Biomedical subjects

R C Barbacane

Publications and source records attributed to R C Barbacane.

At least 19 recordsLinked to original sources

PubMed2003

Oxygen supply modulates MCP-1 release in monocytes from young and aged rats: decrease of MCP-1 transcription and translation is age-related.

Hyper or hypoxia may affect the immune system's chemokine production. Monocyte chemotactic protein-1 (MCP-1), an important chemotactic cytokine can be activated by active oxygen species. Groups of rats were exposed to hypoxic and hyperoxic environmental conditions for 60 h and MCP-1 was determined in their peripheral blood mononuclear cells by Elisa and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). In this study we evaluated if the ability of monocytes to produce MCP-1 under basal conditions or after stimulation with lipopolysaccharide (LPS) or phytohaemagglutinin (PHA) was differently affected by exposure to hyper or hypoxic conditions in young and aged rats. MCP-1 expression and production in monocyte/macrophages from rats at normoxic conditions was reduced in aged subjects. However, spontaneous, LPS or PHA-induced MCP-1 production was up-regulated by exposure to hyperoxic conditions in both young (62 +/- 8, 99 +/- 7, 102 +/- 8 pg/ml, respectively) and aged rats (79 +/- 4, 112 +/- 9, 117 +/- 10 pg/ ml, respectively). We conclude that hyperoxia is an important regulator of MCP-1 release and support the hypothesis that increased % of O2 may serve to initiate MCP-1 production which then serves to recruit and regulate the distribution of mononuclear cells to the sites of inflammation.

Aging↗

PubMed2001

Differential expression and secretion of RANTES and MCP-1 in activated peripheral blood mononuclear cell cultures of atopic subjects.

RANTES and MCP-1 represent a link between the activation of monocytes, lymphocytes, basophils, mast cells and eosinophils in inflammatory disorders, such as the late phase allergic reaction. These C-C chemokines also play a role in regulating Th cell cytokine production and leukocyte trafficking. In this study, we determined the expression and secretion of RANTES and MCP-1 from PHA-activated PBMC of healthy and atopic subjects with no symptoms. Levels of RANTES from PHA-activated PBMC of atopic patients were higher, at 18 and 24 h incubations (42+/-5.5 and 48+/-4), compared to controls (20+/-4 and 35+/-4), respectively; while MCP-1 was not (12+/-3 and 17+/-3) compared to controls (10.5+/-3 and 15+/-2), respectively. This effect was also revealed on RANTES mRNA expression, as determined by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. In addition, PHA-activated PBMC of atopic subjects produce more IL-4 (five times more) than healthy subjects, while IFN-gamma did not vary. RANTES, compared to MCP-1, may have more influence on signal transduction pathways, either in physiologic or inflammatory states and may induce profound effects on the regulation of cell activity. The differential production of RANTES and MCP-1 may lead to diverse regulation of the function and development of cells involved in the allergic response. These studies emphasize the importance of chemokine selectivity during inflammation.

Cells, Cultured↗

PubMed2001

MK-954 (losartan potassium) exerts endothelial protective effects against reperfusion injury: evidence of an e-NOS mRNA overexpression after global ischemia.

BACKGROUND: the cardiac Renin-Angiotensin system (RAS) plays an important role in the regulation of coronary flow and cardiac function and structure in normal and pathological conditions such as ischemia-reperfusion (I/R) injury. The aim of this study was to investigate the effects of the Angiotensin II type 1 (AT-1) receptor antagonist MK-954 (losartan potassium) on postischemic endothelial dysfunction and NOS mRNA expression (inducible nitric oxide synthase, iNOS; endothelial nitric oxide synthase, eNOS) in isolated working rat hearts. METHODS: isolated working rat hearts were subjected to 15 min global ischemia and 180 min reperfusion. MK-954 was added to perfusion buffer (a modified Krebs-Henseleit solution) at 1 microM concentration. We assessed functional parameters, creatin kinase (CK) release, heart weight changes, microvascular postischemic hyperpermeability (FITC-albumin extravasation) and morphological ultrastructural alterations. eNOS and iNOS mRNA levels were also detected by the means of multiplex RT-PCR technique using glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene as internal control; results were expressed as densitometric ratio. RESULTS: in Losartan-treated hearts we observed a significant reduction of postischemic contractile dysfunction, CK release and myocardial ultrastructural damage; postischemic FITC-albumin extravasation was significantly reduced respect to controls. Moreover, 1 microM Losartan produced a significant reduction of eNOS/G3PDH respect to untreated hearts submitted to I/R. Regarding iNOS/G3PDH ratio, no significant changes were detected in Losartan-treated hearts compared with controls. CONCLUSIONS: our study revealed that Losartan treatment before ischemia, and during reperfusion, is able to reduce the reperfusion injury of the rat heart by reducing mechanical and microcirculatory dysfunction and necrotic cell death, ameliorating cardiac ultrastructure and endothelial protection, probably inducing eNOS over-expression and reducing post-ischemic hyperpermeability of coronary microcirculation.

Angiotensin Receptor Antagonists↗

PubMed2001

Expression and secretion of RANTES by human peripheral blood CD4+ cells are dependent on the presence of monocytes.

The cDNA for RANTES (an acronym for "Regulated upon Activation, Normal T cell Expressed and Secreted") was initially discovered by subtractive hybridization as a T cell-specific sequence. Consistent with it being a C-C (beta) chemokine, RANTES is a monocyte chemoattractant. In addition, RANTES can chemoattract unstimulated CD4+/CD45RO+ memory T cells and stimulated CD4+ and CD8+ T cells with the naive and memory phenotypes in immunologically active sites. It has been shown that CD8+ cells are dominant sources of RANTES. Here we attempted to determine if CD4+ cells express and secrete RANTES alone, or if other accessory cells (activated monocytes) are needed to activate them. We found that when autologous monocytes are added to CD4+ cells and then stimulated with phytohaemagglutinin (PHA), the quantity of RANTES, in terms of transcription and translation of the protein, is significantly higher than the amount produced by the PHA-activated monocytes alone; isolated CD4+ cells stimulated with PHA do not produce any appreciable quantity of RANTES. When CD4+ cells are primed overnight with monocytes and then stimulated with PHA, they produce more RANTES compared to PHA-stimulated CD4+ cells alone. The influence that monocytes have on CD4+ cells to produce RANTES was confirmed when the physiological activator, tumor necrosis factor-alpha (TNF-alpha), was used. These data show that CD4+ cells need monocytes to express and secrete appreciable amounts of RANTES.

Blotting, Northern↗

PubMed2000

Endothelial NOS expression and ischemia-reperfusion in isolated working rat heart from hypoxic and hyperoxic conditions.

Induction of endothelial nitric oxide synthase (eNOS) contributes to the mechanism of heart protection against ischemia-reperfusion damage. We analyzed the effects of hypoxia and hyperoxia on eNOS expression in isolated working rat hearts after ischemia-reperfusion damage. Adult male Wistar rats were submitted to chronic hypoxia (2 weeks) and hyperoxia (72 h). The hearts were submitted to 15 min of ischemia and reperfused for 60 min, then we evaluated hemodynamic parameters and creatine phosphokinase (CPK) release. eNOS expression was estimated by RT-PCR; enzyme localization was evaluated by immunohistochemistry and the eNOS protein levels were detected by Western blot. All hemodynamic parameters in hypoxic conditions were better with respect to other groups. The CPK release was lower in hypoxic (P<0.01) than in normoxic and hyperoxic conditions. The eNOS deposition was significantly higher in the hypoxic group versus the normoxic or hyperoxic groups. The eNOS protein and mRNA levels were increased by hypoxia versus both other groups. Chronic hypoxic exposure may decrease injury and increase eNOS protein and mRNA levels in heart subjected to ischemia-reperfusion.

Animals↗

PubMed2000

Localization of the e-NOS enzyme in endothelial cells and odontoblasts of healthy human dental pulp.

Nitric oxide synthases (NOS) are important enzymes present in different cells such as endothelial cells, macrophages, etc. Recently, it has been found that nitric oxide (NO) is responsible for vasodilation, blood pressure regulation, platelet aggregation, cardiac contractility, and the mediation of immunity during bacterial infections and inflammation. However, the production and role of NO in various structures of the oral cavity have not been investigated extensively. The aim of this study was to evaluate the presence of e-NOS in healthy human odontoblasts and endothelial cells of the dental pulp. Twenty healthy human dental pulps were collected and frozen and pulp slices were obtained using a cryostat. The e-NOS enzyme was revealed by immunohistochemical analysis and the enzyme level was detected by Western blotting and mRNA expression by RT-PCR. The immunohistochemical results demonstrated, for the first time, the presence of e-NOS in odontoblasts and in endothelial cells. The presence of e-NOS m-RNA was confirmed by RT-PCR and the expression of the protein by Western blotting. These results clearly show that the e-NOS enzyme is present in both odontoblasts and endothelial cells of healthy human pulp. The presence of e-NOS in the odontoblast and endothelial cells of the dental pulp may mediate local vasodilation and cell proliferation.

Blotting, Western↗

PubMed1999

Monocyte chemotactic protein-1 gene expression and translation in formed granulomatous calcified tissue in vivo.

Monocyte chemotactic protein-1 (MCP-1) and related molecules constitute the C-C class of the beta chemokine supergene family with inflammatory properties. However, the exact role, function, and implication in inflammatory diseases remain to be determined. Here we report that subcutaneous injections (0.2 ml) of a saturated water solution (1:40) of potassium permanganate crystals induces the generation of granuloma tissue at the site of injection in the rat, and reaches its peak of formation after 1 week. The size and weight of the granulomas were increased by i.p. lipopolysaccharide (LPS) (6 microgram/200 microliter) and inhibited by intraperitoneal (i.p.) dexamethasone (Dxs) 300 microgram/200 microliter) treatments in rats, injected 18 hours before sacrifice. Moreover, steady-state levels of MCP-1 mRNA in the granuloma tissue (control), were strongly generated. Rats treated i.p. with LPS produced an increase of MCP-1 mRNA in the granuloma tissue compared with controls (i.p. PBS-treated) whereas in animals treated with Dxs, there was a decrease in (P < 0.05) in formation of mRNA protein. When the granuloma tissues were homogenized the generation of MCP-1 was found in the supernatants. The level of MCP-1 was higher (P < 0.05) in the LPS-treated animals and lower (P < 0.05) in the Dxs group compared with the controls (treated with PBS). Similar results were obtained in the serum and in minced granuloma tissue where samples were further incubated in vitro with LPS (100 ng/ml) overnight. A Strong increase (P < 0.01) in MCP-1 in all samples was detected, but not in the minced granuloma tissue from Dxs-treated animals. Our data demonstrate that calcified tissue from chronic inflammation induced by KMnO4 generates MCP-1 gene expression and translation, an effect increased by LPS and decreased by Dxs.

Animals↗

PubMed1999

Effect of electromagnetic fields on several CD markers and transcription and expression of CD4.

We carried out flow cytometric analysis for multiparametric evaluation of cell surface markers related to cellular functions. Specifically, we studied the expression of CD4, CD8, CD3, CD16, CD19, HLA-DR, and CD14 macrophage receptors expression and cell cycle progression on cells exposed to ELF-EMF. In addition, we tested the effects of ELF-EMF on CD4 mRNA protein transcription and translation and the cell-cycle progression using an immunofluorescence method. Our data show that same CD surface marker expression are weakly influenced by electromagnetic fields, with no differences between cells exposed or not exposed to ELF-EMFs. However, when the CD4 protein generation was studied, an indication of protein production was found in lymphocytes exposed to ELF-EMF, as evidenced by immunofluorescence, Western blotting and RT-PCR analysis. CD16 and CD14 expression were affected by EMF exposure at all times studied (24, 48, 72 h). The results obtained with cell cycle analysis show that after 48 h of exposure to ELF-EMF, PHA-activated and not activated cells in S phase increase with respect to non-exposed cells. The findings from this study demonstrate that under our defined experimental conditions there is evidence that ELF-EMF has a slight effect on CD4, CD14 and CD16 receptor expression, while the other CD receptors are not affected.

Antigens, CD↗

PubMed1999

Vitamin B6-deficient diet plus 4-deoxypyridoxine (4-DPD) reduces the inflammatory response induced by T. spiralis in diaphragm, masseter and heart muscle tissue of mice.

Animals fed diets deficient in vitamin B6 develop microcytic anemia, alterations of growth, and other pathologies. 4-deoxypirydoxine is a potent antagonist of vitamin B6 coenzyme which depresses IL-1, TNF and IL-6 and has anti-inflammatory properties. The aim of this study was to show the anti-inflammatory effects of 4-DPD on chronic inflammation caused by the nematode parasite T. spiralis, specifically on the recruitment and the activation of inflammatory cells. Two groups of mice, 6 weeks of age, were used: one was maintained on a vitamin B6-deficient synthetic pellet diet for 15 days before injection of the nematode, and administered an intraperitoneal injection (i.p.) of 4-DPD (250 microg/mouse) for 15 days (the first, 5 days before infection), and the second group was maintained on a normal diet for the total duration of the experiment. These two groups were then injected with 150 larvae (L1-T7 spiralis) per os. Chronic inflammation was caused by infection of treated or untreated mice with T7 spiralis parasite. After 14 days post-infection all mice developed a chronic inflammatory response. Mice fed with a B6-deficient diet showed a significant decrease in the number of cysts found in the diaphragm when compared to mice treated with normal diet. In addition, in all mice treated with vitamin B6-deficient diet plus 4-DPD the average body weight was significantly lower, compared to the mice on normal diet in all weeks examined. Moreover, in sections of the diaphragm, masseter and myocardium muscles, the infiltration of inflammatory cells, such as macrophages, lymphocytes, and eosinophils were more intense in untreated mice compared to those fed a vitamin B6-deficient diet. These results show that BALB/c mice infected with T. spiralis and fed a vitamin B6-deficient diet plus the vitamin B6 antagonist, 4-DPD, prolong the time of invasion of the larvae in the muscle cells, influence the recruitment of inflammatory cells and the intensity of the inflammatory reaction compared to infected untreated mice (control).

Animals↗

PubMed1999

IgG, IgG1 and IgM response in Trichinella spiralis-infected mice treated with 4-deoxypirydoxine or fed a Vitamin B6-deficient diet.

The aim of this study was to investigate the effect of pyridoxine (Vitamin B6) deficiency on the immunological response of BALB/c mice infected with the parasite T. spiralis. Specific anti-parasite IgM and IgG immunoglobulins were detected by ELISA method in the serum of treated animals at different periods for 60 days post infection. Vitamin B6-deficiency was induced in two separate groups of mice by either (1) maintaining the mice on a Vitamin B6-deficient synthetic pellet diet for 40 days before infection, or (2) by daily intraperitoneal injection of 8 x 10(5) M/100 microl of 4-Deoxypyridoxine (4-DPD), a potent antagonist of Vitamin B6 for 20 days prior to infection. These two groups of mice were then injected with 100 larvae (L1-T. spiralis) per os. Parasite burdens in the mice were observed by light microscopy. Cysts were present in the diaphragms of the mice after 60 days post-infection. Parasite specific IgG, as well as IgG1 levels were determined in the sera of infected mice fed a normal diet. These levels were found to be lower in the 4-DPD-treated mice compared to the untreated mice. The inhibition started from the 10th day and continued to the 60th day, and in the 4-DPD-treated group the inhibition initiated after 24 h to 60 days. IgM level also was depressed by 4-DPD, starting from 24 h after injection of the compound. In mice fed Vitamin B6-deficient diets the levels of IgG were lower than in mice fed normal diets. These results show that BALB/c mice infected with T. spiralis and fed either a Vitamin B6-deficient diet or a diet which included the Vitamin B6-antagonist, 4-DPD, both influence the course of IgG, IgG1 and IgM production.

Animals↗

PubMed1999

Impact of extremely low frequency electromagnetic fields on CD4 expression in peripheral blood mononuclear cells.

There is increasing evidence suggesting that extremely low frequency electromagnetic fields (ELF-EMF) may influence several cell functions. Here the effects of ELF-EMF were studied on the expression of CD4+ cell surface receptors of human peripheral blood mononuclear cells (PBMC) using fluorescence-activated cell sorter (FACScan). The expression of CD4+ in ELF-EMF exposed (24, 48 and 72 h) and not exposed PBMC were not statistically significant. In addition, a flow cytometric analysis was determined by using a fluorescent labeled antibody, at 24 and 72 h incubations. The amount of bound antibody was distributed with a slight difference in the ELF-EMF-exposed PBMC compared to the not exposed cells. Moreover, DNA CD4+ expression in PBMC strongly increased in exposed cells, resting and activated with Phytohaemaglutinin (PHA). When polymerase chain reaction was performed on CD4+ mRNA of PBMC an increase of CD4+ mRNA expression was found after the resting cells were exposed to ELF-EMF at 24 h compared to not exposed cells, while at 48 and 72 h no difference was found. In the cell cycle progression analysis, the PBMC exposed to ELF-EMF presented a significant increase of percentage expression of cell cycle progression in the S phase compared to not exposed cells; while in G1 and G2 phases, there were no differences. Our results provide new evidence that ELF-EMF can affect CD4+ expression in PBMC and describe an additional biological activity for ELF-EMF affecting CD4+ transcription and translation protein and the increase of the percentage expression of the cell cycle progression of the S phase.

Blotting, Western↗

PubMed1998

Expression of lymphocyte subpopulations, cytokine serum levels and blood and urine trace elements in nickel sensitised women.

In this study, 20 non-allergic and 20 non-atopic women sensitised only to nickel (Ni) showed similar levels of urine and serum Ni and serum chromium (Cr). On the contrary, serum copper, a marker of inflammation, was significantly higher in the Ni-sensitised group. Sensitised women also had higher values of blood B CD19+, CD5--CD19+ and B and natural killer CD3--CD25+ lymphocytes, but not alterations of some other lymphocyte subsets and serum cytokines. Urine Ni was correlated with "memory" CD4+-CD45RO+ lymphocytes in the non-allergic women and with T CD4+-CD45RO+ and CD3+-CD25+ cells in the atopic women; these subjects also showed a statistically significant correlation of serum Ni with B CD5+-CD19+ lymphocytes and serum IL-13. Moreover, serum Cr of both groups of women was positively or negatively correlated with activated HLA-DR+ cells and/or serum IL-5 and interferon gamma. These results (confirming in part those of a previous study on non-allergic men) suggest that both Ni and Cr are involved in mechanisms regulating the immune response and that allergy to these metals could be considered an alteration of their physiological role.

Adolescent↗

PubMed1998

Induction of monocyte chemotactic protein-1 (MCP-1) and TNF alpha by Trichinella spiralis in serum of mice in vivo.

MCP-1 is a small (8-10 KDa) protein and a prototype member of the CC chemokine beta subfamily, which plays a critical role in acute and chronic inflammation. Recent evidence suggests an important role for MCP- 1, MCP-2 and MCP-3 in a number of pathological states, including delayed type hypersensitivity conditions, parasitic infections and rheumatoid arthritis. Forty BALB-c mice were treated with the parasite Trichinella spiralis. After the infection the animals were sacrificed at different periods from the initial infection and MCP-1 and TNFalpha were quantified in the mouse serum. The level of MCP-1 in the serum of mice infected with 100 larvae increases from 27.5+/-7.0 pg/ml at day 23, to a maximum level of 31.5+/-5.0 pg/ml at day 33, then decreased to 14.6+/-2.0 pg/ml at day 47. When the mice were infected with 200 larvae of T. spiralis the maximum increase was 34.4+/-2.5 pg/ml found on day 23. From day 33 to day 47 MCP-1 levels were decreased. In addition, in infected mice levels of TNFalpha were detectable in the serum as early as day 1. The level of TNFalpha was maximum at day 35 (3812+/-224 pg/ml). Serum from non-infected mice contained no detectable levels of either MCP-1 or TNFalpha. However, even if MCP-1 seems to be implicated in Trichinellosis, its exact role and function in inflammatory parasitic diseases remains to be determined.

Animals↗

PubMed1998

Mast cell recruitment after subcutaneous injection of RANTES in the sole of the rat paw.

The effect of hrRANTES was studied after the injection in the sole of the rat paw, an area particularly rich in mast cells. Subcutaneous injections of RANTES 50 ng/10 microl produced an erythematous reaction which was inhibited by anti-RANTES antibody 50 microg/rat injected in the tail vein 30 min before hrRANTES 50 ng/10 microl was injected. In another set of experiments the animals were injected subcutaneously in the sole of the paw with PBS 10 microl (control), LPS (100 ng/10 microl) hrRANTES 50 ng/10 microl or anti-RANTES 50 microl/rat injected in the tail vein 30 min before hrRANTES 50 ng/10 microl was injected. The biopsies were analysed after 4 h and counted in an optic field. hrRANTES produced a strong recruitment of mast cells selectively coloured with 0.1% toluidine blue and inhibited by anti-RANTES antibody. In addition to the optical and electron microscope study, in some of the excised tissue Northern blot analysis for histidine decarboxylase (HDC) mRNA was performed to estimate the amount of histamine generation in the tissue of the injection sites. We found that subcutaneous injections of hrRANTES 50 ng/10 microl in the sole of the rat paw produced an accumulation of a great number of mast cells compared to PBS 10 microl (negative control) or LPS 100 ng/10 microl (positive control) after 4 h. The hrRANTES effect was inhibited by anti-RANTES antibody injected in the tail vein 30 min before hrRANTES exposure. Moreover, hrRANTES increased HDC mRNA and histamine generation.

Animals↗

PubMed1998

Intramuscular injection of hrRANTES causes mast cell recruitment and increased transcription of histidine decarboxylase in mice: lack of effects in genetically mast cell-deficient W/WV mice.

RANTES (regulated upon activation, normal T cell expressed and presumably secreted) and other chemoattractant proteins are members of the intercrine or chemokine family of proinflammatory basic polypeptides. RANTES is a prototype of the C-C chemokine subfamily that acts as a selective chemoattractant for human monocytes and CD4-positive lymphocytes and increases the adherence of monocytes to endothelial cells. However, the role of RANTES in white cells is still unclear. We report here that hrRANTES at 20 ng/50 microl in mice causes mast cell recruitment 4 h after intramuscular injection, an effect inhibited by anti-RANTES, as evidenced by 0.1% Toluidine blue, a specific dye for coloring mast cells. Injections of PBS (50 microl) vehicle (negative control) did not produce any appreciable inflammatory response, whereas injection of lipopolysaccharide 20 ng/50 microl (positive control) generated a marked inflammatory state. When RANTES was injected intramuscularly in genetically mast cell-deficient W/Wv mice, the inflammatory effect was not present. The RANTES injection sites were then excised and studied under an optical and electron microscope. A Northern blot analysis was performed using a probe that was prepared to detect mRNA encoding the histidine decarboxylase (HDC) gene on excised muscle tissue. We found that hrRANTES provoked generation of HDC mRNA from muscle tissue after 4 h. These effects were inhibited by an anti-RANTES antibody and were absent in genetically mast cell-deficient mice. The increasing number of mast cells in the RANTES injection sites led to an augmentation of histamine content compared to controls (PBS). The injection of hrRANTES 20 ng/20 microl into the sole of a rat paw confirmed the inflammatory and the mast cell recruitment potential of this chemokine. In these studies, hrRANTES injections in muscle tissue provided direct in vivo evidence that RANTES has a significant effect on mast cell recruitment and HDC mRNA generation.

Animals↗

PubMed1997

Influence of interleukin-1 receptor antagonist on [3H]serotonin and histamine release by rat basophilic leukemia-2H3 cells.

Mast cells located in connective tissues are a potent source of vasoactive and inflammatory mediators, such as cytokines. They accumulate in tissues in a wide variety of diseases where their function in most cases in unclear. In this report we provide evidence that rat basophilic leukemia cells (RBLC) cultured with a natural inhibitor of IL-1, interleukin-1 receptor antagonist (IL-1RA) (500 ng/ml) for 48 h, strongly inhibited the spontaneous release of serotonin (5HT) (from 25.2 to 29.9%), and histamine (from 22.50 to 43.49%), compared to untreated cells (control). When IL-IRA-treated and -untreated RBLC were stimulated with a secretagogue (anti-IgE), no difference was found in the percent of 5HT and histamine release. The present studies describe an additional biological activity of IL-1RA, inhibiting histamine and 5HT spontaneous release from RBLC cultures.

Animals↗

PubMed1997

Impact of Rantes and MCP-1 chemokines on in vivo basophilic cell recruitment in rat skin injection model and their role in modifying the protein and mRNA levels for histidine decarboxylase.

RANTES and related molecules, constitute the C-C class of chemokine supergene family and a group of cytokines produced by hematopoietic cells constitute the MCP-1 or C-X-C class. The roles of most of these chemokines are not well known, although members of the C-X-C family are inflammatory agents. Here, we report that intradermal injection of RANTES 10 ng/50 microL subcutaneously in the abdominal skin produced a strong inflammatory reaction, as evidenced by Evans blue dye, greater than FMLP (10(-6) mol/L) (approximately 57%); while MCP-1, 10 ng/50 microL was less effective than FMLP (10(-6) mol/L) (approximately 54%). Moreover, the histologic analysis of the cells stained with Toluidine blue (0.1%) were analyzed at a magnification of x40). RANTES 10 ng/50 microL and LPS produced higher numbers (142 +/- 11 and 193 +/- 21 of cells/200 mm2, respectively) of basophilic cell accumulation in the skin injection sites compared with FMLP (10(-6) mol/L) (127 +/- 14/200 mm2), while MCP-1 10 ng/50 microL was less effective (88 +/- 10/200 mm2). Electron microscopy (x13,800) studies of skin injection sites revealed that RANTES was chemoattractant for mast cells. In a Northern blot analysis from homogeneous tissue biopsy from the intradermal injection sites, RANTES was more potent than MCP-1 in increasing histidine decarboxylase (HDC) mRNA, the sole enzyme responsible for the production of histamine from histidine. Since PGD2 is formed by mast cells on cell activation, we also studied the effect of RANTES and MCP-1 on PGD2 production in inflamed tissue in vivo. RANTES (20, 10, and 5 ng) and MCP-1 (20, 10, and 5 ng) strongly stimulated PGD2, in a dose-dependent manner, with a potency rank order of RANTES (10 ng/mL) approximately two times greater than MCP-1 (10 ng/mL).

Animals↗

PubMed1997

RANTES is a pro-inflammatory chemokine and chemoattracts basophil cells to extravascular sites.

RANTES (regulated upon activation normal T expressed and secreted) is another member of the intercrine beta subfamily which acts as a selective chemoattractant for human monocytes and CD4-positive lymphocytes and increases the adherence of monocytes to endothelial cells. In this work, the effect of RANTES was studied on rat skin injection sites. Rats were intradermally injected with 50 microliters of RANTES, at different concentrations, fMet-Leu-Phe (FMLP), or LPS (positive controls) or PBS vehicle (negative control). The animals were then injected with 0.6 ml of Evans' blue in the tail vein in order to obtain a blue colour in the areas where the compounds were injected. After 4 h the rats were killed and the maximum diameter of the blue extravasation area was measured. The coloured areas were then excised and optical and electron microscopic studies were performed. In addition, in some of the excised tissue, a Northern blot analysis for histidine decarboxylase (HDC) mRNA was performed along with an estimation of the amount of histamine generated in the tissue injection sites. In these studies it was found that intradermal injections of 5, 2.5, and 1.25 x 10(-5) M RANTES produced a strong inflammatory response with the accumulation of a great number of basophil cells compared with the PBS (50 microliters) negative control, or FMLP (10(-6) M/50 microliters) or LPS (10 ng/50 microliters) positive control, after 4 h. Moreover, 5, 2.5, 1.25 x 10(-5) M RANTES produced a dose-response stimulation of HDC mRNA in the tissues of skin injection sites. The increasing number of basophils in the RANTES inflamed tissues led to augmentation of histamine content, compared with the PBS control. In conclusion, the pro-inflammatory chemokine RANTES stimulates the generation of HDC mRNA in skin injection sites.

Animals↗