Isolation of healthy individuals' and rheumatoid arthritis patients' peripheral blood neutrophils by the gelatin and Ficoll-Hypaque methods: comparative efficiency and impact on the neutrophil oxidative metabolism and Fcγ receptor expression.

In vitro assessment of the functional responses of leukocytes sometimes requires their isolation from blood, joint and tissues. In this study, we compared the efficiency of two procedures - the gelatin method and Ficoll-Hypaque density centrifugation gradient - to isolate peripheral blood neutrophils of healthy individuals and patients with active rheumatoid arthritis (RA). We also assessed whether these procedures affect the neutrophil activation status. Both purification procedures were concluded in 90min, and yielded cell populations with similar degrees of purity (80-90%), number of neutrophils (1-2×10(6) cells per mL of blood), and viability (97-100%). In vitro neutrophil priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly increased the reactive oxygen species producing ability of the cells stimulated with n-formyl-methionyl-leucyl-phenylalanine (n-fMLP), soluble immune complexes (s-ICs), and insoluble immune complexes (i-ICs). Isolated neutrophils not treated with GM-CSF responded to n-fMLP and i-IC, but not to s-IC. Almost all of the neutrophils (98-100%) purified by both methods expressed FcγRII/CD32 and FcγRIII/CD16, but they did not express significant levels of FcγRI/CD64. Similar results were obtained for healthy individuals' and RA patients' neutrophils. In summary, the gelatin method was comparable to Ficoll-Hypaque gradient in terms of purity, yield, and viability of the neutrophil preparations. Both methods neither primed or activated the neutrophils, nor affected their functional responsiveness. Therefore, both methods are suitable to isolate peripheral blood neutrophils of healthy individuals and RA patients.

Assessment of antioxidant activity of spray dried extracts of Psidium guajava leaves by DPPH and chemiluminescence inhibition in human neutrophils.

This work evaluated the physicochemical properties and antioxidant activity of spray dried extracts (SDE) from Psidium guajava L. leaves. Different drying carriers, namely, maltodextrin, colloidal silicon dioxide, Arabic gum, and ß -cyclodextrin at concentrations of 40 and 80% relative to solids content, were added to drying composition. SDE were characterized through determination of the total phenolic, tannins, and flavonoid content. Antioxidant potential of the SDE was assessed by two assays: cellular test that measures the luminol-enhanced chemiluminescence (LumCL) produced by neutrophils stimulated with phorbol myristate acetate (PMA) and the DPPH radical scavenging (DPPH∗ method). In both assays the antioxidant activity of the SDE occurred in a concentration-dependent manner and showed no toxicity to the cells. Using the CLlum method, the IC50 ranged from 5.42 to 6.50 µg/mL. The IC50 of the SDE ranged from 7.96 to 8.11 µg/mL using the DPPH(•) method. Psidium guajava SDE presented significant antioxidant activity; thus they show high potential as an active phytopharmaceutical ingredient. Our findings in human neutrophils are pharmacologically relevant since they indicate that P. guajava SDE is a potential antioxidant and anti-inflammatory agent in human cells.

Galectin-3 is essential for reactive oxygen species production by peritoneal neutrophils from mice infected with a virulent strain of Toxoplasma gondii.

Toxoplasma gondii stimulates a potent pro-inflammatory response and neutrophils are involved in early infection. Galectin-3 (Gal-3) is an endogenous modulator of inflammatory processes and anti-infective agents, but its interaction with neutrophils in T. gondii infection is still unclear. Here, we evaluated the role of Gal-3 in peritoneal inflammation, reactive oxygen species (ROS) production by neutrophils and survival, after in vivo T. gondii infection with virulent RH strain, using Gal-3 deficient and wild type mice. Animals were inoculated with thioglycollate or tachyzoites, and peritoneal cells were harvested for analysis of the influx of leukocytes. Neutrophils were isolated from peritoneal exudates from infected mice and stimulated with phorbol myristate acetate (PMA) to evaluate ROS production by luminol-dependent chemiluminescence assay. Our results showed that: (1) Gal-3 upregulates peritoneal inflammation, with enhanced recruitment of neutrophils and lymphocytes after thioglycollate stimulation, but does not influence the enhanced neutrophil influx after early T. gondii infection; (2) Gal-3 upregulates ROS generation by inflammatory peritoneal neutrophils from infected mice, but downregulates its production in non-infected mice and (3) Gal-3 does not influence the survival of mice after infection with the virulent T. gondii strain. In conclusion, Gal-3 is essential for ROS generation by neutrophils in the initial acute phase of T. gondii infection and this phenomenon may constitute an attempt to control parasite growth during in vivo infection with the T. gondii virulent strain.

Alternative complement pathway and factor B activities in rats with altered blood levels of thyroid hormone

Evaluating the activity of the complement system under conditions of altered thyroid hormone levels might help elucidate the role of complement in triggering autoimmune processes. Here, we investigated alternative pathway (AP) activity in male Wistar rats (180 ± 10 g) after altering their thyroid hormone levels by treatment with triiodothyronine (T3), propylthiouracil (PTU) or thyroidectomy. T3 and thyroxine (T4) levels were determined by chemiluminescence assays. Hemolytic assays were performed to evaluate the lytic activity of the AP. Factor B activity was evaluated using factor B-deficient serum. An anti-human factor B antibody was used to measure factor B levels in serum by radial immunodiffusion. T3 measurements in thyroidectomized animals or animals treated with PTU demonstrated a significant reduction in hormone levels compared to control. The results showed a reduction in AP lytic activity in rats treated with increasing amounts of T3 (1, 10, or 50 µg). Factor B activity was also decreased in the sera of hyperthyroid rats treated with 1 to 50 µg T3. Additionally, treating rats with 25 µg T3 significantly increased factor B levels in their sera (P < 0.01). In contrast, increased factor B concentration and activity (32 percent) were observed in hypothyroid rats. We conclude that alterations in thyroid hormone levels affect the activity of the AP and factor B, which may in turn affect the roles of AP and factor B in antibody production.

Alternative complement pathway and factor B activities in rats with altered blood levels of thyroid hormone.

Evaluating the activity of the complement system under conditions of altered thyroid hormone levels might help elucidate the role of complement in triggering autoimmune processes. Here, we investigated alternative pathway (AP) activity in male Wistar rats (180 ± 10 g) after altering their thyroid hormone levels by treatment with triiodothyronine (T3), propylthiouracil (PTU) or thyroidectomy. T3 and thyroxine (T4) levels were determined by chemiluminescence assays. Hemolytic assays were performed to evaluate the lytic activity of the AP. Factor B activity was evaluated using factor B-deficient serum. An anti-human factor B antibody was used to measure factor B levels in serum by radial immunodiffusion. T3 measurements in thyroidectomized animals or animals treated with PTU demonstrated a significant reduction in hormone levels compared to control. The results showed a reduction in AP lytic activity in rats treated with increasing amounts of T3 (1, 10, or 50 µg). Factor B activity was also decreased in the sera of hyperthyroid rats treated with 1 to 50 µg T3. Additionally, treating rats with 25 µg T3 significantly increased factor B levels in their sera (P < 0.01). In contrast, increased factor B concentration and activity (32%) were observed in hypothyroid rats. We conclude that alterations in thyroid hormone levels affect the activity of the AP and factor B, which may in turn affect the roles of AP and factor B in antibody production.

The role of seasonality on the inhibitory effect of Brazilian green propolis on the oxidative metabolism of neutrophils.

The reactive oxygen species (ROS) produced by neutrophils are involved in the pathogenesis of several diseases, for which the intake of antioxidants could benefit patients either as a prophylactic or therapeutic treatment. Propolis is among the known antioxidants, and its chemical composition may vary under the influence of seasonality, which may interfere in its biological properties. This work evaluates the role of seasonality on the production of some important compounds of propolis samples produced monthly from November 2001 through October 2002 as well as the effect of these samples on the oxidative metabolism of stimulated neutrophils, by using both luminol and lucigenin to produce chemiluminescence (CLlum and CLluc, respectively). The cytotoxicity of the most active extracts to neutrophils was also investigated. The inhibitory effect of the propolis samples varied significantly during the studied period for both assays (3.4 ± 1.1 to 16.0 ± 1.1 µg/mL for CLlum and 6.2 ± 2.0 to 30.0 ± 5.0 µg/mL for CLluc), which was also observed in the quantitative profile of the main analyzed compounds (aromadendrin-4'-methyl ether, artepillin C, and baccharin). This effect started to become more prominent during the fall and, among all the studied extracts, the one obtained in May displayed the highest inhibitory effect on CL production (3.4 ± 1.1 µg/mL for CLlum and 6.2 ± 2.0 µg/mL for CLluc). The HPLC qualitative profiles of the extracts of propolis samples were quite similar, but there was a huge variation in terms of quantitative profile. It seems that aromadendrin-4'-methyl ether and baccharin play an essential role in the antioxidant activity, while artepillin C is not very important for this effect. The extracts presenting the highest antioxidant activity were produced in May, June, and August, and they did not display cytotoxicity at 25 µg/mL; quercetin, used as control, was not toxic to neutrophils at 8.5 µg/mL.

Antioxidant activity and cytotoxicity as mediators of the neutrophil chemiluminescence inhibition by butylated hydroxytoluene.

The tissue damage found in some inflammatory and autoimmune diseases has been shown to be mediated by an increased activation of neutrophil effector functions. In this study, we investigated the inhibitory effect of the phenolic compound butylated hydroxytoluene (BHT) on reactive oxygen species (ROS) generation by opsonized zymosan-stimulated neutrophils, assessed by luminol- and lucigenin-enhanced chemiluminescence (CL-lum and CL-luc, respectively), and some aspects of its mechanism of action. BHT showed concentration-dependent: (a) inhibitory effect on CL-lum and CL-luc; (b) cytotoxic effect, expressed by increased lactate dehydrogenase leakage by the cells; (c) interaction with neutrophil membranes; (d) ROS scavenger activity. These biological effects were observed in the same range of concentrations (0-5 x 10(-5) mol/l). Taken together, the results suggest that inhibition of neutrophil chemiluminescence by BHT was a result of multiple mechanisms, especially a cytotoxic effect probably mediated by BHT interaction with neutrophils membranes, and the ROS scavenging effect.

Antibody responses in hyperthyroid rats.

This study evaluated antibody production against sheep red blood cells (SRBC) in hyperthyroid rats during treatment with triiodothyronine (T(3)). The immune response was evaluated by measuring plaque forming cells (PFC) in the spleen and by enzyme-linked immunosorbent assay (ELISA) in serum of male Wistar rats (180+/-10 g) treated with 25 mug/day of triiodotironine (T(3)) during 7-12 days and immunized with SRBC at the 8th day of treatment. The results showed that anti-SRBC antibody production was significantly decreased in animals treated for 12 days when compared to normal rats immunized with the same antigen, as evaluated by the two assays. These results show that in this experimental model hyperthyroidism decreases antibody response. We previously observed the opposite effect, that is, an increase in this response in hypothyroid rats resulting from the treatment with propylthyouracil, a blocker of thyroid hormone biosynthesis. It is suggested that antibody production is affected by thyroid hormone levels.

CR1 on erythrocytes of Brazilian systemic lupus erythematosus patients: the influence of disease activity on expression and ability of this receptor to bind immune complexes opsonized with complement from normal human serum.

Hypocomplementaemia and low expression of CR1 on erythrocytes (E) of patients with systemic lupus erythematosus (SLE) are associated with defective clearance of circulating immune complexes (IC) and so they may have pathogenic significance. Here, we investigated whether the reduced CR1/E in SLE patients per se might affect the binding of IC to CR1/E. First, we analysed the expression of CR1 on E of active (n=30) and inactive (n=34) SLE patients using a FITC-conjugated mouse anti-CR1 monoclonal antibody E11 and flow cytometry. Both groups of patients had a significantly reduced CR1/E expression compared with healthy controls (n=40). It was also observed that the number of E bearing CR1 was reduced in both groups of SLE patients studied. Second, we determined the functional activity of CR1/E by measuring the binding to E of FITC-bovine serum albumin (BSA)/rabbit anti-BSA complexes, formed at equivalence, which were opsonized with complement from normal human serum (NHS). On the other hand, we did not find differences between the patient and control groups in the ability of E to bind IC/NHS. There was also a positive correlation between the CR1/E expression and the number of E bearing CR1 in control and inactive SLE groups, which was not observed in the group of active SLE patients. Considering the involvement of low levels of complement and CR1/E expression on complex processing, in this in vitro model the results show that an effective coating of the complexes with complement is sufficient to bind them preferentially to CR1 over normal levels of receptor expression.

Neutrophils from Brazilian patients with Graves' disease: some biochemical and functional aspects.

Graves' disease shows important systemic inflammatory complications and has been considered to be systemic autoimmune thyroid, skeletal muscle and connective tissue syndrome. Neutrophils participate in the pathophysiology of the two major immune and inflammatory manifestations of the disease, ophthalmopathy and myxedema, and may worsen the inflammatory picture. In this study we analysed some biochemical and functional aspects of neutrophils in Graves' disease patients to assess their participation in these processes. The results show that the complement and/or Fcgamma receptor-mediated oxygen radical production by neutrophils was increased when patient cells were compared with controls. However the percentage of cells expressing complement and IgG receptors and the per-cell fluorescence, were similar, indicating that the increased oxidative burst was not due to an abnormal expression of mediating receptors. The production of hydrogen peroxide was also increased in hyperthyroid patient neutrophils as compared to controls. Conversely, antioxidant defences (superoxide dismutase activity and reduced glutathione content) in neutrophils from patients were not significantly different from healthy controls. The liberation of potent oxidative compounds together with the absence of adequate quenching of them by antioxidant mechanisms could be responsible for greater tissue damage in inflammatory conditions, as is the case in ophthalmopathy and myxedema patients. Considering our results and those of other workers, we encourage and suggest an associated antioxidant therapy to complement the conventional anti-thyroid therapy, especially in obvious inflammatory cases and in individuals who smoke.

Effect of Brazilian green propolis on the production of reactive oxygen species by stimulated neutrophils.

The activity of a crude ethanol extract of green propolis and its fractions obtained by partition with hexane, chloroform and n-butanol was assessed on luminol- and lucigenin- enhanced chemiluminescence (CL) produced by rabbit neutrophils (PMNs) stimulated with particles of serum-opsonized zymosan (OZ). The total production of reactive oxygen species (ROS) by PMNs was measured by the luminol-enhanced CL (LumCL) assay and the production of the superoxide anion (O2*-) by the lucigenin-enhanced CL (LucCL) assay. All evaluated propolis samples had inhibitory effect on the LumCL and LucCL, which was concentration dependent. The n-butanol and chloroform fractions displayed the highest inhibitory effect on the LumCL produced by PMNs stimulated with OZ, in comparison with both the ethanol extract and the hexane fraction. Besides, the hexane fraction was the one which presented the highest effect for the LucCL assay. Some isolated compounds from both n-butanol and chloroform fractions were also assessed, including kaempferide, isosakuranetin, aromadendrine-4'-methyl-ether and 3-prenyl-p-coumaric acid. Kaempferide presented the highest inhibitory effect on the LumCL in comparison with the other compounds. Moreover, under the conditions assessed, the studied green propolis samples and isolated compounds were not toxic to the rabbit PMNs.

The complement-fixing activity of immune complexes containing IgG antibodies of different functional affinities: effects on superoxide production by rabbit neutrophils.

When neutrophil phagocytes are stimulated by IgG containing immune complexes (IgG-IC), with or without the participation of the complement system, they show a sharp increase in oxygen uptake and begin to release large quantities of superoxide anions (O2-) and hydrogen peroxide (H2O2) into the surrounding medium. The aim of the present investigation was to provide insights into the production and release of O2- by rabbit neutrophils activated with immune complexes (IC) containing IgG antibodies of different functional affinity, opsonized and not opsonized by complement system components. For this purpose, two populations of polyclonal anti-ovalbumin (OVA) IgG antibodies with different functional affinity, 5 x 10(8) M(-1) and 2 x 10(7) M(-1), were prepared. The production of O2- was measured spectrophotometrically by a method using the superoxide dismutase-inhibited reduction of ferricytochrome C to the ferrous form. The activation of complement by different IgG-IC was determined by estimating the total residual haemolytic activity of the alternative and classical pathways in sera treated with different concentrations of anti-OVA IgG/ OVA immune complexes formed at equivalence. The results showed that: 1) antibody functional affinity influenced O2- production and the complement-fixing activity induced by the IC. In general, the higher functional affinity antibodies were more efficient in stimulating the respiratory burst of neutrophils and in activating complement by the classical and alternative pathways than the lower functional affinity antibodies at all IC concentrations tested; 2) complement components incorporated into the immune complex lattice caused an increase in the stimulatory activity of both IgG antibodies to produce O2- (approximately equal to 15% for the IC of IgG with Ka = 5 x 10(8) M(-1) and approximately equal to 7% for the IC of IgG with Ka = 2 x 10(7) M(-1)). This effect was dependent on antibody affinity and concentration; 3) there was a direct relationship between the overall level of complement activation, antibody affinity and superoxide production by neutrophils. Thus, we conclude that antibody affinity influences immune complex lattice formation, modulating its three-dimensional structure and the disposition of Fc fragments interfering with the antibody's biological properties. These results can help understand the precise role of antibody functional affinity in antigen-antibody complex diseases and define the immunochemical characteristics of pathogenic complexes.

Effect of the period of treatment with a single dose of propylthiouracil on the antibody response in rats.

Propylthiouracil (PTU) was employed in a fixed quantity to evaluate the effect of the period of treatment with this drug on the antibody response to sheep red blood cells in Wistar rats. Animals were treated for 8, 16, 30 and 90 days by intragastric route with 5 mg/day, and immunized 24 h following the end of treatment; other groups were treated for 21 days, and immunized on the 17th day of treatment. Animals were sacrificed 5 or 6 days following immunization; the primary response was evaluated by the number of plaque-forming spleen cells and in some cases also by enzyme-linked immunosorbent assay (ELISA). Secondary response of PTU-treated rats, immunized and boostered after 15 days, was evaluated by ELISA 3 days following the booster. RIA performed the measurement of T3 in animals treated for 16 days, immunized, and sacrificed after 1, 2, 3, 4 and 5 days following immunization. Results showed that treatment for 8 and 16 days increased production of antibodies, and for 30 and 90 days decreased this response. Thus, according to the period of treatment, the same dose of PTU stimulates or suppresses the antibody response. This biphasic effect of a single dose of PTU was independent of alterations of serum levels of T3 during the buildup of the immune response. These results contributes towards the understanding of the literature controversy regarding the effects of this drug on the immune response, and could be of interest for studies involving autoimmune processes in thyroid.

Fcgamma and complement receptors: expression, role and co-operation in mediating the oxidative burst and degranulation of neutrophils of Brazilian systemic lupus erythematosus patients.

We have investigated the individual role of FcgammaR and CR, as well as their cooperation, in mediating the oxidative burst and degranulation of neutrophils of Brazilian systemic lupus erythematosus (SLE) patients. Neutrophils were stimulated with the immune complexes (IC)-IgG or -F(ab')2, opsonized or not with normal or SLE human serum. The oxidative burst was decreased in neutrophils of active SLE patients compared to healthy controls when this response was mediated by FcgammaR and/or CR, while the degranulation was unaffected. The SLE hypocomplementemia did not affect the oxidative burst mediated only by CR. FcgammaRII and CR1 expression on neutrophils of active SLE patients was reduced, while the expression of FcgammaRIII and CR3 was unaffected. These results suggest that the different FcgammaR and CR may be involved or cooperate in different ways in the mediation of the oxidative burst and the degranulation. Moreover, the decreased oxidative burst of neutrophils of active SLE patients may not depend only on SLE hypocomplementemia for IC opsonization. These observations are directed at the understanding of how each of these immune system components (FcgammaR, CR and complement) influences the precise biological neutrophil responses both in physiological and pathological conditions. Since the Brazilian population comprises many races, these results are important because they are directed at a specific population of SLE patients.

Effect of low-dose prednisone in vivo on the ability of complement receptor to mediate an oxidative burst in rat neutrophils.

Glucocorticoids have been used in the treatment of a variety of inflammatory processes including autoimmune diseases. However, the influence of low-dose glucocorticoids on the respiratory burst activity of neutrophils has not been studied. The aim of this work was to study the effect of treatment with low-dose prednisone on the oxidative burst of rat peripheral blood neutrophils. Wistar male rats were treated with prednisone by gavage (28, 87 or 257 microg/animal/day) for 7 or 15 days. These doses are equivalent to 10, 30 or 90 mg/adult human ( approximately 70 kg)/day, respectively. Sera from normal rats were used to opsonize zymosan (opZy). Neutrophils (1x10(5)) were stimulated by opZy and the oxidative burst of control or treated rat cells was measured by luminol-dependent chemiluminescence (CL). Prednisone did not affect the CL of rat neutrophils for either period of treatment, or any studied doses, when compared with controls. These results suggest that the low-dose prednisone has no effect on the oxidative burst mediated by complement receptors during the rat neutrophil phagocytosis of complement-opZy.

The effect of the antithyroid drug propylthiouracil on the alternative pathway of complement in rats.

The effect of propylthiouracil (PTU) on the lytic activity of complement in rat serum was investigated in vivo. Rats (180+/-10 g) were treated daily by gavage with PTU doses of 1-50 mg/200 g body weight for time intervals ranging from 1 to 30 days. Serum classical pathway (CP) and alternative pathway (AP) activities were determined 24 h after the last dose. A single dose of 50 mg/200 g body weight was administered to additional groups and the animals were sacrificed after periods of 1-48 h. The results showed a relatively small reduction ( approximately 30%) in CP activity, evident only in animals treated with 50 mg of PTU for three weeks. However, a clear and opposite effect of PTU, an increase in lytic activity reaching values up to 180% of controls, was observed on AP activity. This effect was seen at all PTU doses used, and occurred within 4 days of treatment with the highest dose. Maximum activity was observed at intermediate intervals, depending on the PTU dose, with a return to control levels occurring after the longer periods of treatment. The lytic activity of serum from animals treated with a single PTU dose of 50 mg/200 g body weight and sacrificed 1-48 h after dosing did not differ from controls. Serum levels of thyroid hormone (triiodo L-thyronine, T3, and thyroxine, T4) were determined in representative groups of treated animals (injected with 5 mg of PTU/200 g body weight/day). These were either undetectable or considerably lower than those of controls. The serum PTU levels of these rats increased for up to 22 days, reaching values of 2-4 microg/ml.PTU is described in the literature as a modulator of both cellular immune responses and antibody production. Upon complement activation fragments of complement components bind to immune complexes and to specific receptors on cells of the immune system. Thus, alteration in AP activity caused by PTU treatment suggests a possible mechanism by which the drug exerts its modulatory effect. Increased complement AP activity might affect events as antigen presentation and hence the onset and course of the immune response.

Correlation of alternative pathway (AP) lytic activity and AP-dependent neutrophil phagocytosis with factor B levels and consumption in serum.

This work investigated the correlation between serum levels of factor B, AP-lytic activity, ratio of factor B activation by zymosan, and AP-dependent neutrophil phagocytosis in samples of normal human serum (NHS). In addition, since the antithyroid drug propylthiouracil (PTU) induces increased levels of AP lytic activity in rats, groups of these animals were treated with this drug in order to increase AP titers and to evaluate those parameters also in this condition. The results showed no correlation between factor B concentration and AP lytic activity in 18 samples of NHS or between factor B concentration and proportion of consumption by zymosan. Interestingly, this consumption was also not correlated with phagocytosis as measured by the chemiluminescence (CL) response of neutrophils to the opsonized particles. The two biological properties of phagocytosis and lytic activity, dependent of AP, were not correlated to each other in the NHS samples. In the samples of rat serum with increased AP lytic levels a different result was observed. A positive correlation between CL response and lytic activity occurred in serum of animals receiving a low PTU dose, but not in serum of animals receiving a high dose, where CL responses were lower than those of controls. The results are compared to literature data and discussed in terms of individual differences in resistance or susceptibility to infections and or diseases involving the complement system.

The influence of antibody functional affinity on the effector functions involved in the clearance of circulating immune complexes anti-BSA IgG/BSA.

A systematic study was carried out to investigate the role of antibody functional affinity in the capacity of immune complexes (IC) to activate the complement system and to trigger subsequently the molecular events involved in the handling of IC by providing a clearance mechanism. For this purpose, two populations of polyclonal anti-BSA IgG antibodies of different affinities were prepared, with values of 1.89x10(8) M(-1) and 4.94x10(8) M(-1). First we studied the capacity of IC formed at equivalence with both antibodies to activate the classical and the alternative pathways of human complement and the ability of the complexes to bind to erythrocyte C3b-C4b receptors (CR1; CD35). The data showed that the highest affinity antibodies were more efficient in activating complement by both pathways. However, their binding to erythrocyte CR1 was significantly lower compared to the binding of the lowest affinity IgG. Second we compared these IC in terms of their ability to stimulate the respiratory burst of neutrophils (PMN) and to induce the release of PMN lysosomal enzymes. In general, both of these PMN functions were better stimulated by the IC prepared with the IgG antibodies having a highest affinity, although the effects were variable for different IC concentrations. The suggestion to be drawn from the data is that the antibody affinity has an influence on the formation of the immune complex lattice, modulating its three-dimensional structure and the arrangement of the antibody Fc fragments, interfering with complement activation and access to the neutrophil IgG receptors. The significance of these observations for the understanding of how affinity influences the precise biological mechanism that participates in the fate of IC is discussed.

Sheep erythrocyte sensitisation and restriction to lysis by the complement system from equine and sheep serum

Neste trabalho foi analisado o efeito do grau de sensbilizaçäo de hemácias de carneiro por anticorpo, na resistência à lise pelo complemento do soro equino e de carneiro. Os resultados mostraram que, à medida que houve um aumento do nível de sensibilizaçäo, ocorreu também um aumento da atividade do complemento homólogo, levando a 100 por cento de lise; o mesmo foi observado para o complemento do soro equino, geralmente considerado ineficiente na lise destas células. A restriçäo à lise pode, assim, ser sobrepujada por este tratamento. Estes dados podem ser de interesse para a compreensäo dos mecanismos envolvidos na modulaçäo do fenômeno de restriçäo, na saúde e em doenças envolvendo o sistema complemento em animais domésticos e no homem, bem como em diagnóstico laboratorial, onde säo utilizados ensaios hemolíticos para medida de atividade do complemento e pesquisa de anticorpos em amostras de soro

Effect of the in vitro ageing on the restriction of sheep erythrocyte lysis by horse serum.

Fresh sheep erythrocytes are resistant to lysis by horse serum but gradually became susceptible to this source of complement after storage (ageing) in the blood at 4 degrees C. This occurs faster when the fresh cells are stored in isotonic buffer. The supernatant of the buffer/red cell suspension stored at 4 degrees C for 10-15 days restrict lysis by horse serum when incubated back at 37 degrees C or 43 degrees C with the 'aged' sheep cells or fresh guinea pig red cells. In assays using fresh guinea pig erythrocytes this effect is described by reincubation of the cells in buffer, and is specific to horse serum when compared to human serum.