylakoid membrane. Moreover, treatment of dimers with PL-A2 increased the free level of this fatty acid specific to PG compared with untreated dimers. Further evidence that PG is involved in stabilizing the dimeric state of photosystem II comes from reconstitution experiments. Using size exclusion chromatography, it was shown that PG containing C16:1Delta3tr, but not other lipid classes, induced significant dimerization of isolated photosystem II monomers. Moreover, this dimerization was observed by electron crystallography when monomers were reconstituted into thylakoid lipids containing PG. The unit cell parameters, p2 symmetry axis, and projection map of the reconstituted dimer was similar to that observed for two-dimensional crystals of the native dimer.     

Hypersensitivity to acute ANG II in female growth-restricted offspring is exacerbated by ovariectomy

Female growth-restricted offspring are normotensive in adulthood. However, ovariectomy induces a marked increase in mean arterial pressure (MAP) that is abolished by renin angiotensin system (RAS) blockade, suggesting RAS involvement in the etiology of hypertension induced by ovariectomy in adult female growth-restricted offspring. Blockade of the RAS also abolishes hypertension in adult male growth-restricted offspring. Moreover, sensitivity to acute ANG II is enhanced in male growth-restricted offspring. Thus, we hypothesized that an enhanced sensitivity to acute ANG II may contribute to hypertension induced by ovariectomy in female growth-restricted offspring. Female offspring were subjected to ovariectomy (OVX) or sham ovariectomy (intact) at 10 wk of age. Cardio-renal hemodynamic parameters were determined before and after an acute infusion of ANG II (100 ng·kg(-1)·min(-1) for 30 min) at 16 wk of age in female offspring pretreated with enalapril (40 mg·kg(-1)·day(-1) for 7 days). Acute ANG II induced a significant increase in MAP in intact growth-restricted offspring (155 ± 2 mmHg, P < 0.05) relative to intact control (145 ± 4 mmHg). Ovariectomy augmented the pressor response to ANG II in growth-restricted offspring (163 ± 2 mmHg, P < 0.05), with no effect in control (142 ± 2 mmHg). Acute pressor responses to phenylephrine did not differ in growth-restricted offspring relative to control, intact, or ovariectomized. Furthermore, renal hemodynamic responses to acute ANG II were significantly enhanced only in ovariectomized female growth-restricted offspring. Thus, these data suggest that enhanced responsiveness to acute ANG II is programmed by intrauterine growth restriction and that sensitivity to acute ANG II is modulated by ovarian hormones in female growth-restricted offspring.     

Microinjection of ANG II into paraventricular nucleus enhances cardiac sympathetic afferent reflex in rats

The aims of present study were to determine whether angiotensin II (ANG II) in the paraventricular nucleus (PVN) is involved in the central integration of the cardiac sympathetic afferent reflex and whether this effect is mediated by the ANG type 1 (AT(1)) receptor. While the animals were under alpha-chloralose and urethane anesthesia, mean arterial pressure, heart rate, and renal sympathetic nerve activity (RSNA) were recorded in sinoaortic-denervated and cervical-vagotomized rats. A cannula was inserted into the left PVN for microinjection of ANG II. The cardiac sympathetic afferent reflex was tested by electrical stimulation (5, 10, 20, and 30 Hz in 10 V and 1 ms) of the afferent cardiac sympathetic nerves or epicardial application of bradykinin (BK) (0.04 and 0.4 microg in 2 microl). Microinjection of ANG II (0.03, 0.3, and 3 nmol) into the PVN resulted in dose-related increases in the RSNA responses to electrical stimulation. The percent change of RSNA response to 20- and 30-Hz stimulation increased significantly at the highest dose of ANG II (3 nmol). The effects of ANG II were prevented by pretreatment with losartan (50 nmol) into the PVN. Microinjection of ANG II (0.3 nmol) into the PVN significantly enhanced the RSNA responses to epicardial application of BK, which was abolished by pretreatment with losartan (50 nmol) into the PVN. These results suggest that exogenous ANG II in the PVN augments the cardiac sympathetic afferent reflex evoked by both electrical stimulation of cardiac sympathetic afferent nerves and epicardial application of BK. These central effects of ANG II are mediated by AT(1) receptors.