Posible papel de Porphyromonas gingivalis en el desarrollo de la artritis reumatoide / Possible role of Porphyromonas gingivalis in the develpment of rheumatoid arthritis

Varios estudios han sugerido una asociación entre la periodontitissevera, la prevalencia de la bacteria Porphyromonas gingivalis y el desarrollo de artritis reumatoide. Como fundamento de esta relación, se ha observado que esta bacteria secreta una enzima, peptidil-arginina deiminasa, que es capaz de citrulinar proteínas del hospedero y así favorecer una respuesta autoinmune. Sin embargo, debido a la heterogeneidad de diseños experimentales, selección de pacientes y valoración de los desenlaces, los resultados no han mostrado la reproducibilidad deseada. Asimismo, observaciones recientes apuntan a que la actividad enzimática podría ser generada por otras especies bacterianas, lo que hace más compleja su relación. Sin embargo, por otro lado, algunos estudios sugieren que el tratamiento periodontal puede limitar el desarrollo de la artritis reumatoide.

Various studies have suggested a link between severe periodontitis,the prevalence of Porphyromonas gingivalis, and the development ofrheumatoid arthritis. As evidence of this relationship, P. gingivalis hasbeen found to secrete an enzyme, peptidyl arginine deiminase, which isable to citrullinate host proteins and thus help activate an autoimmuneresponse. However, due to the heterogeneity of experimental designs,patient selection, and assessment of clinical outcomes, the results havenot shown the desired reproducibility. Furthermore, recent fi ndingsindicate that the enzymatic activity may be produced by other species ofbacteria, which suggests the relationship is more complex. However, anumber of studies have shown that periodontal treatment could inhibitthe development of rheumatoid arthritis.

Optimization of a flow cytometric assay to evaluate the human neutrophil ability to phagocytose immune complexes via Fcγ and complement receptors.

INTRODUCTION: This study aims to optimize some experimental conditions of a flow cytometric assay to examine the human neutrophil ability to phagocytose immune complexes (ICs) via Fcγ and complement receptors (FcγR and CR, respectively). The parameters assessed were: number of cells, concentration of ICs, reaction time, pH and concentration of the Trypan Blue quenching solution. METHODS: Neutrophils were isolated from peripheral blood of healthy volunteers. Precipitated ICs composed of IgG and fluorescein isothiocyanate (FITC)-labeled ovalbumin, opsonized or not with serum complement, were used to trigger the neutrophil phagocytosis via FcγR, CR, and FcγR+CR. Fluorescence of the internalized ICs was measured by flow cytometry, after quenching the extracellular fluorescence with Trypan Blue. RESULTS: The optimal experimental conditions established for the phagocytosis assay were: 1 × 10(6) cells mL(-1) and 40 µg mL(-1) FITC-labeled ICs, incubated for 30 min, at 37°C, in 0.5 mL of reaction volume. Trypan Blue solution at 1.25 mg mL(-1) pH4.4 was the best fluorescence quencher of FITC-labeled ICs attached to the outer surface of neutrophils. DISCUSSION: The selected experimental conditions were viable to evaluate IC phagocytosis by neutrophils; they are also suitable to compare the efficiency of IC phagocytosis mediated by FcγR and CR classes of membrane receptors, alone or in combination. This method finds application in studies of (i) the receptor-specific phagocytic function of normal and pathogenic neutrophils as well as (ii) the impact of drugs and therapies on this essential effector function of neutrophils.

Inhibitory activity of liposomal flavonoids during oxidative metabolism of human neutrophils upon stimulation with immune complexes and phorbol ester.

CONTEXT AND OBJECTIVE: The massive production of reactive oxygen species by neutrophils during inflammation may cause damage to tissues. Flavonoids act as antioxidants and have anti-inflammatory effects. In this study, liposomes loaded with these compounds were evaluated as potential antioxidant carriers, in attempt to overcome their poor solubility and stability. MATERIALS AND METHODS: Liposomes containing quercetin, myricetin, kaempferol or galangin were prepared by the ethanol injection method and analyzed as inhibitors of immune complex (IC) and phorbol ester-stimulated neutrophil oxidative metabolism by luminol (CLlum) and lucigenin-enhanced (CLluc) chemiluminescence (CL) assays. The mechanisms involved this activity of liposomal flavonoids, such as cytotoxicity and superoxide anion scavenging capacity, and their effect on phagocytosis of ICs were also investigated. RESULTS AND DISCUSSION: The results showed that the inhibitory effect of liposomal flavonoids on CLlum and CLluc is inversely related to the number of hydroxyl groups in the flavonoid B ring. Moreover, phagocytosis of liposomes by neutrophils does not seem to necessarily promote such activity, as the liposomal flavonoids are also able to reduce CL when the cells are pretreated with cytochalasin B. Under assessed conditions, the antioxidant liposomes are not toxic to the human neutrophils and do not interfere with IC-induced phagocytosis. CONCLUSION: The studied liposomes can be suitable carriers of flavonoids and be an alternative for the treatment of diseases in which a massive oxidative metabolism of neutrophils is involved.

Inhibition of immune complex-mediated neutrophil oxidative metabolism: a pharmacophore model for 3-phenylcoumarin derivatives using GRIND-based 3D-QSAR and 2D-QSAR procedures.

In this study, twenty hydroxylated and acetoxylated 3-phenylcoumarin derivatives were evaluated as inhibitors of immune complex-stimulated neutrophil oxidative metabolism and possible modulators of the inflammatory tissue damage found in type III hypersensitivity reactions. By using lucigenin- and luminol-enhanced chemiluminescence assays (CL-luc and CL-lum, respectively), we found that the 6,7-dihydroxylated and 6,7-diacetoxylated 3-phenylcoumarin derivatives were the most effective inhibitors. Different structural features of the other compounds determined CL-luc and/or CL-lum inhibition. The 2D-QSAR analysis suggested the importance of hydrophobic contributions to explain these effects. In addition, a statistically significant 3D-QSAR model built applying GRIND descriptors allowed us to propose a virtual receptor site considering pharmacophoric regions and mutual distances. Furthermore, the 3-phenylcoumarins studied were not toxic to neutrophils under the assessed conditions.

Immune complexes inhibit differentiation, maturation, and function of human monocyte-derived dendritic cells.

The interaction between immune complexes (IC) and the receptors for the Fc portion of IgG (FcgammaRs) triggers regulatory and effector functions in the immune system. In this study, we investigated the effects of IC on differentiation, maturation, and functions of human monocyte-derived dendritic cells (DC). When IC were added on day 0, DC generated on day 6 (IC-DC) showed lower levels of CD1a and increased expression of CD14, MHC class II, and the macrophage marker CD68, as compared with normally differentiated DC. The use of specific blocking FcgammaR mAbs indicated that the effect of IC was exerted mainly through their interaction with FcgammaRI and to a lesser extend with FcgammaRII. Immature IC-DC also expressed higher levels of CD83, CD86, and CD40 and the expression of these maturation markers was not further regulated by LPS. The apparent lack of maturation following TLR stimulation was associated with a decreased production of IL-12, normal secretion of IL-10 and CCL22, and increased production of CXCL8 and CCL2. IC-DC displayed low endocytic activity and a reduced ability to induce allogeneic T cell proliferation both at basal and LPS-stimulated conditions. Altogether, these data reveal that IC strongly affect DC differentiation and maturation. Skewing of DC function from Ag presentation to a proinflammatory phenotype by IC resembles the state of activation observed in DC obtained from patients with chronic inflammatory autoimmune disorders, such as systemic lupus erythematosus disease and arthritis. Therefore, the altered maturation of DC induced by IC may be involved in the pathogenesis of autoimmune diseases.

The role of complement in the modulation by fluid-phase IgG of the production of reactive oxygen species by polymorphonuclear leukocytes stimulated with IgG immune complexes.

The production of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMN) can be induced by immune complexes and is an important component of phagocytosis in the killing of microorganisms, but can also be involved in inflammatory reactions when immune complexes are deposited in tissues. We have observed that fluid-phase IgG can inhibit the generation of ROS by rabbit PMN stimulated with precipitated immune complexes of IgG (ICIgG) in a dose-dependent manner, acting as a modulatory factor in the range of physiological IgG concentrations. This inhibitory effect is compatible with the known affinity (Kd) of monomeric IgG for the receptors involved (FcRII and FcRIII). The presence of complement components in the immune complexes results in a higher stimulation of ROS production. In this case, however, there is no inhibition by fluid-phase IgG. The effect of complement is strongly dependent on the presence of divalent cations (Ca2+ or Mg2+) in the medium, whereas the stimulation of ICIgG (without complement) does not depend on these cations. We have obtained some evidence indicating that iC3b should be the component involved in the effect of complement through interaction with the CR3 receptor. The absence of the inhibitory effect of fluid-phase IgG in ROS production when complement is present in the immune complex shows that complement may be important in vivo not only in the production of chemotactic factors for PMN, but also in the next phase of the process, i.e., the generation of ROS.

Platelet-activating factor and eicosanoids are mediators of local and systemic changes induced by immune-complexes in mice.

A passive Arthus reaction (AR) induced in the peritoneal cavity of mice was followed by increased local vascular permeability and haemoconcentration. The intensity of the increased vasopermeability was higher in BALB/c compared with C3H/HePas mice despite the latter being 10 times more sensitive to platelet-activating factor (PAF). C3H/HePas mice however, exhibited higher levels of haemoconcentration and shock-like symptoms. Both events were inhibited by the PAF antagonist, WEB 2170. Indomethacin reduced both pathological events whereas L663,536, that inhibits leukotrienes synthesis reduced haemoconcentration but only in BALB/c mice. PAF was released into the peritoneal cavity, peak release being at 10 min after induction of AR. Prostaglandin E2 (PGE2), thromboxane B2 (TXB2), leukotriene B4 (LTB4), and leukotriene C4/D4 (LTC4/D4) were also released at this time. Similar levels of PAF and eicosanoids were found in BALB/c and C3H/HePas mice except for LTB4, which was higher in C3H/HePas. It is concluded that PAF and eicosanoids are mediators of local and systemic changes induced by immune complexes in the peritoneal cavity of mice.

Modulation of human neutrophil apoptosis by immune complexes.

In the present study we examined whether immune complexes (IC) are able to modulate human neutrophil apoptosis. We observed different effects depending on the type of IC employed. Precipitating IC (pIC) and Ab-coated erythrocytes (E-IgG) triggered a marked stimulation of apoptosis, while heat-aggregated IgG and soluble IC, significantly delayed spontaneous apoptosis. Blocking Abs directed to Fcgamma receptor type II (FcgammaRII), but not to FcgammaRIII, markedly diminished the acceleration of apoptosis triggered by either pIC or E-IgG, supporting a critical role for FcgammaRII in apoptosis stimulation. This phenomenon, on the other hand, does not appear to involve IC phagocytosis or the participation of CR3. Acceleration of neutrophil apoptosis triggered by either pIC or E-IgG seems to require the activation of the respiratory burst, as suggested by 1) the ability of catalase to prevent apoptosis stimulation; 2) the effect of azide, an heme enzyme inhibitor, which dramatically enhanced apoptosis induced by pIC or E-IgG; and 3) the inability of pIC or E-IgG to accelerate apoptosis of neutrophils isolated from CGD patients. It is well established that IC affect the course of inflammation by inducing the release of inflammatory cytokines, proteolytic enzymes, oxidative agents, and other toxic molecules. Our results suggest that IC may also affect the course of inflammation by virtue of their ability to modulate neutrophil apoptosis.

Activation of human neutrophils induced by immune complexes prepared with cationic and anionic fractions of normal IgG antibodies.

The authors have recently shown that the ability of immune complexes (IC) to trigger Fc gamma R-dependent cell responses can be dramatically enhanced when the isoelectric point (pI) of normal IgG antibodies is increased from 5.8-8.5 to 8.5-9.8 by treatment with 1-ethyl-3-2(3-dimethylaminopropyl) carbodiimide HCl and ethylene diamine. In the current work the authors analyse whether differences in the charge of normal IgG antibodies may also affect IC activity. Soluble IC (sIC) were prepared with (a) rabbit IgG antibodies to human IgG and anionic or cationic fractions of human IgG; and (b) bovine serum albumin (BSA) and anionic or cationic fractions of rabbit IgG anti-BSA antibodies. Similar abilities to bind to neutrophil surface were observed for sIC prepared with both anionic (anIC) and cationic fractions of IgG (catIC). Moreover, no differences were found when neutrophil shape change, chemiluminescence (CL) emission and elastase release were induced by either anIC or catIC. As in the case of sIC, particulate IC prepared with erythrocytes (E) and anionic or cationic fractions of specific IgG antibodies (IgG-E) showed no differences in their abilities to trigger either CL emission or ADCC. Taken together, these results suggest that the pI of normal IgG antibodies do not affect the ability of IC to trigger neutrophil responses mediated by receptors for the Fc portion of IgG antibodies (Fc gamma R).

Impairment of the murine mononuclear phagocyte system function by antigenic stimulation.

This study examined the mononuclear phagocyte system (MPS) capacity to eliminate IgG-sensitized syngeneic erythrocytes (EA) after antigenic challenge. Survival data of EA in normal and preimmunized mice showed that a single dose of T-dependent antigen was able to delay Fc gamma R-dependent clearance. This impairment in EA elimination was dependent on the dose of antigen injected. On the other hand, T-independent antigens, B cell mitogen, and the inflammatory agent Freund's incomplete adjuvant were ineffective in modulating MPS function. As expected, the liver and the spleen were the main sites of EA trapping, but the spleen of immunized mice sequestered significantly less EA than that of control mice. Impaired clearance capacity was observed as soon as 24 hr after immunization and was persistent up to the seventh day after antigenic stimulation. Moreover, mice decomplementation by cobra venom factor treatment did not prevent the impairment of MPS by antigenic stimulation, suggesting that it is strictly Fc gamma R dependent. Our results indicate that stimulation of the immune system by T-dependent antigens can diminish the Fc gamma R-mediated clearance capacity of the MPS. The possible mechanisms involved in this regulation are discussed.

Immunosuppressive effect of paracoccidioidomycosis sera on the proliferative response of normal mononuclear cells. Identification of a Paracoccidioides brasiliensis 34-kDa polypeptide in circulating immune complexes.

In this paper we relate that sera from paracoccidioidomycosis patients inhibited the mitogen-induced proliferative responses of normal mononuclear cells. Treatment of these sera with 2.5% polyethyleneglycol (PEG), a method classically used to precipitate immune complexes, significantly reduced their inhibitory activity. Immunoblot analysis of the PEG precipitates identified a 34-kDa polypeptide, recognized by rabbit anti-P. brasiliensis IgG. Patient mononuclear cells showed partial restoration of their proliferative capacity after 24 h culture in medium alone, which suggests release of membrane-bound molecules in the culture medium. These findings indicate that circulating P. brasiliensis antigens, complexed or not with antibodies, may play a negative immunoregulatory effect in the mitogen-induced proliferative responses of paracoccidioidomycosis patients.

Interferon-gamma enhances PAF-acether production by stimulated human polymorphonuclear leucocytes.

Human polymorphonuclear leucocytes (PMN) stimulated with either immune complexes (IC), phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (FMLP) generate platelet-activating factor (PAF-acether). The present study demonstrates that treatment of PMN with recombinant human interferon-gamma (IFN-gamma) significantly enhanced the production of PAF-acether by stimulated cells, in a concentration-dependent mode. On the contrary, alpha and beta IFN were completely unable to increase PAF-acether synthesis by stimulated PMN. The significance of these results is discussed.

Neutrophil erythrotoxicity induced by phorbol myristate acetate: mechanisms involved in neutrophil activation.

The capacity of phorbol myristate acetate (PMA) to prime neutrophil cytotoxic responses induced by a second stimulus was investigated. Treatment of neutrophils with low concentrations of PMA (0.2-0.5 ng/ml) for 18 hr at 37 degrees C markedly enhanced cytotoxicity triggered by Ca2+ ionophore A23187, N-formyl-methionyl-leucyl-phenylalanine (FMLP) and PMA. Pretreatment with PMA also enabled neutrophils to mediate significant cytotoxicity when triggered by platelet-activating factor (PAF), a stimulus unable to induce untreated cells to display cytotoxicity. Conversely, neutrophil cytotoxicity triggered by immune complexes (IC) was not modified by PMA treatment, whereas cytolytic activity of neutrophils against antibody-sensitized target cells was significantly increased. Treatment with PMA concentrations higher than 1 ng/ml directly triggered neutrophil cytotoxicity. Interestingly, we found that PMA-triggered neutrophils were able to sustain maximal levels of cytotoxicity for at least 8 hr after stimulation. With regard to the mechanisms involved in neutrophil activation by PMA, we found that catalase but not superoxide dismutase (SOD) prevented neutrophil activation measured as 1) induction of cytotoxic responses, 2) increase of neutrophil adhesiveness to cell-free surfaces, and 3) inhibition of chemotactic responses to FMLP. These findings suggest that H2O2 may play a major role in neutrophil activation induced by PMA.

The role of complement in the stimulation of lysosomal enzyme release by polymorphonuclear leucocytes induced by immune complexes of IgG and of IgM.

The effect of complement components incorporated into precipitated immune complexes (IC) of IgG or of IgM on their capacity for stimulating lysosomal enzyme release from rabbit polymorphonuclear leucocytes was studied in vitro. We have found that: (i) complement causes an amplification in the stimulatory capacity of both classes of IC, the effect being dependent on the concentration of the IC, and higher for the IgM class; (ii) dose-effect experiments of competition by fluid-phase immunoglobulins have shown that IgG (in physiological or smaller concentrations) can inhibit greatly the stimulation by this class of immune complex; this inhibition can be prevented, however, by the presence of complement in the IC (a situation expected to occur in vivo); for IgM immune complexes there was no such competitive inhibition, so complement would not be necessary; (iii) the relevant complement factors must be located in the range C1-C3. These results help us to understand the importance of complement (besides the well-known generation of chemotactic factors) in the mechanisms of tissue injury produced by neutrophils in immune complex diseases.

Evaluation of neutrophil activity and circulating immune complexes levels in diabetic patients.

We have studied chemiluminescence produced by neutrophils stimulated by opsonized zymosan in insulin dependent (IDD) and non insulin dependent (NIDD) diabetic patients. Chemiluminescence was evaluated as the integral and maximum peak, total time and time to maximum peak of the response curve to opsonized zymosan. These values were then compared with circulating immune complexes (CIC) and glucose levels. Both IDD and NIDD patients had significantly higher values of chemiluminescence and CIC than normal controls. We also observed that patients who had the highest values of CIC and chemiluminescence levels were the ones with clinical microvascular complications.

Papel del complemento en la recuperación funcional de los receptores para IgG bloqueador por complejos inmunes / Role of complement in the functional recovery of IgG receptors blocked by immune complexes

La interacción de complejos inmunes (CI) con receptores para el fragmento Fc de IgG (RFcgama) expresados en leucocitos pone en marcha mecanismos efectores y regulatorios de suma relevancia en el curso de la respuesta inmune. En trabajos anteriores, empleando la citotoxicidad celular dependiente de anticuerpos (ADCC) como expresión funcional de los RFcy, hemos demostrado que las células monocucleares periféricas humanas (CMPH), previamente bloqueadas en CI, recuperan la capacidad de mediar la ADCC a través de la activación de la vía alternativa del complemento (VAC). El objetivo de este trabajo fue analizar los mecanismos de recuperación funcional de los RFcgama cuando éstos han sido bloqueados por CI no fijadores de complemento (C). A tal fin, la IgG usada para preparar los CI se trató con carbodiimida (CDI), procedimiento que modifica su capacidad para fijar C, sin alterar mayormente su sitio de combinación con el antígeno, ni su habilidad para unirse a los RFcgama. Los resultados obtenidos demostraron que el C sólo es eficiente para revertir el bloqueo de la ADCC por CI, cuando éstos son capaces de fijar C

Papel del complemento en la recuperación funcional de los receptores para IgG bloqueador por complejos inmunes / Role of complement in the functional recovery of IgG receptors blocked by immune complexes

La interacción de complejos inmunes (CI) con receptores para el fragmento Fc de IgG (RFcgama) expresados en leucocitos pone en marcha mecanismos efectores y regulatorios de suma relevancia en el curso de la respuesta inmune. En trabajos anteriores, empleando la citotoxicidad celular dependiente de anticuerpos (ADCC) como expresión funcional de los RFcy, hemos demostrado que las células monocucleares periféricas humanas (CMPH), previamente bloqueadas en CI, recuperan la capacidad de mediar la ADCC a través de la activación de la vía alternativa del complemento (VAC). El objetivo de este trabajo fue analizar los mecanismos de recuperación funcional de los RFcgama cuando éstos han sido bloqueados por CI no fijadores de complemento (C). A tal fin, la IgG usada para preparar los CI se trató con carbodiimida (CDI), procedimiento que modifica su capacidad para fijar C, sin alterar mayormente su sitio de combinación con el antígeno, ni su habilidad para unirse a los RFcgama. Los resultados obtenidos demostraron que el C sólo es eficiente para revertir el bloqueo de la ADCC por CI, cuando éstos son capaces de fijar C (AU)