Zsolt Lohinai

Inosine Inhibits Inflammatory Cytokine Production by a Posttranscriptional Mechanism and Protects Against Endotoxin-Induced Shock

Extracellular purines, including adenosine and ATP, are potent endogenous immunomodulatory molecules. Inosine, a degradation product of these purines, can reach high concentrations in the extracellular space under conditions associated with cellular metabolic stress such as inflammation or ischemia. In the present study, we investigated whether extracellular inosine can affect inflammatory/immune processes. In immunostimulated macrophages and spleen cells, inosine potently inhibited the production of the proinflammatory cytokines TNF-α, IL-1, IL-12, macrophage-inflammatory protein-1α, and IFN-γ, but failed to alter the production of the anti-inflammatory cytokine IL-10. The effect of inosine did not require cellular uptake by nucleoside transporters and was partially reversed by blockade of adenosine A1 and A2 receptors. Inosine inhibited cytokine production by a posttranscriptional mechanism. The activity of inosine was independent of activation of the p38 and p42/p44 mitogen-activated protein kinases, the phosphorylation of the c-Jun terminal kinase, the degradation of inhibitory factor κB, and elevation of intracellular cAMP. Inosine suppressed proinflammatory cytokine production and mortality in a mouse endotoxemic model. Taken together, inosine has multiple anti-inflammatory effects. These findings, coupled with the fact that inosine has very low toxicity, suggest that this agent may be useful in the treatment of inflammatory/ischemic diseases.

Protective effects of mercaptoethylguanidine, a selective inhibitor of inducible nitric oxide synthase, in ligature-induced periodontitis in the rat

Excessive production of nitric oxide (NO), and the generation of peroxynitrite have been implicated in various proinflammatory conditions. In the present study, using mercaptoethylguanidine (MEG), a selective inhibitor of iNOS and a peroxynitrite scavenger, we investigated the role of iNOS and peroxynitrite in a rat model of periodontitis. Periodontitis was produced in rat by a ligature of 2/0 braided silk placed around the cervix of the lower left 1st molar. Animals were then divided into two groups: one group of rats was treated with MEG (30 mg kg−1, i.p., 4 times per day for 8 days), animals in the other group received vehicle. At day 8, the gingivomucosal tissue encircling the mandibular 1st molars was removed on both sides from ligated and sham operated animals for inducible nitric oxide synthase (iNOS) activity assay and for immunocytochemistry with anti‐iNOS serum. Plasma extravasation was measured with the Evans blue technique. Alveolar bone loss was measured with a videomicroscopy. Ligation caused a significant, more than 3 fold increase in the gingival iNOS activity, whereas it did not affect iNOS activity on the contralateral side, when compared to sham‐operated animals. Immunohistochemical analysis revealed iNOS‐positive macrophages, lymphocytes and PMNs in the connective tissue and immunoreactive basal layers of epithelium on side of the ligature, and only a few iNOS‐reactive connective tissue cells on the contralateral side. Ligation significantly increased Evans blue extravasation in gingivomucosal tissue and alveolar bone destruction compared to the contralateral side. MEG treatment significantly reduced the plasma extravasation and bone destruction. The present results demonstrated that ligature‐induced periodontitis increases local NO production and that MEG treatment protects against the associated extravasation and bone destruction. Based on the present data, we propose that enhanced formation of NO and peroxynitrite plays a significant role in the pathogenesis of periodontitis.

Nitric Oxide Synthase in Healthy and Inflamed Human Dental Pulp

Nitric oxide synthase (NOS) plays a significant role in the pathogenesis of pulpitis. In this study, we hypothesized the existence of endothelial (eNOS) and inducible (iNOS) enzyme isoforms in human dental pulp. Extracted third molar pulps were divided into groups based on clinical diagnosis: healthy, hyperemic, and irreversible pulpitis. We have localized the eNOS and iNOS by immunohistochemistry and have tested their mRNA expression by RT-PCR and protein levels by Western blots. eNOS is present in the endothelial cells and odontoblasts of the healthy pulp, but an elevation of eNOS mRNA and protein levels with a concomitant dilation of vessels was characteristic under pathological conditions. Healthy pulp tissue failed to exhibit any iNOS; however, acute inflammation enhanced the mRNA and protein levels of iNOS, mainly in the leukocytes. There are differences in localization and expression between eNOS and iNOS in healthy and inflamed dental pulp.

Evidence for the expression of cyclooxygenase-2 enzyme in periodontitis.

We investigated the role of the inducible isoform of cyclooxygenase (COX-2) in a rat model of periodontitis using a selective COX-2 inhibitor NS-398. Periodontitis was produced by a silk ligature placed around the lower left 1st molar. Animals were treated with NS-398 (3 mg kg(-1) i.p., 2 times per day for 7 days) or vehicle. At Day 8, the gingivomucosal tissues encircling the mandibular 1st molars were removed on both sides for COX-2 immunohistochemistry, measurement of plasma extravasation by the Evans blue technique, and alveolar bone loss by videomicroscopy. Immunohistochemical analysis revealed numerous strongly COX-2-positive cells in the subepithelial tissues in the ligated side and only a few COX-2-reactive cells in the contralateral (control) side. Ligation significantly increased Evans blue extravasation in the gingivomucosal tissue and alveolar bone destruction compared to the control side. NS-398 treatment significantly reduced the plasma extravasation and alveolar bone resorption of the ligated side compared to vehicle administration. The present results suggest that COX-2 is induced by periodontitis, and plays an important role in gingival inflammation and alveolar bone destruction. In a previous study (Br J Pharmacol 1998;123:353-60) we found the expression of the inducible isoform of nitric oxide synthase in this model. Therefore, based on our own data and the literature, we propose that selective inhibition of these inducible enzymes might be a basis for adjunctive therapy, or new therapeutic approaches in periodontitis.

Evidence for reactive nitrogen species formation in the gingivomucosal tissue.

An increase in nitric oxide production has been demonstrated in periodontitis. Here we investigated the potential role of nitric-oxide-derived nitrating species (such as peroxynitrite) in a rat model of ligature-induced periodontitis. Formation of 3-nitrotyrosine, the stable product formed from tyrosine reacting with nitric-oxide-derived nitrating species, was detected in the gingivomucosal tissue. 3-Nitrotyrosine immunohistochemical analysis revealed a significant elevation in the number of immunopositive leukocytes, and higher immunoreactivity of the gingival ligaments and epithelium in the ligated than in the contralateral (control) side. On both sides, several 3-nitrotyrosine-positive bands and, on the ligated side, a unique 52-kDa 3-nitrotyrosine-positive band were detected by Western blot. However, in the sterile gingivomucosal tissue of rat pups, no 3-nitrotyrosine or inducible nitric oxide synthase immunoreactivity was found. Analysis of these data suggests that resident bacteria of the gingivomucosal tissue induce an increase in reactive nitrogen species, which is greatly enhanced by plaque formation in periodontitis.

Nitric oxide synthase containing nerves in the cat and dog dental pulp and gingiva

In a previous study we found that nitric oxide (NO) plays an essential role in the hemodynamic regulation of the feline dental pulp. However, no evidence for the presence of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) containing nerve fibers was found in the rat and cat dental pulps. In the present study, we are first to report the presence of a small number of NADPH-d positive and/or NO synthase immunoreactive perivascular and solitary varicose axons in the dental pulp and abundant number of similar axons in the gingiva of cats and dogs. These fibres may travel within the inferior alveolar nerve and might participate in sensory (i.e. pain) as well as in autonomic (i.e. regulation of blood flow) innervation of the dental pulp and gingiva.

Evidence for the Role of Nitric Oxide in the Circulation of the Dental Pulp

Many authors have studied the hemodynamics of the dental pulp; however, there are scarcely any data regarding the involvement of the L-arginine/nitric oxide pathway in the regulatory mechanism. Thus, we have examined the physiological effects of (1) NG-nitro-L-arginine as an inhibitor of nitric oxide synthesis and (2) the nitric oxide donor 3-morpholinosydnonimine on blood flow and vascular resistance in the canines of anesthetized cats to study the potential involvement of nitric oxide in the regulation of dental vascular homeostasis. Mean arterial blood pressure, heart rate, blood gases, pH, cardiac output, and tissue blood flow were determined prior to and 15 min after i.v. administration of either NG-nitro-L-arginine (30 mg/kg, n = 9) or 3-morpholinosydnonimine (1 mg/kg, n = 7). Blood flow was measured by radioactive-labeled microspheres. There were no significant differences in baseline parameters between the two groups of cats. The dental pulp blood flow decreased to 53 ± 13% (p < 0.01) of the control level after NG-nitro-L-arginine administration, while it decreased only slightly (to 82 ± 12%) after 3-morpholinosydnonimine administration. The dental pulps vascular resistance increased to 367 ± 69% (p < 0.01) of the control level after NG-nitro-L-arginine, while it decreased to 73 ± 10% (p < 0.05) of control after 3-morpholinosydnonimine. We found that the L-arginine/nitric oxide pathway plays an important role in the regulation of pulpal blood circulation. A nitric-oxide-dependent basal vasodilator tone exists in the dental pulp; furthermore, since the dental pulp circulation is sensitive to exogenous nitric oxide, the stimulated release of endogenous nitric oxide may also be involved in the control of the dental pulp vascular tone.

Nitric oxide modulates salivary amylase and fluid, but not epidermal growth factor secretion in conscious rats

The involvement of the L-arginine/NO pathway in the control of salivary fluid, amylase and epidermal growth factor (EGF) secretion was investigated in conscious rats. For the collection of saliva, an oesophageal cannula was implanted. To obtain steady secretion, submaximal carbachol background infusion was given. Different treatments included NO synthase inhibitor N(G)-nitro-L-arginine (NOLA; with or without phentolamine, propranolol), L-arginine, D-arginine and NO donor 3-morpholinosydnonimine (SIN-1) administration. Volume, amylase activity and EGF output in the secreted fluid were determined in 30 min mixed saliva samples. Carbachol infusion alone produced a modest, sustained salivary fluid and amylase secretion. NOLA (30 mg/kg) further increased both fluid (p<0.001) and amylase outputs (p<0.001). These latter effects were prevented by L-arginine but not by D-arginine or by phentolamine. Propranolol administration decreased both fluid and amylase secretion below the carbachol plateau, and NOLA did not modify this suppressed secretory rate. SIN-1 did not alter either volume or amylase secretion. Interestingly, NOLA given without carbachol did not modify salivation. Neither carbachol nor NOLA changed salivary EGF output. The present results suggest that the L-arginine/NO pathway has a modulatory role in the cholinergic control of salivary amylase secretion, but not in EGF output. The mechanisms of inhibitory action of NO on salivary fluid and amylase secretion remain to be identified.

Gingival endothelial and inducible nitric oxide synthase levels during orthodontic treatment: A cross-sectional study

This study uses a cross-sectional design to examine the endothelial and inducible nitric oxide synthase (eNOS and iNOS, respectively) levels of gingival tissue. Fifteen subjects, 10 female and 5 male individuals (aged 14.6-21.2 years; mean 17.4 +/- 1.8 years), who needed extraction of the four first premolars for orthodontic reasons and who had indications for a gingivectomy were enrolled in the study. In each patient, two maxillary/mandibular premolars were extracted, and two months later an orthodontic appliance was placed in the same arch. A canine undergoing treatment for distal movement served as the test tooth (TT), whereas its contralateral canine was used as the control tooth (CT). The CT was included in the orthodontic appliance but was not subjected to the orthodontic force. Two weeks after the orthodontic appliance placement, clinical data consisting of the presence of supragingival plaque, bleeding on probing, and probing depth were collected from each experimental tooth. Immediately after, gingival tissue was collected from the distal aspect of each TT and CT for immunohistochemistry, messenger RNA reverse transcription by polymerase chain reaction, and Western blot analysis for both eNOS and iNOS. The results showed that no differences in clinical conditions occurred between the experimental teeth. On the contrary, both the eNOS and iNOS levels and the expression of the TTs were significantly greater than those of the CTs (all comparisons significant to P < .01). Our results indicate a role for gingival eNOS and iNOS during the early phases of orthodontic treatment in humans.

Role of the Activation of the Nuclear Enzyme Poly(ADP-Ribose) Polymerase in the Pathogenesis of Periodontitis

We have investigated the role of the activation of nuclear poly(ADP-ribose) polymerase (PARP) enzyme, a mediator of downstream nitric oxide toxicity, using a combined approach of pharmacological inhibition and genetic disruption in a ligature-induced-periodontitis model in rats and mice. Immunohistochemical analysis revealed significantly increased poly(ADP-ribose) nuclear staining (indicative of PARP activation) in the subepithelial connective tissue of the ligated side compared with the non-ligated side. Ligation-induced periodontitis resulted in marked plasma extravasation in the gingivomucosal tissue and led to alveolar bone destruction compared with the non-ligated side, as measured by the Evans blue technique and by videomicroscopy, respectively. PARP inhibition with PJ34, as well as genetic PARP-1 deficiency, significantly reduced the extravasation and the alveolar bone resorption of the ligated side compared with controls. Thus, PARP activation contributes to the development of periodontal injury. Inhibition of PARP may represent a novel host response modulatory approach for the therapy of periodontitis.