RESUMO
BACKGROUND: We have previously explored a therapeutic strategy for specifically targeting the profibrotic activity of IL-13 during experimental pulmonary fibrosis using a fusion protein comprised of human IL-13 and a mutated form of Pseudomonas aeruginosa exotoxin A (IL13-PE) and observed that the intranasal delivery of IL13-PE reduced bleomycin-induced pulmonary fibrosis through its elimination of IL-13-responsive cells in the lung. The aim of the present study was to determine whether the presence of an immune response to P. aeruginosa and/or its exotoxin A (PE) would diminish the anti-fibrotic properties of IL13-PE. METHODOLOGY/PRINCIPAL FINDINGS: Fourteen days after P. aeruginosa infection, C57BL/6 mice were injected with bleomycin via the intratracheal route. Other groups of mice received 4 doses of saline or IL13-PE by either intranasal or intraperitoneal application, and were challenged i.t. with bleomycin 28 days later. At day 21 after bleomycin, all mice received either saline vehicle or IL13-PE by the intranasal route and histopatological analyses of whole lung samples were performed at day 28 after bleomycin. Intrapulmonary P. aeruginosa infection promoted a neutralizing IgG2A and IgA antibody response in BALF and serum. Surprisingly, histological analysis showed that a prior P. aeruginosa infection attenuated the development of bleomycin-induced pulmonary fibrosis, which was modestly further attenuated by the intranasal administration of IL13-PE. Although prior intranasal administration of IL13-PE failed to elicit an antibody response, the systemic administration of IL13-PE induced a strong neutralizing antibody response. However, the prior systemic sensitization of mice with IL13-PE did not inhibit the anti-fibrotic effect of IL13-PE in fibrotic mice. CONCLUSIONS: Thus, IL13-PE therapy in pulmonary fibrosis works regardless of the presence of a humoral immune response to Pseudomonas exotoxin A. Interestingly, a prior infection with P. aeruginosa markedly attenuated the pulmonary fibrotic response suggesting that the immune elicitation by this pathogen exerts anti-fibrotic effects.
Assuntos
ADP Ribose Transferases/farmacologia , Toxinas Bacterianas/farmacologia , Exotoxinas/farmacologia , Exotoxinas/uso terapêutico , Imunotoxinas/uso terapêutico , Interleucina-13/uso terapêutico , Fibrose Pulmonar/tratamento farmacológico , Fatores de Virulência/farmacologia , ADP Ribose Transferases/imunologia , Animais , Toxinas Bacterianas/imunologia , Bleomicina/toxicidade , Exotoxinas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/induzido quimicamente , Proteínas Recombinantes de Fusão , Fatores de Virulência/imunologia , Exotoxina A de Pseudomonas aeruginosaRESUMO
Previous reports have shown that angiotensin II and oxidative stress may be important features in acute poststreptococcal glomerulonephritis (APSGN) and that streptococcal erythrogenic toxin type B (ETB) and its precursor (ETBP) may have an important role in the pathogenesis of APSGN. The aim of this study was to determine the effect of ETBP on the production of angiotensin II and oxidative stress in rat mesangial cells and human mononuclear leukocytes. Mesangial cells and leukocytes were isolated from digested glomeruli and by histopaque gradient, respectively, while ETBP was isolated from nephritogenic streptococcus cultures using a cation exchange column. Angiotensin II was determined by an enzyme-linked immunosorbent assay and by cytometrics. Superoxide anion, reduced glutathione, nitrites, lipid peroxidation and catalase activity were determined by cytochemical, biochemical and enzymatic assays. Inducible nitric oxide synthase expression was determined by cytometrics. An increased production of angiotensin II was observed in ETBP-treated mesangial cell and leukocyte cultures. The ETBP induced an elevated production of superoxide anions and nitrites in mesangial cells and superoxide anions in leukocytes, while this streptococcal protein decreased the expression of inducible nitric oxide synthase in leukocytes. The ETBP was capable of inducing an increased production of angiotensin II and increased oxidative stress, both of which may be important mediators of inflammatory events in the renal tissue and during APSGN.
Assuntos
Angiotensina II/biossíntese , Proteínas de Bactérias/farmacologia , Exotoxinas/farmacologia , Mesângio Glomerular/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Animais , Catalase/metabolismo , Separação Celular , Células Cultivadas , Mesângio Glomerular/metabolismo , Mesângio Glomerular/patologia , Glutationa/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Superóxidos/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Previous reports have shown the presence of streptococcal erythrogenic exotoxin type B (ETB), leukocyte infiltration, interleukin-8 (IL-8), transforming growth factor-beta (TGF-beta) and glomerular proliferation in renal biopsies from patients with acute post-streptococcal glomerulonephritis (APSGN). In addition, increased levels of plasma interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha), and urinary IL-6, have also been reported in this disease. To determine the effect of streptococcal proteins on leukocyte proliferation and leukocyte production of IL-6, TNFalpha, IL-8 and TGF-beta1, we cultured human mononuclear leukocytes with ETB or ETB precursor (ETBP). After 24 h, 48 h and 96 h, culture supernatants were assessed for cytokines by enzyme-linked immunosorbent assay (ELISA), and for leukocyte proliferation by a monoclonal antibody anti-proliferating cellular nuclear antigen (PCNA). A significant increase in all cytokines was found in ETB- or ETBP-treated cultures when compared with controls. A polyclonal anti-ETB antibody diminished the cytokine stimulatory effect of ETB. An increased number of PCNA-positive cells was observed in ETB or ETBP treated cultures at 48 h and 96 h. Cytokine production and proliferation were not correlated. The stimulatory effect of streptococcal exotoxin B on leukocyte cytokine production may be relevant in renal tissue during the course of APSGN.
Assuntos
Proteínas de Bactérias/farmacologia , Exotoxinas/farmacologia , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Fator de Crescimento Transformador beta1/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Células Cultivadas , HumanosRESUMO
At fertilization, the sea urchin egg undergoes an internal pH (pHi) increase mediated by a Na+ -H+ exchanger. We used antibodies against the mammalian antiporters NHE1 and NHE3 to characterize this exchanger. In unfertilized eggs, only anti-NHE3 cross-reacted specifically with a protein of 81-kDa, which localized to the plasma membrane and cortical granules. Cytochalasin D, C3 exotoxin (blocker of RhoGTPase function), and Y-27632 (inhibitor of Rho-kinase) prevented the pHi change in fertilized eggs. These inhibitors blocked the first cleavage division of the embryo, but not the cortical granule exocytosis. Thus, the sea urchin egg has an epithelial NHE3-like Na+ -H+ exchanger which can be responsible for the pHi change at fertilization. Determinants of this pHi change can be: (i) the increase of exchangers in the plasma membrane (via cortical granule exocytosis) and (ii) Rho, Rho-kinase, and optimal organization of the actin cytoskeleton as regulators, among others, of the intrinsic activity of the exchanger.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Óvulo/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ouriços-do-Mar/citologia , Trocadores de Sódio-Hidrogênio/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Amidas/farmacologia , Animais , Citocalasina D/farmacologia , Exotoxinas/farmacologia , Concentração de Íons de Hidrogênio , Masculino , Piridinas/farmacologia , Espermatozoides/efeitos dos fármacos , Quinases Associadas a rhoRESUMO
BACKGROUND/AIMS: Previous reports have shown the presence of streptococcal erythrogenic toxin type B (ETB), IL-8, transforming growth factor-beta (TGF-beta) and glomerular proliferation in renal biopsies from patients with acute poststreptococcal glomerulonephritis (APSGN). In addition, increased levels of plasma IL-6 and tumor necrosis factor-alpha (TNFalpha) and urinary IL-6 have also been reported in this disease. To determine the effect of ETB in mesangial cell cytokine production and proliferation, the concentration of several cytokines (IL-6, IL-1beta, TNFalpha, IL-10, IL-4, RANTES), soluble TNF receptor I (STNFR-I), soluble TNF receptor II (STNFR-II) and proliferation were measured in rat mesangial cells cultures after treatment with ETB or its precursor (ETBP). METHODS: To analyze the levels of cytokines and production of soluble receptors as well as proliferation, rat mesangial cells were cultured with ETB or ETBP (50 microg/ml). After 24, 48 and 96 h of incubation, culture supernatants were assessed for cytokines and receptors by ELISA and for proliferation by incorporation of radioactive thymidine. RESULTS: A significant increase in IL-6 levels was found in mesangial cell cultures treated with either ETBP or ETB when compared with controls. Streptococcal proteins treated mesangial cells also showed elevated levels of proliferation at 96 h. Increased production of IL-6 was not correlated with proliferation. A polyclonal anti-ETB antibody abolished the IL-6 stimulatory effect of ETB on mesangial cells. ETB/ETBP failed to increase the levels of other cytokines and cytokine soluble receptors. CONCLUSION: Streptococcal ETB/ETBP is capable of inducing increased production of IL-6 and proliferation on mesangial cells. These findings could be relevant in a possible early interaction of streptococcal proteins with mesangial cells and during the course of APSGN.
Assuntos
Proteínas de Bactérias/farmacologia , Exotoxinas/farmacologia , Interleucina-6/análise , Células Mesangiais/química , Células Mesangiais/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/análise , Células Mesangiais/citologia , Ratos , Ratos Sprague-Dawley , Receptores do Fator de Necrose Tumoral/análiseRESUMO
Pseudomonas syringae pv. phaseolicola synthesizes a non-host-specific toxin, phaseolotoxin, and also synthesizes a phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) to protect itself from its own toxin. ROCT is encoded by argK, which is expressed coordinately with phaseolotoxin synthesis at 18 degrees C. To investigate the regulatory mechanisms of this system, null mutants were constructed for argK, argF (encoding the phaseolotoxin-sensitive OCTase [SOCT]), and amtA (encoding an amidinotransferase involved in phaseolotoxin synthesis). The argF mutant did not exhibit arginine auxotrophy when grown in M9 medium at 28 degrees C, because under this condition SOCT was replaced by ROCT. This loss of thermoregulation of argK was apparently caused by accumulation of carbamoylphosphate, one of the substrates of SOCT. Carbamoylphosphate, which has a structure similar to that of the inorganic moiety of phaseolotoxin, was used in induction assays with wild-type P. syringae pv. phaseolicola and was shown to be able to induce argK expression in M9 medium at 28 degrees C. These results indicate that argK expression is independent of temperature and is regulated directly by a compound resembling the inorganic moiety of phaseolotoxin.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Ornitina Carbamoiltransferase/metabolismo , Precursores de Proteínas/metabolismo , Temperatura , Proteínas de Bactérias/metabolismo , Carbamoil-Fosfato/metabolismo , Meios de Cultura , Exotoxinas/biossíntese , Exotoxinas/farmacologia , Mutação , Ornitina/análogos & derivados , Ornitina Carbamoiltransferase/genética , Pseudomonas syringae/efeitos dos fármacos , Pseudomonas syringae/genética , Pseudomonas syringae/crescimento & desenvolvimento , Pseudomonas syringae/metabolismoRESUMO
BACKGROUND: Previous reports have demonstrated the presence of streptococcal erythrogenic toxin type B (ETB) as well as proliferation and expression of adhesion molecules along with leukocyte infiltrations in biopsies from patients with acute post-streptococcal glomerulonephritis (APSGN). The purpose of the present study was to correlate infiltrative and proliferative events with interactions between ETB or its precursor (ETBP) and intrinsic mesangial cells. METHODS: Rat mesangial cells were cultured with ETB or ETBP (50 micro g/ml) while measuring production of monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) and while examining proliferation and expression of intercellular adhesion molecule-1 (ICAM-1). After 24, 48 and 96 h of incubation, MCP-1 and MIP-2 in culture supernatants were assessed by enzyme-linked immunosorbent assay (ELISA). Cells were assessed for proliferation by incorporation of radioactive thymidine and expression of ICAM-1 was measured by indirect immunofluorescence and by cellular ELISA. RESULTS: Compared with controls, treatment with either ETBP or ETB significantly increased MCP-1 and MIP-2 levels in mesangial cell cultures. Mesangial cells also showed elevated proliferation at 96 h of culture when treated with streptococcal proteins. Although production of MCP-1 and MIP-2 was not correlated with proliferation, treatment with ETBP resulted in a significant correlation between MCP-1 production and proliferation. Immunofluorescence studies revealed an increased expression of ICAM-1 in ETBP/ETB-treated mesangial cells. In addition, cellular ELISA studies showed increased absorbance in cultures treated with ETBP/ETB. Finally, low serum concentrations in the culture medium potentiated the stimulatory effect of ETB on MCP-1 production. CONCLUSIONS: Our findings, by demonstrating a role for cationic streptococcal ETB or ETBP in the induction of chemotactic molecules as well as the proliferation and expression of adhesion molecules, delineate an additional possible pathway for the pathogenesis of APSGN.
Assuntos
Proteínas de Bactérias , Quimiocina CCL2/biossíntese , Exotoxinas/farmacologia , Molécula 1 de Adesão Intercelular/biossíntese , Rim/efeitos dos fármacos , Proteínas de Membrana , Monocinas/biossíntese , Streptococcus , Animais , Quimiocina CXCL2 , Modelos Animais de Doenças , Glomerulonefrite/imunologia , Glomerulonefrite/metabolismo , Glomerulonefrite/microbiologia , Rim/citologia , Rim/imunologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Previous reports have shown the presence of erythrogenic toxin type B (ETB), apoptosis, proliferation, and leukocyte infiltration in biopsies from patients with acute poststreptococcal glomerulonephritis (APSGN). METHODS: Attempting to correlate the apoptotic and proliferative events with the interaction of ETB or its precursor (ETBP) with leukocytes, mononuclear leukocytes from 12 healthy subjects were cultured with ETB or ETBP to analyze the levels of apoptosis, proliferation, expression of modulatory apoptosis gene products, and oxidative metabolism. After four days of incubation, cells were assessed for apoptosis by morphological criteria, annexin V assay, and terminal deoxy transferase uridine triphosphate nick end-labeling (TUNEL) assay. The expression of regulatory apoptosis genes was assessed by relevant monoclonal antibodies; proliferation was by incorporation of radioactive thymidine; and oxidative metabolism was by oxidation of 2',7'-dichlorofuorescein diacetate to 2',7'-dichlorofuorescein. Neutralization of Fas-L and cysteine protease activity of ETB were performed by incubation of ETB-treated leukocyte cultures with anti-human Fas-L mAb or with E64, respectively. RESULTS: Elevated levels of apoptosis in ETBP/ETB-treated leukocytes were found when compared with controls: morphological criteria (P < 0.01), Annexin V (control, 5.01 +/- 0.61; ETBP, 10.60 +/- 1.98%, P = 0.0005), and TUNEL (control, 12.5 +/- 2.6; ETBP, 20.56 +/- 3.06%, P = 0.001; ETB, 30.69 +/- 5.05%, P = 0.001). Increased expression of apoptosis was accompanied by increased expression of Fas (control, 20.15 +/- 5.28; ETBP, 43.51 +/- 5.6%, P = 0.03; ETB, 47.16 +/- 5.54%, P = 0.01), Fas ligand (control, 5.64 +/- 2.38; ETBP, 11.66 +/- 3.65%, P = 0.04; ETB, 16.39 +/- 5.05%, P = 0.02) and p53 products (control, 9.22 +/- 3.44; ETBP, 22.82 +/- 5.72%, P = 0.01; ETB, 24.60 +/- 5.20%, P = 0.01). Treatment of ETB-leukocyte cultures with anti-human Fas-L exhibited 2.2-fold lower apoptosis expression. Treatment with E64 significantly abrogated the apoptotic effect of ETB. There was no increment on leukocyte oxidative metabolism. Mononuclear leukocytes also showed elevated levels of proliferation when treated with different concentrations (from 50 to 6.2 microg/mL) of streptococcal proteins (Stimulation index ranging: ETBP, 5.6 +/- 1.9 to 6.4 +/- 1.9; ETB, 9.9 +/- 2.8 to 13.9 +/- 3.8). CONCLUSIONS: These results delineate an additional pathway for the pathogenesis of APSGN related to the role of cationic streptococcal ETB or ETBP on the induction of apoptosis and proliferation during the course of the disease.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Bactérias , Exotoxinas/farmacologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/fisiologia , Proteínas de Membrana , Anticorpos Monoclonais/farmacologia , Morte Celular/genética , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Proteína Ligante Fas , Expressão Gênica , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Glicoproteínas de Membrana/imunologia , Oxirredução , Pró-Fármacos/farmacologiaRESUMO
BACKGROUND: Leukocyte infiltration is a common feature in renal biopsies from patients with acute poststreptococcal glomerulonephritis (APSGN). Cationic streptococcal erythrogenic toxin type B (ETB) and its precursor (ETBP) have been implicated in the pathogenesis of the disease, and the presence of ETB has been evidenced in renal biopsies from patients with APSGN. The present studies were performed to determine the effect of the ETBP and ETB on renal leukocyte infiltration and the mechanism(s) implicated in the phenomenon. METHODS: Male Sprague-Dawley rats were injected intrarenally with 100 microg of ETB or ETBP. Animals were sacrificed at 1, 6 and 24 h after injection and renal samples were studied by indirect immunofluorescence for the presence of leukocyte common antigen (LCA+) cells, C3, monocyte chemotactic protein-1 (MCP-1) and intercellular adhesion molecule-(ICAM-1), and by direct immunofluorescence for the presence of immunoglobulins. ETB and ETBP were tested for chemotactic effect and migration inhibition factor (MIF) activity by chemotaxis under agarose and agarose microdroplet methods, respectively. Streptococcal proteins were also tested for the capacity to induce MIF activity in rat glomerular cultures. To test for the influence of cationic charge on renal LCA+ cell infiltration, rats were injected with cationized ferritin or polyethyleneimine (PEI) and sacrificed 1 h later. RESULTS: An increased number of LCA+ cells was found in glomeruli and interstitial areas in ETB- or ETBP-injected animals. ETB and ETBP showed chemotactic and MIF activity on neutrophils and macrophages, and ETBP induced MIF activity in supernatants of glomerular cultures. Data obtained from C3, MCP-1, ICAM-1 or immunoglobulin renal staining in experimental animals were not significantly different when compared to control values. Cationized compounds failed to induce LCA+ cell infiltration; however, an increased number of glomerular LCA+ cells was observed after PEI perfusion. CONCLUSIONS: ETB and ETBP induce renal LCA+ cell infiltration during a short period after intrarenal injection, and this finding could be mediated by chemotactic and MIF activities. These observations could be relevant in the early events of pathogenesis of APSGN.
Assuntos
Proteínas de Bactérias , Exotoxinas/farmacologia , Rim/citologia , Rim/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Proteínas de Membrana , Pró-Fármacos/farmacologia , Animais , Técnica Indireta de Fluorescência para Anticorpo , Contagem de Leucócitos/efeitos dos fármacos , Leucócitos/citologia , Leucócitos/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , Valores de ReferênciaRESUMO
The gene coding for the phaseolotoxin-insensitive ornithine carbamoyltransferase (OCTase) from Pseudomonas syringae pv. phaseolicola has been cloned and sequenced. The gene has a deduced coding capacity for a polypeptide with a calculated Mr of 36,520 daltons. Comparison of the amino acid sequence of the OCTase enzymes encoded by the P. aeruginosa argF and the Escherichia coli argI and argF genes with the deduced sequence of the newly identified gene shows that 79 amino acid residues are strictly conserved in all four polypeptides; among these 7 out of 9 residues are involved in enzyme function. Of three amino acid regions that have been implicated in substrate binding or catalysis, two are strictly conserved, and the third involved in carbamoylphosphate binding differs. This correlates well with published data showing that phaseolotoxin competes for the carbamoylphosphate binding site in the phaseolotoxin-sensitive OCTases. We propose that the gene be named argK.