A B Schütz
Dentist, Pharmacist, Master in Oral Pathology at the Federal University of Rio de Janeiro (UFRJ) and Doctor in Oral Pathology at the Faculty of Dentistry at the University of Sao Paulo (FOB/USP). Address reprint request to Dr. Antônio Beltrão Schütz, Conde de Porto Alegre, Santa Maria, RS, Brasil, Cep: 97010-100.
Key words: anti-inflammatory, cytokines, growth factors, granuloma, methotrexate (MTX)
Formation of nodular inflammatory lesions induced by dental plaque may also present in other inflammatory disease such as in Bowen disease (BD), Rheumatoid Arthritis (RA), Dental Granuloma, Wegener' granulomatosis and in malign neoplasm. With the objective of verifying whether there was the participation of immune and/or inflammatory responses on the phlogogenic mechanisms induced by dental plaque and corn oil, we studied the acute and chronic anti-inflammatory effects of the methotrexate (MTX) in rats administered in the dose of 1.66 mg/kg (edemogenic test) and 0.71 mg/kg per day (granuloma inhibition assays). The anti-inflammatory effect of MTX was evaluated in vivo though the vascular permeability inhibition (edemogenic test) and the granuloma inhibition test. The determination of the inflammatory exudates was performed by dye-extraction method (Udaka, Takeuchi and Movat, 1970) at 1 h, 3 h and 6 h time periods.
MTX anti-granuloma effect was evaluated at nodular inflammatory lesions induced in the subcutaneous tissue of rats, by the number of nucleus per multinucleate giant cells; number of vessels per microscopic fields; number (Ahern, 1967) and monocytes/macrophages and collagen fibers volume density; as well as by the volume density occupied by granulomatous tissue and necrotic region (Chalkley, 1943; Catanzaro Guimarães, 1967).
MTX inhibited the plasmatic exudation at 1 h and 6 h (p<0.05); decreased the number of monocytes/macrophages (p<0.01) and number of nucleus per giant cells (p<0.01); as well as decreased the volume density occupied by collagen fibers at 14 days (p<0.05). However, apparently, increased the number of vessels per microscopic field in this same experimental period (p<0.05). MTX does not decrease the volume density of the granuloma and central necrotic region during all the experiment period (p>0.05). These results essentially confirm the immunomodulator and/or anti-inflammatory effects of MTX suggesting that the immune and inflammatory responses inhibited by MTX have participation on phlogogenic mechanism induced by dental plaque, decreasing the rate of growth of nodular inflammatory lesions induced by this agent. The decrease of the number of monocytes/macrophages and volume density of these cells in the granuloma may have been promoted by the MTX anti-proliferative effect, via activation apoptosis, or necrosis induction, while the imnunomodulator and/or anti-inflammatory effect may be attributed to inhibition of pro-inflammatory cytokines.
MTX, when administered in low concentration (0.1-0.3 mg/kg), modulates the function of inflammatory cells; angioblast and/or endothelial cells implicated in Rheumatoid Arthritis (RA) (Cronstein, 1993); and the angiogenesis necessary for the formation of nodular inflammatory lesions.
One complication of the therapy with MTX in RA is the increase of rheumatoid nodules, whose formation seems to occur in spite of the synovial inflammation suppression.
In vivo, MTX inhibits aminoimidazole carboxamidoribonucleotide (AICAR) transformylase resulting in the increase intracellular of AICAR. The activity of this enzyme promotes the release of adenosine, principally from fibroblasts and endothelial cells. This autacoid by mean of a mechanism dependent of its concentration binds itself on specific membrane receptors, which are expressed on multiple subtypes in a single cell (Cronstein 1997).
Adenosine A1 receptors have been demonstrated in neutrophils and macrophages - but not in peripheral blood mononuclear cells. These receptors, when active, mediate the increased of chemotaxis and phagocytes of immunoglobulin-coated particles (Cronstein, 1993).
In vivo, a low concentration of adenosine, induced by a low doses MTX, causes activation of A1 receptors promoting the following effects: "immunomodulator" effect, modulator effect of the mononuclear cell function; inhibitor effect of the production of LTB4 by synovial cells and neutrophils; and reducer effect of the angiogenesis (Cronstein, 1992). In vitro, this same concentration of adenosine active neutrophils and macrophages, causing the release of superoxide anion, phagocitosis via FC receptors, and adhesion to endothelial cells. The activation of A1 receptors on monocytes/macrophages too promotes cell fusion and formation of multinuclear giant cells (Cronstein, 1997).
Adenosine A2 receptors are also presents on neutrophils, monocytes, lymphocytes and basophils and, when occupied by high adenosine concentration, generally, suppress the inflammatory and immune function of these cells (Cronstein, 1993, 1997). However, recently, the anti-arthritic effect of adenosine was questioned, because it did not decrease the effect that was obtained by association of MTX and adenosine antagonists (Andersson, Lexmuller, Ekstron, 2000). In addition, in solid neoplasm, the adenosine has showed to stimuli the tumor growth and angiogenesis, as well as to inhibit the synthesis of cytokines; the adhesion of immune cells on the vascular endothelium; and the function of macrophages, T cells and natural killer cells (NK) (Spychala, 2000). MTX by means of one dose-dependent mechanism may affect the neovascualrization as in vivo as in vitro. In vivo, a low dose of MTX caused the suppression of visualization, while in vitro it was showed that in culture of rheumatoid synovial cells, MTX did not affect VEGF production (Nagashima et al., 1999; Hirata et al., 2000).
In this experiment we used 52 Wistar rats of 150 g ( mean) maintained during the experiment with balanced alimentation and "ad libitum" water.
Preparation of the Microbial Dental Plaque
The microbial dental plaque was collected from students between 7 and 14 years old, deposited in sterile assay tube containing physiological saline (NaCl 0,9%), arranged in recipient accommodate ground ice, and transported to pathology laboratory at the FOB/USP.
Afterwards, the dental plaque was suspended in automatic mixer B-1 (Tayo Bussan Co. LTD), and the homogenate centrifuged (Centrifuge International - USA) at 4 C for 10 minutes at 12,100 g. The supernatant, after having been separated by elutriation, was submitted to a biochemical analysis, and the sediment (nuclear fraction) was packed in a freezer at the temperature of -20 C, until preparation . At this time, the assay tube with this material was introduced for 10 min into a cylinder with liquid nitrogen, and afterwards introduced in fervent water ( 82 C) for 10 min (thermal chock).
After adding SDS (L4509-Sigma) 2% in double of weight of dental plaque, it stayed in ambient temperature for 2 h, when then, was again centrifuged for 20 min at 12,100 g. The supernatant was separated and submitted biochemical analyze. This stage was repeated tree times and auditioned 20 mL of FPT (phenolate phosphate buffered) at ph = 7,5 and 10 mg of ribonuclease (R7003 - Sigma) and 20 mg of deoxiribonuclease (EC. 220.127.116.11-Sigma) remaining for 12 h at 37 C. The suspension was centrifuged for 30 min at 12,100 g. Then, the supernatant was separated and submitted biochemical analyze. The sediment was washed three times with FPT and auditioned 20 mL of SDS-phenol solution, staying in ambient temperature for 2 h, and centrifuged for 20 min at 3.000 rpm. The supernatant was separated and submitted at biochemical analyze. The precipitated was suspended in FPT for 20 min. This stage was repeated for 4 times. The supernatant was separate and submitted at biochemical analyze. Subsequently, it was suspended in alcohol-ether the 50% and centrifuged 30 min at 12,100 g. This last stage was repeated 3 times.
Afterwards, this material was suspended in 10 mL of distilled water divided and collected in 1.0 mL plastic tubes and stored in freezer prompt for using. In the moment experiment, of these material was heaved (E. Mettler type H15 - Zurich) the equivalent in mg necessary for the inoculation in each animal.
The protein and carbohydrate determinations were carried in supernatant fractions. Experiment I: vascular permeability inhibition assay In the three lots of 6 rats (control group) was inoculate 100 mg of dental plaque on subcutaneous tissue of dorsal region of the rats a constant volume 0,2 mL of the preparation of microbial dental plaque, on concentration of 100 mg/mL of Hanks solution (H6136 - Sigma). This preparation was injected immediately after the administration of Evans blue dye (E-2129- Sigma) for intravenous via, on the dose of 20 mg/kg of weight corporal. In the 18 animals of the experimental group, was administered methotrexate (Methotrexate - Lederle) intramuscularly on the dose of 1,66 mg/kg equivalent (0.833 mg/kg or 25 mg/day) in humans. The anti-inflammatory effect of the drug was carried on 1 h, 3 h and 6 h time periods, after Phlogogenous stimulation. In these experimental periods, each animal of the control and experimental groups was killed by ethyl ether inhalation, and the tissue marked by dye Evans blue, around of injection of the dental plaque and Hanks, was carefully removed with aid of a standard steel punch of 23 mm in diameter.
The pieces removed were incised in
small fragments, which after had been re-collect in flask of glass contend 4
ml of formamide for the extraction of Evans blue dye, remained in stove to 45
C during 3 days. The evaluation of the inflammatory exsudate (expressed in micrograms
of the total extravased dye) was calculated by mean of spectrophotometry (absorbance)
in 630 nm according to the dye-extraction method (Dakar, Takeuchi, Movat, 1970).
Experiment II: Test of inhibition granuloma This assay was performed to evaluate the anti-inflammatory effect of medicament in the development of experimental granuloma. In 2 lots of 4 rats (control group), 0,2 mL of the suspension of microbial dental plaque in corn oil (10 mg/mL) was inoculated in an air pouch (high concentration of oxygen) inside subcutaneous tissue of the dorsal region of rats. In the other 2 groups of 4 rats (experimental group), was injected. Methotrexate in the dose identical (1.66 mg/kg in rats) the used in vascular permeability inhibition assay (0.833 mg/kg or 25 mg/day in human) injected tree times per week (0.71 mg/kg per day in rats).
The first dose was injected immediately after the inoculation of the phlogogenous agent. At the experimental periods of 7 and 14 days, the animals of the control and methotrexate groups were killed with ethyl ether, and the granulomas after have been carefully removed, were fixed in formol the 10% for 24 hours, embedding in paraffin, cutting with 6 m of thickness and stained with hematoxylin and eosin (H&E) and Mallory Trichromic. Monocytes/macrophages total number
This analysis was determinate by the counting of the total number of monocytes/macrophages according to method II of Aherne (Aherne, 1967) at 7 and 14 days. For this couting was used a Zeiss II integrated grid fitted adapted in a microscope Olimpus, with objective in increase of x 40. In 60 microscopic fields selected at random for each animal, was counted the number of images (n) and crosses (c) between the margins of the profiles of the nuclear images and parallel lines of the test system. Knowing the total area examined in mm2 (A), the distance between the lines of the test system (d), the thickness of the section (t) and the gland processed volume (Vp), was obtained the total number of macrophages nuclei, using the following equation: N = 2n x Yp/A (c/n x d+2t). To determine whether MTX decreased or not the formation multinucleated giant cells, was realized the courting of the number of nuclei per giant cell in each microscopic field, while to verify whether MTX fall or not the angionenesis, was realized the simple counting of the number of vessels per microscopic field. Stereological evaluation of volume density For evaluating the density of volume occupied by tissue granulomatous, central region of necrosis, macrophages and collagen fibers was utilized a microscope in augment x 40, with an ocular Zeiss Kpl x 8 adapted, containing a grating of integration II of the Zeiss with 10 lines and 100 points.
In each one of the 80 histopathologic fields, for the group and experimental periods, as well as selected for systematic causality, and previous studied under descriptive aspect was counted the number of points above of the structures studied (Pei), and the number of points above of the total structure of granuloma (Pt), according to the method of Chalkley's point counting (Chalkley, 1943). For the calculation of the density of volume, was applied the following equation: Vvi = Pei/Pt x 100, which informed the percentage occupied by the analyzed structures in the granuloma. Statistical analysis As the statistical analysis realized had as basis numerical data, was used the parametric test of Student (unpaired t-test), with objective of verifying whether the treatment promoted by MTX presented significant difference statistic to the control group (comparison of means of two groups) at level of significance of 1 (p<0.01) and 5% (p<0.05). As there was also the interest of studying the effect of MTX in the two experimental periods, was realized an interval-to-ordinal transformation of numerical to ordinal data (ranked), and applicated Friedman test (nonparametric variance analysis), whose results are similar to the parametric analysis of variance (ANOVA), besides presenting the advantage of diluting possible digitations errors in the processing of data.
The Friedman test was considered significant at a level of significance of 5% (p=0.05).
Vascular permeability inhibition In tree experimental periods tested, MTX reduced the plasmatic exsudation to the control group with statistical significance (p<0.05) at 1 h and 6 h (table 1).
Number of monocytes/macrophages At 7 and 14days, MTX decreased the number these cells with statistical significance at level of 1% (p<0.01) (table 2). At 14 days, MTX decreased the number of nuclei per giant cell (p<0.01) (table 5). Volume density monocytes/macrophage in the granulomatous tissue At 7 and 14 days, MTX decreased the volume density of these cells with not statistical significance to the control group (p>0.05). Volume density occupied by collagen fibers At 14 days, MTX decreased the volume density of collagen fibers inside the granulommatous tissue (p<0.05) (table 4). In this time period, MTX increased the number of sanguineous vessels per microscopic field, with statistical significance to the control group (p<0.05) (table 6). Volume density occupied by granulommatous tissue. MTX decreased the volume density of this tissue during the experiment, with not statistical significance (p>0.05), according to table 7. Volume density occupied by central necrotic region. MTX increased the necrosis tissue, with not statistical significance (p>0.05) to the control group (table 8). MTX effect and time variable MTX effect presented statistical significance with relation the volume density of monocytes/macrophages in the granuloma (granulomatous tissue) between 7 and 14 days (p<0.05). In this time period, MTX effect did not present statistic difference with relation to volume density of the collagen, necrosis and granulomatous tissue between 7 and 14 days (p>0.05) (table 9).
Due to the fact of the dental plaque has an infectious nature, and contains a great number of noxious substances, it may be considered as a natural substitute to the conventional irritants (kaolin, formalin, carrageenan) used to study inflammation, because it better reproduces the inflammatory physiopathologic mechanism caused by infectious agents (Schütz, 1996).
In our study, the inflammation was induced by injection of dental plaque in an air pouch, localized in the subcutaneous tissue of the dorsal region of the rats, whose initial stage is characterized for alterations vascular, causing vasodilatation and increase of vascular permeability on the microcirculation and resulting at edema or inflammatory exudate, which in laboratory animals may be easily demonstrated through injection of vital dyes such as Evans blue dye. This vital dye, injected intravenously, bounds itself on plasma albumin forming protein-bound dye complex, which is a plasma marker capable of detecting the leakage of proteins into edema (Swingle, 1974).
In this study, the anti-exudates activity was evaluated by means of the vascular permeability inhibition assay (edematous test). MTX was administered in a dose of 0. 833 mg/kg in human which was adapted to the metabolism of the rat at 1.66 mg/kg. In this dose, it inhibited the acute inflammatory exudate at 1 h (initial permeability phase) and 6 h (late permeability phase), with statistical significance to the control group (p<0.05). At 1 h time period, the dose used in the present study (1.66 mg/kg in rats) possibly increased adenosine concentration in the inflammation area activating the A2 receptors, and inhibiting the adhesion neutrophils and monocytes on the endothelial cells, as well as decreasing the generation of free radicals, and inhibiting the phagocytosis of immunocomplexes capable of damaging the blood vessels via activation of Fc receptors in monocytes/macrophages and neutrophils (Merril et al., 1977). Other inhibitor mechanism of generation of toxic oxygen metabolites might have been originated by increase of adenosine concentration in the inflammatory tissue, because this autacoid is capable of inducing the conversion from adenosine to ionosine, which originate a liposoluble antioxidant (uric acid) (Cronstein, 1992). MTX may also suppress the generation of reactive oxygen species (ROS) induced by IL-6 at fibroblast (Sung et al., 2000).
The inhibition of the initial phase of peroxidation of protein and lipids, promoted by MTX, might too decrease the generation of toxic oxygen radicals because inhibits the activation of methyl group connected to conversion from B-12 coenzyme to CH4 and CH3COOH, which are generators of methyl (-CH3 and -CH2COOH) and peroxyl radicals, decreasing the edema, respectively (Schütz, 1996). The activation of xanthine dehydrogenase/xanthine oxidase enzymatic system presents on the endothelium vascular is other generation via of toxic oxygen metabolites. Both enzymes catalyze, among other enzymatic reactions, the conversion from xanthine dehydrogenase to xanthine oxidase, as well as the transformation from xanthine to uric acid. These reactions are accompanied by decrease of (NAD+) cofactor in the case of XD, and reduction of O2 in the case of XO, resulting on the formation of superoxide anions (Hassoun, 1994). Therefore, as the MTX is an inhibitor of the nucleotide coenzymes synthesis necessaries to the functioning of this enzymatic system, it may inhibit the activation of XD/XO enzymatic system, decreasing the generation of free radicals (Schütz, 1996). These radicals may be generated during the oxidation from the folic acid to N5metil-FH4, methionine and S-adenosylmethionine (Nesher, Moore, Dorner, 1991). Therefore, the deficiency of S-adenosylmethionine induced by MTX dihidrofolase reductase enzymatic inhibition may decrease the conversion from putrescine to spermidine (Watanabe, Sato, Negaste, Shimosato et al., 1999), as well as may reduce the oxidation of these and other polyamines, decreasing the production of toxic oxygen radicals and nitric oxide vasodilator (Schütz, 1996; Li et al., 2001). In addition, MTX may also cause inhibition of the synthesis of polyamines in consequence of the inhibition of NADH coenzyme, necessary to reduce the carboxilic group of the glutamic acid, as well as to decrease the production of acetyl-CoA, which is indispensable to the acetylation reaction from glutamic acid to N-acetylglutamic acid, which is considered other generation via of ornithine.
The decrease of the polyamines concentration may also inhibit the activation of latent metalproteinases and of the nitric-oxide-synthase enzyme (NOs), with subsequent diminution on the generation of nitric oxide and other vasodilators agents (Blantz, Satriano, Gahbat, kelly, 2000). This effect was observed in endothelial cells presenting low concentration of L-arginine and high concentration of argirase I. These cells showed a decrease of 60% in the NO (nitric oxyde) production, comparatively to endothelial cells, which presented argirase II and diminution of 47% on the NO production (Li et al., 2001). This effect may also be attributed at the blockade of the mRNA necessary for the synthesis of nitric-oxide-synthase (NOs) and/or inhibition of cytokines such al TNF- and IL-1 that are inductors of the increase of activity of this enzyme (Milano, 1995, Cronstein, 1996). In addition, because MTX inhibits essentials cofactors (NADH, FADH, FMN and BH4) necessaries to the functioning of the NOs, it inhibits the nitric oxide production decreasing the vascular permeability. As these nucleotides are cofactors necessaries to the activity of plasmatic proteases, which are related to the kinins, coagulation and fibrinolytic systems localized on extravasated plasma, the inhibition of these proteases by MTX might too inhibit these enzymatic systems. Considering the relation existing between the synthesis and release of PGE2, the functioning of these systems, as well as the fact that during the conversion from aracdonic acid to prostaglandins also occurs an accentuate generation of reactive oxygen radicals (ROS); the inhibition of the PGE2 synthesis promoted by MTX might decreased the generation of ROS, and vasodilatators agents release by activation of those enzymatic systems present in the extravasated plasma (Schütz, 1996).
The occupation of adenosine receptors in basophils, neutrophils, macrophages and lymphocytes may suppress the inflammatory and immune activity of these cells, decreasing the formation and phagocytosis of immune complexes deposited on the sanguineous vessels, as well as decreasing the secretion of histamine and serotonin vasodilatators by basophils and mast cells. Moreover, has been also suggested that the competitive occupation of receptor for histamine by adenosine might decrease the permeability vascular. In the other hand, MTX not affect the VEGF production by endothelial cells (Nagashima et al., 1999). In addition, MTX possess in vivo and in vitro citotoxic effect to endothelial cells that by mean of activation of the apoptosis in these cells causes microvascular perturbation (Fuskevag et al., 2000, Meckle et al., 2000). This effect might partially explicate the decrease MTX effect in the inhibition of initial plasmatic exudation, when compared to the effect promoted by steroid and non-steroid anti-inflammatory (Schütz, 1996). Because in the initial and late phases of vascular permeability, the dental plaque induces increase of production of prostaglandins and leukotrienes (Schütz, 1996), is possible that the inhibition of edema induced by MTX at 3 h and 6 h time period had been caused through the decrease of the leukotrienes synthesis in neutrophils and/or diminution of the capacity of these cells to adhere at endothelial cells and fibroblast, resulting in the decrease of chemotaxis, which is an event characteristic of the late vascular permeability phase. This effect might have been induced by occupation of specific cell surface receptors (A1) by adenosine. In addition, the decrease of transmigration endothelial and chemotaxis of neutrophils might have been caused by inhibition of synthesis or release of cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor (TNF- ), which are stimulators of the expression adhesion molecules (E-selectin, VLA4, CD18). In vitro, was demonstrated that MTX- -LIPO and MTX- -LIPO (4.5 mol/105), as well as MTX-di-LIPO (6.5 mol/10) inhibited PG2 release from LPS-stimulated rat macrophages at a percentage of 85.3 3.7 (Williams; Topley; Williams, 1993).
Therefore, other mechanism involved in the inhibition PGE2 synthesis may be the inhibition of plasmatic proteases participants of the generations of kinins, which increase the activity of PGE-9-Ketoredutase enzyme (Terragno, 1975). However, would be also possible MTX to inhibit the synthesis of coenzymes necessaries to the activity of the enzymatic systems related to conversion from aracdonic acid to prostanoids to decrease the synthesis of PGE2 (Schütz, 1996)? When macrophages, epitelioid cells and multinucleates giant cells predominate in the site of inflammation, this lesion is termed nodular chronic inflammation or granuloma, whose formation mechanisms may be separated in exsudative and proliferatives (Catanzaro-Guimarães, 1967). The former is represented by the edema, which is formed in the early phases of development of granuloma, or in consequence of the immune reactions that participate of the pathogenesis of these lesions. These reactions originate immuno complexes that activate the complement system generate vasodilatators mediators. The latter mechanism is represented for cells, collagen fiber, and neoformed bloods vessels. In our model inflammatory, MTX demonstrated to reduce the number monocytes/macrophages, with statistical significant to control group in 7 and 14 days (p<0.01). The inhibition of the number of these cells was more accentuate in 14 days (p<0.01). This resulted suggest that MTX possesses effect anti-proliferative to monocyte/macrophages, or to its progenitors hematopoietic cells (Schütz, 1996). This inference was confirmed at in vitro study, whose results schowed the effec antiproliferative myeloid monocytic cells (THP-1). This effect might have been caused by activation of the apoptosis in monocytes, as well as by inhibition of citokines pro-inflammatory (IL-1, TNF) (Cutolo et al., 2000). Other possibility might be the inhibition of the synthesis of polyamines related to modulation of activity of lymphocites (Schütz, 1996; Nesher, Osborn, Moore, 1996). The inhibition of the polyamines synthesis may also decrease the synthesis of essential cofactors (NADH, Acetyl-CoA, AMPc, metals ions), which participate of the ornithine synthesis. The inhibition of this synthesis might also inhibit the decarboxylase ornithine enzyme, because the ornithine is an essential substrate for the activity of this enzyme (Schütz, 1996). Other mechanism responsible for decreasing the concentration of polyamines (spermidine and spermine) in the granuloma might be the inhibition of the dihydrofolate reductase (DHFR) enzyme, causing the inhibition of N-methyl-FH4, methionine, Sadenosilmethionine as well as decreasing the cellular proliferation (Schütz, 1996).
In consonance with the antiproliferative effect presented by MTX, the data presented on table 6 demonstrated the reduction of the number of nucleus per giant cells (p<0.01). This result suggests that the adenosine concentration induced by MTX (0.71 mg/kg per day in rats), used in a dose greater than one indicated to treat rheumatoid arthritis and other inflammatory diseases (0.1-0.3 mg/kg), caused the saturation of A2 receptors (Cronstein, 1997). In spite of MTX to have decreased the number of monocytes/macrophages, it decreased the volume density occupied by monocytes/macrophages on the granulomatous tissue, with not statistical significance (p>0.05) at 7 and 14 days (table III). This resulted suggested that occurred the decrease of the volume density occupied by other components of granuloma, considering that the volume density of the granulomatous tissue did not present statistical significance to the control group (p>0.05) table 7. Our results confirmed this inference because MTX decreased the volume density occupied by collgen fibbers at 14 days experimental period with statistical significance (p<0.05). As the lymphocites have participation in the control of the fibrillogenesis, the reduction of volume density occupied for these fibbers might be partially explicated by the MTX cytotoxic effect in lymphocites, capable of inhibiting the replication of activated lymphocytes, and consequently to inhibit the release of lymphokines able to stimul the proliferation of cells directlly involved in the collagen synthesis such as fibroblast, macrophages and endothelial. The decrease in the number of these cells also decrease the release of cytokines (IL-1) and growth factor (PDGF, TGF- and FGF, TNF) capable of stimuling the synthesis of collagen (Neurath et al., 1999). As the formation of collagen fibber too depend of the enzymatic reaction of hydroxilation of lysine and proline aminoacids, respectively, by the procolagen-lisine-hidroxilase and procollagen-proline-hydroxylase enzymes, as well as needs of O2, Fe+2, -ketoglutarate and ascorbic acid, the inhibition coenzymes (NAD+, NADPH and CoA) necessary to the synthesis of lisine and proline (NADH), and to the inhibition of -ketoglutarato (NAD+ and NADPH) might have contributed for the inhibition of these fibers (Schütz, 1996).
This MTX effect was recently proofed in vitro (Renner et al., 2000). As the volume density of the granulomatous tissue did not decrease (p>0.05), comparatively at the control group, despite of MTX to have decreased the number of monocytes/macrophages, as well as the volume density occupied by collagen fibers in the granulommatous tissue, is possible that other components of this tissue might have increased. According to our study, in spite of having been realized the counting of the blood vessels in only 80 microscopic fields, and not to have been evaluated the volume density occupied by these vessels in the granulomatous tissue, nor to have been used fixion, or specific couloring for their identification, was demonstrated the increase of the number of vessels per microscopic fields at 14 days, indicating that MTX - in the dose used (0.71 mg/kg per day in rats) - increased the angiogenesis in this experimental period (p<0.05). This result, at least in part, may be explicated by the inference of MTX not to inhibit VEGF production in macrophages and fibroblast (Nagashima et al., 1999). In addition, the fact of agonist of A1 and A2 receptors, such as 5-N- ethycarboxamida adenosine and 2-5 dideoxyadenosine does not produce agiogenic effedt, as well as the fact of it have not been inhibited by 8-phenyltheophyline, which is one antagonist of these receptors, suggested that the angiogenic mechanism might not be related to activation of A1 and A2 receptors for adenosine (Van-Daele et al, 1992). However, recently, was suggested that the activation of the A (2b) adenosina receptors in endothelial cells by high adenosine concentration might promotes proliferation, EVGF expression, and angiogenesis (Spychala, 2000; Grant et al., 2001). Other mechanisms that might be also related to the MTX agiogenic effect might be the decrease of oxygen tension induced by inhibition of toxic oxygen radicals. In addition, because the tissue hypoxia may increase the synthesis and release of tibroblastic growth factor (FGF) by smooth muscular cells of vessels, is also possible that the hypoia induce the proliferation of endothelial cells (Schütz, 1996). Recent study proofed that the hypoxia induces the VEGF and IL-1 expression in fibroblast (Jackson et al., 1997).
Because MTX increases the genic transcription and stabilization of mRNA PTHrP, which is a peptide ables to bounding itself to receptors localized on surface of smooth muscular cells, this medicament may increase the proliferation of these cells, as well as may increase the agiogenesis, considering that this peptide induces the mRNA VEGF expression (Schütz, 1996). The mechanism of stimulation angiogenic promoted by PTHrP may be via activation of the protein kinase A (PKA) induced by inibition of sialoproteins (cytokines) such as IL-1 and IL-8, among others (Oyang et al., 2000). As in vivo, MTX did not influence the synthesis of PGE2 (Omata et al., 1997), the activation of receptors to PGE2 in fibroblast might stimule the production of PTHrP (Yoshita et al., 2001). Recently, was schowed that macrophages, THP-1 cells (promonocytic cell line) and myofibroblasts express this protein (Bloome et al., 1999). Therfore, PGE2 receptors activation in inflammatory and vascular cells might increase the synthesis of PTHrP inducing the increase of angiogenesis at 14 days. In addition, as the TNF 1 alfa synthesis is not affected by MTX (Williams, Topley, Williams, 1994), the increase of concentration of this cytokine in the inflammatory foccus might stimule the angiogenesis at 14 days (Schütz, 1996). Was also proofed that various cytokines, such as IL-1b, TNF-alfa and IL-6 increased the expression of mRNA PTHrP, while IL-2 and IL-8 seem not to effect the expression of this protein (Ichizucha K et al., 1999; Uemura Y et al. 2000).
Possibly, one other mechanism that might explicate not statistical significance presented by MTX group, with relation to volume density of the granulomatous tissue to control group, would be the increase of edema induced of AVP secretion (vasopressin) and excretion renal such as is observed in malign neoplasm (Fram, Von Hust, 1988). The edema might also be attributed to not inhibition of PGE2, when MTX is given in high dose (Weiler, Moser, Gerok, 1990). However, the results of the differential weight inhibition test, which is one indicative of the presence of inflammatory edema, suggested that MTX (0.71 mg/kg at day) decreased the edema, possibly by mean of a mechanism time-dependent, and related to inhibition the nitric oxide synthase, and inhibition of other enzymatic systems connected to production of vasodilatadors (Schütz, 1996). This inference was maked with basis in the fact that MTX can inhibit in vitro the IL-1 and PGE2 (Williams et al. 2000).
The decrease of volume density of the necrotic central region at 7 days did not present statistical significance to the control group (p>0.05). This effect may be partialy explicated by the inhibition of the adhesion of neutrophils and monocytes/macrophages on endothelial cells, induced by the occupation of A2 receptors induced by a high adenosine concentration, via inhibition of citokines and chemokines attractants, decreasing the phagocitosis of necrotic products and extravased fibrin, which accumulated themselves in the central region of granuloma (Gadangi et al., 1996, Schütz, 1996). This results is in concordance with the results obtained on rat periapical lesions induced by pulpal expossure, whose results did not schow significant statistic difference between the control and the group treated whit methotrexate (group B) at 2 weeks with relation to formation abscess area, in spite of having occurred the inhibition of the neutrophils number into the lesion (Yamasaki et al., 1994). Our results essentially confirm the decrease of the chemotaxis of neutrophils and monocytes/macrophages, considering that the vehicle used (corn oil) is rich in n-6 PUFA linoleic acid (18:3n-3), which stimules the chemotaxis of monocytes and neutrophils via increse of the production of aracdonic acid (Calder 1998). After this period, data not presented, the volume density of this region increased with not significant statistic difference for control group (p>0.05) such as reported by Yamasaki et al. (1994) and Yoshinari (1990), in spite of the initial phogogenic stimulation to have been possibly more intense in our study.
MTX decreased the number of monocytes/macrophages in the granuloma by mean of antiproliferative and inhibitor of chemotaxis effects, as well as presented immunomodulador and/or antiinflammatory effects by mean of the inhibition of cytokines pro-inflammatory, considering that the phlogogenic stimulation by dental plaque induces the accentuate release of cytokines, growth factors and products resultants of the enzymatic convertion from aracdonic acid to its final products (leucotrienes and prostaglandins), which also present chemotatic, proliferative, inflammatory, and immune synergistic effects to cytokines. In addition, these effects were increased by (n-6) PUFA linoleic acid (18:3n-3) present in the corn oil (Rusyn I et al, 1999; Carleto et al., 1996; Kreuzer J et al., 1996; Scheik C, Spiteller, Frohlich, 1996; Tmisikas et al., 1999).
Professor. S A Catanzaro Guimarães (CNPq grant 50707691-7) - by orientation always safe and sound; Juracy Nascimento and Maria Cristina Carrara by the technical assistance; and CAPES by scholarship.
Aherne W (1967) Methods of counting discrete tissue components in microscopical sections. J Roy Micr Soc 87: 493-508
Andersson SE, Johanson LH, Lexmuller K, Ekstrun GM (2000) Anti-arthritic effect of methotrexate: is it really mediated by adenosine? Eur J Pharmacol 9: 333-343.
Blantz RC, Satriano J, Gabbai F, Kelly C (2000) Biological effects to arginine metabolites. Acta Physiol Scand 160: 21-25.
Bloome EA, Zou H, Kartsogiannis V, Capenn CC (1999) Spatial and temporal expression of parathyroid hormone-related protein during the healing J Invest Dermatol 112: 788-795.
Carleto A, Belavite P, Guarini P, Biasi D (1996) Changes in the fatty acid composition and oxidative composition neutrophils migrating into an inflammatory exudate. Inflammation 20: 123-137.
Chalkley HW (1943) A method for quantitative morphologic analysis of tissue. J Nat Cancer Inst 4: 47-53.
Calder PC Immunoregulatory and anti-inflammatory effect of n-3 polyunsaturated fat acids. Braz. J Med Biol Res 31: 467-490.
Catanzaro Guimarães AS (1967) Histometric determination of collagen fibers in granulating wounds of alloxan diabetic rats. Experentia (Basel) 24: 1168-1169.
Cronstein BN (1993) Molecular mechanism of methotrexate action in inflammation. Inflammation 5: 411-423.
Cronstein BN, Naime D, Ostad E (1993) The antiinflammatory mechanism of methotrexate. J Clin Invest 92: 2675-2682.
Cronstein BN, Naime D, Firestein G (1995) The anti-inflammatory effects of an adenosine kinase inhibitor are mediated by adenosine. Arthritis & Rheumatism 8: 1040-1045
Cronstein BN (1996) Methotrexate and its mechanism of action. Arthritis & Rheumatism 39: 1951-1960.
Cuolo M, Bisso A, Sulli A, Felli L et al. (2000) Antiproliferative and anti-inflammatory in the methotrexate on cultured differentiating myeloid monocytic cells (THP-1) but not on synovial macrophages from patients with rheumatoid arthritis (RA). J. Rheumatoid 27: 2551-2557, 2000.
Frahn H, Von Hulst M Increase of secretion of vasopressin and edema in high dose of methotrexate therapy. Z Gesamte Inn Med 43: 411-414.
Furskevag OM, Kristesen C, Olsen R, Aarbakke J. (2000) Microvascular perturbation in rats receiving the maximum tolerable dose of methotrexate or its major metabolite 7-hidroxymethotrexate. Ultrastruct Pathol 24:325-332.
Gadangi G, Longaker M, Naime D, Levin RI et al. (1996) The anti-inflammatory mechanism of sulfazalazine is related to adenosine release at inflamed sites. J Immunol 156: 1937-1941.
Grant MB, Davis MI, Cabalero S, Feokistov I et al (2001) Proliferation, migration, and ERK activation in human retinal endothelial cells through A2 adenosine receptor stimulation. Invest Ophtalmol Vis Sis 42: 2068-2073.
Hassoun PM, YU FS, hedd AL, Zulueta JJ et al (1994) Regulation of endothelial cell xanthine dehydrogenase/xanthine oxidase gene expression by oxygen tension. Amer Phys Soc 10: L164-L171.
Hirata S, Matsubara T, Satura R, Tateishi H et al. (1989) Inhibition in vitro vascular endothelial cellular proliferation and in vivo neovascularization by low dose of methotrexate. Arthrits Rheumatoid 32:1065-1073.
Ichizucha K, Marimoto T (1999) The effect of cytokines on the parathyroid hormone related protein in human amnion cells. Endocr J 46: 479-486.
Jakson JR, Milton JA, Wo ML, Wei N (1997) Expression of vascular endothelial growth factor in synovial fibroblast is induced by hypoxia and Interleukin 1beta. J Rheumatol 24: 1253-1259.
Keuzer J, Denger S, Jahn L, Bader J LDL stimulate the chemotaxis of human through a ciclooxigenase-dependent pathway. Artherioesc Tromb Vasc Biol 16: 1481-1487.
Li H, Meininger CJ, Hawker JR Jr, Haynes TH, Kepker-Lenhart D et al. (2001) Regulatory role of argirase I and II in nitric oxide, polyamine and proline synthesis in endothelial cells. Am J Physiol Endocrinol Metabol 280: E75-82.
Merkle CJ, Moore IM, Penton BS, Torres BJ et al (2000) Methotrexate cause apoptosis in posmitotic endothelial cells. Biol Res Nurs 2: 5-14.
Merril A, Salmon J, Cronstein BN, Shen C et al (1997) Adenosine A1 receptor promotion of multinucleated giant cell formation by human monocytes, Artritis & Rheumatism 40: 1308-1315.
Milano S, Arcoleo F, Dieli M, D'Agostino P et al. (1995) Prostaglandin E2 regulates inducible nitric oxide syntheses in the murine macrophage cell line 1774. Prostaglandins 49: 207-213.
Nagashima M, Yoshino S, Aono H, Takat M et al. (1999) Inhibitory effects of anti-rheumatic drugs in the endothelial growth factor in cultured rheumatoid synovial. Clinic Exp Immunol 116: 360-365, 1999.
Neuraty MF, Hilder K, Becher C, Schlaak JF et al. Methotrexate specifically modulates (CIA): a mechanism for methotrexate mediated immunosupression the citokines production by B cell and macrophages in murine collagen induced arthritis. Clin Exper Immunol 115: 42-55.
Omata T, Segawa Y, Inoue N, Tzuizuki N et al (1997) Methotrexate supresses nitric oxide production ex vivo in macrophages of rats with adjuvant-induced arthritis. Resp Exp Med (Berl) 197: 81-90.
Oyniang H, Franceschi RT, Mccaule LK, Wang D et al. (2000) Parathyroid hormone-related protein down regulates bone syaloprotein gene expression in cementoblast: role of the protein kinase A pathway. Endocrinology 141: 4671-4680.
Renner S, Kuci Z, d'Cruze H, Niethammer D, Bruchelt G et al. (2000) Isotachophoretic analysis of the dihidropholato redutase reaction in the presence of methotrexate and ascorbic acid. Electrophoresis 21: 2828-2833.
Rusyn I, Bradhan CA, Cohn L, Schoohonhoven R (1999) Corn oil rapidly actives the factor Kappa B in hepatic kupper cells by oxidant dependent mechanism. Carciongenesis 20: 2095-2195.
Schütz, AB (1996) Comparative study of tenoxican, indhometacine, dexamethasone and methotrexate on acute and chronic inflammatory process (thesis). Bauru, Dentistry School of Bauru. Sao Paulo University.
Scheick C, Spiteller G, Frohlich W (1996) Human neutrophil chemotaxis in response to dienpox of linoleic acid. Z Naturforsch 51: 877-872.
Spycala J (2000) Tumor-promoting function of adenosine. Pharmacol Ther.87: 161-173, 2000.
Sung JY, Hong JH, Kang HS, Choi I et al. (2000) Methotrexate suppresses the interleukin-6 generation of reactive oxygen species in synoviocytes of rheumatoid arthritis. Immunolpharmacology 47: 35-44.
Swingle KF (1974) Evaluation for antiinflammatory activity. In: Antiinflammatory agents. Chemistry and Pharmacology. Scherrer RA and Whitehouse MW, editors. Academic Press, London, pp. 33-109.
Tmisikas S, Philis-Tmisikas A, Alexopulos S, Sigare F et al. (1999) LDL isolates from Greek subjects on a typical diet or from American subjects on an oleate-supplemented diet induces less monocyte chemotaxis and adhesion when exposes to oxidative stress. Artherioesc Thomb Vasc Biol 19: 22-130
Uemeura Y, Nakata H, Kobayash M, Harada R (2000) Regulation of granulocyte colony stimulating factor by parathyroid hormone relationed protein in lung carcinoma cells line OK-C-1 Jpn Cancer Research 91: 911-917.
Van Daele CG et al (1992) Effects of adhenine nucleotides on proliferation of endothelial cells. Circ Res 70: 82-90
Watanabe S, Sato S, Negase S, Shimosato K et al. (1999) Effects of methotrexate and cyclophosfamide on polyamide level in various tissues of rats. J Drug Target 7: 197-205.
Williams AS, Topley N, Williams BD (1994) Effect of liposomally encapsulated MTX-DMPE conjugates upon TNF and PGE2 release by lipopolysaccharide stimulated rat peritoneal macrophages. Biochimica Biophysica Acta 1225: 217-222.
Williams A, Goodfellow R, Topley N, Amos N (2000) The suppression of rat collagen induced arthritis and inhibition of macrophages derived mediator release by lipossomal methotrexate formulations. Inflammation Res 49: 149-161.
Yamasaki M, Kumazawa M, Kohsaka T, Nakamura H (1994) Effect of methotrexate-induced neutropenia on rat periapical lesion. Oral Surg Oral Med Oral Pathol 77: 655-661.
Yoshida T, Sakamoto H, Horiuchi T, Yamamoto S (2001) Involviment of prostaglandin E (2) in Interleukin-1alfa induced parathyroid hormone related peptide in rheumatoid in the synovial fibroblast of patients with arthritis rheumatoid. J Clin Endocr 86:3272-3278.
Yoshinari N. (1990) Effect of long-term-methotrexate-induced neutrophenia on experimental lesions in rats. Aichi Gaigui Dagaku Shigakai Shi 28:345-366. [Abstract]
Table 1: Evans blue dye ( g) concentration in the inflammatory focus on the experiment I.
(Dental Plaque + Hanks - 100mg/mL)
(1,66 mg/kg in rats)
(Dental Plaque + Hanks - 100mg/mL)
a Mean and SEM
b t-test significant (p<0.05).
C t- test not significant (p>0.05).
Table 2: Total number of monocytes/macrophages (Aherne II) present at granulomas induced by microbial dental plaque in the Experiment II.
177,1 x 106 4,61a
305.1 x 106 14,63a
127,5 x 106 2.90a
210.84 x 106 4.98a
Unpaired t- test
a Mean and SEM
b t-test significant (p<0.01).
Table 3: Volume density (Vvi) occupied by monocytes/macrophages in granulomatous tissue on the Experiment II
(Dental Plaque + Hanks - 100mg/mL)
(1,66 mg/kg in rats)
(Dental Plaque + Hanks - 100mg/mL)
a Mean and SEM
b t-test not significant (p>0.05).
Table 4: Density of volume (Vvi) occupied by collagen fibbers in the granulomatous tissue on the experiment II.
7 days 14 days
a Mean and SEM
b t-test significant (p<0.05)
Ct-test not significant (p>0.05).
Table 5: Nuclei per cell multinucleated giant cell (n/cell) at the14 days on the experiment II.
a Unpaired t-test significant (p<0.01)
Table 6: Number of vassels in the control group and test group on the experiment II.
a Mean and ESM
b t-test not significant (p<0.05)
C t-test significant (p>0.05)
Table 7: Density of volume (%) occupied by granulomatous tissue in the experiment II.
Group Experimental Period
7 days 14 days
a Mean and SEM
b t-test not significant (p>0.05).
Table 8: Density of volume (%) occupied by necrosis central region in the experiment II.
7 days 14 days
a Mean and SEM
b t-test not significant (p>0.05)
Table 9: Volume density (%) and time (MTX group)
Friedman test (X2R)
Multiple comparation test
a Mean and SEM
b Friedman test significant (p<0.05)
c Friedeman test not significant (p>0.05)
d Multiple comparision test significant (p<0.05)
Copyright Dentistry On-Line 2001
First Published: December 1st 2001