Armando Ialenti

Modulation of acute inflammation by endogenous nitric oxide

The role of endogenous nitric oxide (NO) in acute inflammation was investigated using two inhibitors of NO synthase (NG-nitro-L-arginine methyl ester(L-NAME) and NG-monomethyl-L-arginine (L-NMMA)) as well as L- or D-arginine. The effect of test compounds was studied on the carrageenin-induced increase in vascular permeability in rat skin and in dextran- and carrageenin-induced paw oedema. Both L-NAME and L-NMMA dose dependently inhibited the increase in vascular permeability and oedema formation. L- but not D-arginine increased these inflammatory responses and reversed the inhibitory effects of L-NAME and L-NMMA. In dexamethasone-treated rats L-arginine enhanced the dextran-induced oedema and the early phase of carrageenin-induced oedema but did not modify the inhibition by dexamethasone of the late phase of carrageenin-induced oedema. These results suggest that endogenous NO is released at the site of acute inflammation and modulates oedema formation. Depending on the time course or on the type of inflammation, NO may be predominantly generated by the constitutive or by the inducible NO synthase.

Modulation of adjuvant arthritis by endogenous nitric oxide

1 The role of endogenous nitric oxide (NO) in adjuvant arthritis in Lewis rats has been studied by use of l‐arginine, the amino acid from which NO is synthesized, and NG‐nitro‐l‐arginine methyl ester (l‐NAME), an inhibitor of NO synthase. Prolonged modulation (35 days) of the l‐arginine: NO pathway in rats was achieved by dissolving test compounds in the drinking water (l‐arginine: 3, 10 and 30 mg ml−1; l‐NAME: 0.1, 1 and 10 mg ml−1). 2 Arthritis was exacerbated by l‐arginine and suppressed by l‐NAME in a dose‐related fashion. Combined treatment with l‐NAME (1 mg ml−1) and l‐arginine (30 mg ml−1) did not modify the arthritis. 3 Reduced weight gain, which is a feature of adjuvant arthritis, was modified by these compounds so that l‐arginine reduced weight gain whereas l‐NAME increased weight gain compared with that in control animals. 4 d‐Arginine (30 mg ml−1), NG‐nitro‐d‐arginine methyl ester (d‐NAME: 1 mg ml−1) and l‐lysine (30 mg ml−1), an amino acid not involved in the generation of NO, were without effect on either arthritis or body weight gain. 5 . Antigen‐stimulated proliferation of T‐lymphocytes as well as generation of nitrite (NO2−) and release of acid phosphatase from macrophages were all enhanced in l‐arginine‐treated arthritic rats and reduced in l‐NAME‐treated animals. 6 These results suggest that endogenous NO modulates adjuvant arthritis, possibly by interfering with the activation of T‐lymphocytes and/or macrophages.

Phytochemical compounds involved in the anti-inflammatory effect of propolis extract

Two ethanolic propolis extracts (EPE) with and without the caffeic acid phenethyl ester (CAPE), CAPE and galangin (major components of propolis) were investigated for anti-inflammatory activity in rats using carrageenin foot oedema, carrageenin pleurisy and adjuvant arthritis. In our experiments, EPE with CAPE and CAPE alone significantly inhibited carrageenin oedema, carrageenin pleurisy and adjuvant arthritis. In contrast EPE without CAPE and galangin did not exhibit anti-inflammatory effects in acute and chronic inflammation. These results suggest that the anti-inflammatory activity of propolis is due to CAPE.

Modulation by nitric oxide of prostaglandin biosynthesis in the rat.

1 Modulation of prostaglandin biosynthesis in vivo by either exogenous or endogenous nitric oxide (NO) has been studied in the rat using arachidonic acid (AA)‐induced paw oedema and measuring both the foot volume and the amount of 6‐keto‐prostaglandin F1α (6‐keto‐PGF1α), the stable metabolite of prostacyclin (PGI2), in the oedematous fluid recovered from inflamed paws. 2 Paw injections of 150 or 300 nmol of AA were virtually inactive whereas 600 nmol produced a moderate oedema which was greatly reduced by the NO synthase inhibitor l‐NG‐nitro arginine methyl ester (l‐NAME, 100 nmol/paw) and the NO scavenger haemoglobin (Hb, 30 μmol/paw), but unaffected by the inhibitor of the soluble guanylate cyclase, methylene blue (Mb, 3 μmol/paw) and L‐arginine (15 μmol/paw). 3 The NO‐donors (10 μmol/paw) 3‐morpholino‐sydnonimine‐hydrochloride (SIN‐1), S‐nitroso‐N‐acetyl‐d, l‐penicillamine (SNAP) and sodium nitroprusside (SNP) significantly potentiated the paw oedema induced by AA (300 nmol/paw). 4 SIN‐1 (2.5, 5 and 10 μmol/paw) produced a significant dose‐dependent increase of the oedema induced by AA which was correlated with increased amounts of 6‐keto‐PGF1α in the fluid recovered from inflamed paws. 5 Both oedema and prostaglandin biosynthesis induced by the combination AA + SIN‐1 were greatly suppressed by either Hb (30 μmol/paw) or indomethacin (3 μmol/paw or 5 mg kg−1 s.c.) but unaffected by Mb (3 μmol/paw). 6 In LPS‐treated rats (6 mg kg−1, i.p.) doses of AA inactive in normal animals produced a remarkable oedema which was reduced by l‐NAME or Hb, unaffected by Mb and increased by l‐arginine. 7 These results demonstrate that NO increases prostaglandin biosynthesis in vivo through a guanosine 3′:5′‐cyclic monophosphate (cyclic GMP)‐independent mechanism and suggest that the interaction between NO synthase and cyclo‐oxygenase (COX) pathways may represent an important mechanism for the modulation of the inflammatory response.

Plasmacytoid Dendritic Cells Play a Key Role in Promoting Atherosclerosis in Apolipoprotein E–Deficient Mice

Objective—Clinical studies have identified that reduced numbers of circulating plasmacytoid dendritic cells (pDCs) act as a predictor of cardiovascular events in coronary artery disease and that pDCs are detectable in the shoulder region of human atherosclerotic plaques, where rupture is most likely to occur. Results from animal models are controversial, with pDCs seen to inhibit or promote lesion development depending on the experimental settings. Here, we investigated the role of pDCs in atherosclerosis in apolipoprotein E−deficient mice. Methods and Results—We demonstrated that the aorta and spleen of both apolipoprotein E−deficient and C57BL/6 mice displayed similar numbers of pDCs, with similar activation status. In contrast, assessment of antigen uptake/presentation using the E&agr;/Y-Ae system revealed that aortic pDCs in apolipoprotein E−deficient- mice were capable of presenting in vivo systemically administered antigen. Continuous treatment of apolipoprotein E−deficient mice with anti−mouse plasmacytoid dendritic cell antigen 1 (mPDCA-1) antibody caused specific depletion of pDCs in the aorta and spleen and significantly reduced atherosclerosis formation in the aortic sinus (by 46%; P<0.001). Depletion of pDCs also reduced macrophages (by 34%; P<0.05) and increased collagen content (by 41%; P<0.05) in aortic plaques, implying a more stable plaque phenotype. Additionally, pDC depletion reduced splenic T-cell activation and inhibited interleukin-12, chemokine (C-X-C motif) ligand 1, monokine induced by interferon-&ggr;, interferon &ggr;−induced protein 10, and vascular endothelium growth factor serum levels. Conclusion—These results identify a critical role for pDCs in atherosclerosis and suggest a potential role for pDC targeting in the control of the pathology.

Artery tertiary lymphoid organs control aorta immunity and protect against atherosclerosis via vascular smooth muscle cell Lymphotoxin β receptors

Summary Tertiary lymphoid organs (TLOs) emerge during nonresolving peripheral inflammation, but their impact on disease progression remains unknown. We have found in aged Apoe−/− mice that artery TLOs (ATLOs) controlled highly territorialized aorta T cell responses. ATLOs promoted T cell recruitment, primed CD4+ T cells, generated CD4+, CD8+, T regulatory (Treg) effector and central memory cells, converted naive CD4+ T cells into induced Treg cells, and presented antigen by an unusual set of dendritic cells and B cells. Meanwhile, vascular smooth muscle cell lymphotoxin β receptors (VSMC-LTβRs) protected against atherosclerosis by maintaining structure, cellularity, and size of ATLOs though VSMC-LTβRs did not affect secondary lymphoid organs: Atherosclerosis was markedly exacerbated in Apoe−/−Ltbr−/− and to a similar extent in aged Apoe−/−Ltbrfl/flTagln-cre mice. These data support the conclusion that the immune system employs ATLOs to organize aorta T cell homeostasis during aging and that VSMC-LTβRs participate in atherosclerosis protection via ATLOs.

Hyaluronic acid modulates acute and chronic inflammation.

We have studied the effect of hyaluronic acid (HA) in two standard models of acute and chronic inflammation in the rat, i.e. carrageenin oedema and adjuvant arthritis. Our results show that HA was able to inhibit, in a dose-related fashion, both types of inflammatory reactions. These findings suggest that HA may have a modulatory role in the inflammatory process and may explain, at least in part, the protective role of HA in the inflammatory cartilage damage.

Role of cyclopentenone prostaglandins in rat carrageenin pleurisy.

In this study, using rat carrageenin‐induced pleurisy, we found that treatment of rats with either indomethacin or NS‐398 suppressed the pleurisy at 2 h but significantly exacerbated this reaction at 48 h. Exacerbated inflammation was associated with reduced prostaglandin D2 levels, decreased heat shock factor 1 (HSF1) activation, reduced hsp72 expression and increased activation of nuclear factor κB (NF‐κB). Replacement of cyclopentenone prostaglandins by treating rats with either prostaglandin J2 or prostaglandin D2 reversed the exacerbating effects of cyclooxygenase inhibitors leading to the resolution of the reaction. In conclusion, we demonstrate that cyclopentenone prostaglandins may act as anti‐inflammatory mediators by inducing in inflammatory cells HSF1‐dependent hsp72 expression and NF‐κB inhibition, two crucial events for the remission of inflammation.

Relationship between nitric oxide and prostaglandins in carrageenin pleurisy

The correlation between endogenous nitric oxide (NO) generation and prostaglandin biosynthesis was studied in rat carrageenin pleurisy induced by the injection of 0.2 mL of 1% lambda-carrageenin into the pleural cavity. The pleural exudate was collected at 4 hr and the amounts of NO2- + NO3- (NOx) and prostaglandin E2 (PGE2) measured. The NOx present in the inflammatory exudate was determined by measuring the NO2- with the Griess reaction, after the reduction of NO3- to NO2- using acid-washed cadmium powder. PGE2 was measured by radioimmunoassay. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 1-3-10 mg/kg subcutaneously) reduced NOx by 20 +/- 7%, 41 +/- 6% and 55 +/- 9% (P < 0.01) and PGE2 by 9 +/- 6%, 41 +/- 11% and 74 +/- 9% (P < 0.001). Conversely, L-arginine (300 mg/kg SC) increasedNOx by 39 +/- 7% (P < 0.01) and PGE2 by 78 +/- 6% (P < 0.001). The NO scavenger haemoglobin (Hb), coinjected into the pleural cavity (3 mg/site) with carrageenin, produced a parallel inhibition of NOx (65 +/- 16%, P < 0.001) and PGE2 (71 +/- 18%, P < 0.001). The soluble guanylate cyclase inhibitor methylene blue (Mb; 2 mg/site) had no effect. Moreover haemoglobin, but not methylene blue, was able to significantly suppress the L-arginine-induced increase of both NOx and PGE2. In each pleural exudate, independently from the animal treatment, the amount of NOx was highly correlated to the amount of PGE2 (r = 0.93, P < 0.001). These results suggest that in rat carrageenin pleurisy the modulation of the L-arginine:NO pathway results in a parallel modulation of prostaglandin biosynthesis. The interaction between cyclooxygenase and the NO pathway may represent an important mechanism for the modulation of the inflammatory response.

Endogenous nitric oxide increases prostaglandin biosynthesis in carrageenin rat paw oedema

The influence of endogenous nitric oxide (NO) on prostaglandin biosynthesis in rat carrageenin oedema was studied. Oedema formation and prostaglandin E2 generation in the inflamed paw were both increased by L- but not D-arginine and reduced by the NO synthase inhibitor, L-NG-nitro arginine methyl ester, and the NO scavenger, haemoglobin. Methylene blue, an inhibitor of the soluble guanylate cyclase, had no effect. These results suggest that endogenous NO modulates carrageenin oedema by increasing prostaglandin biosynthesis at the inflammatory site through a cGMP-independent mechanism.