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PubMed2003

Neutrophil function and plasma opsonic capacity in colostrum-fed and colostrum-deprived neonatal kittens.

OBJECTIVE: To determine whether passive transfer of IgG in neonatal kittens affects plasma opsonic capacity and neutrophil phagocytic and oxidative burst responses to bacteria in vitro. ANIMALS: 22 kittens from 6 specific pathogen-free queens. PROCEDURE: Kittens were randomized at birth into the following treatment groups: colostrum-fed, colostrum-deprived, or colostrum-deprived supplemented with feline or equine IgG. Blood samples were collected at intervals from birth to 56 days of age. Plasma IgG concentrations were determined by radial immunodiffusion assay. Neutrophil function was assessed by a flow cytometry assay providing simultaneous measurement of bacteria-induced phagocytosis and oxidative burst. The opsonic capacity of kitten plasma was determined in an opsonophagocytosis assay with bacteria incubated in untreated or heat-inactivated plasma. RESULTS: Among treatment groups, there were no significant differences in neutrophil phagocytic and oxidative burst responses to bacteria or opsonic capacity of plasma. In all samples of plasma, inactivation of complement and other heat-labile opsonins significantly reduced the opsonic capacity. Plasma IgG concentrations in kittens did not correlate with neutrophil function or plasma opsonic capacity before or after inactivation of complement. CONCLUSIONS AND CLINICAL RELEVANCE: The plasma opsonic capacity and neutrophil phagocytic and oxidative burst responses in vitro of kittens receiving passive transfer of IgG via colostrum intake or IgG supplementation and those deprived of colostrum were similar. The alternate complement pathway or other heat-labile opsonins may be more important than IgG in bacterial opsonization and phagocytosis.

Animals↗

PubMed2003

Assessment of the opsonic activity of purified bovine sIgA following intramammary immunization of cows with Staphylococcus aureus.

The phagocytosis of Staphylococcus aureus by bovine polymorphonuclear neutrophils (PMN) requires the presence of antibodies. Among the major isotypes of bovine antibodies, IgG2 and IgM are considered opsonic for bovine PMN. However, the role of purified bovine secretory IgA (sIgA) as an opsonin has not been assessed. In the present study, IgG2 were obtained from serum and sIgA, IgG1, and IgM were purified from the colostrums of three cows intramammarily immunized with heat-killed Staphylococcus aureus. The Ig preparations were assayed for specific antibodies, and the opsonic capacity of every isotype was investigated. Despite the presence of antibodies, we observed no distinct chemiluminescence response of PMN stimulated with sIgA- or IgG1-opsonized S. aureus, whereas IgM or IgG2 bound to bacteria induced a marked chemiluminescence response. Moreover, the counting of internalized bacteria per PMN after phagocytosis revealed a low uptake of S. aureus opsonized with sIgA or IgG1, in contrast to IgM or IgG2, which triggered efficient ingestion of bacteria. Priming of neutrophils by TNF-alpha, IFN-gamma, or C5adesArg did not promote an oxidative burst or uptake of sIgA-opsonized S. aureus to a greater extent than with IgG1-opsonized bacteria. Furthermore, analysis of uningested bacteria by flow cytometry after incubation with PMN showed a preferential uptake of IgM-opsonized S. aureus by PMN and only few sIgA-positive stained bacteria were PMN-associated. These experiments indicate that sIgA, like IgG1 and unlike IgM or IgG2, could not be considered as a major opsonin for phagocytosis of S. aureus by bovine blood PMN.

Animals↗

PubMed1975

Opsonic characteristics of chylomicron-bound protein.

A blood protein in complex with native chylomicrons was labeled with radiogold colloid, thus indirectly identifying the protein material in the complex as opsonin and suggesting the possibility that native chylomicrons may utilize the opsonin system for its clearance from the circulation. The protein in question has been shown to be at least divalent, in that the protein in combination with one type of particle could still react with another. It is felt that these studies provide further insight into the complex surface events that result during the process of opsonization.

Agglutination↗

PubMed1991

Mechanisms of host defense against Candida species. I. Phagocytosis by monocytes and monocyte-derived macrophages.

We studied the biochemical basis of phagocytosis of Candida albicans, a serious pathogen, and Candida parapsilosis, which is rarely pathogenic, by human monocytes (Mo) and monocyte-derived macrophages (MDM). Optimal phagocytosis of both species by Mo required the presence of extracellular Ca2+ and opsonization through both the classic and alternative complement pathways. Serum-opsonized Candida were ingested equally by Mo and MDM; unopsonized Candida were phagocytosed only by macrophages, and uptake began slowly. This opsonin-independent phagocytosis required Ca2+ and could be blocked by yeast mannan or mannose-BSA conjugate, suggesting a role for the mannose receptor. Opsonized Candida elicited a vigorous increase in the concentration of [Ca2+]i in Mo and MDM, but no Ca2+ transient was detected in MDM stimulated with unopsonized Candida. Pretreatment of MDM with ionomycin to increase [Ca2+]i had no effect on phagocytosis of unopsonized Candida. Addition of 5 mM EGTA completely inhibited changes in [Ca2+]i in Mo and MDM, suggesting that the Ca2+ transient induced by opsonized Candida is due to an influx of extracellular Ca2+. Differences in pathogenicity between the two Candida species could not be explained by differences in any aspect of phagocytosis. Uptake mediated by the macrophage mannose receptor could play a role in clearance of Candida under opsonin-poor conditions.

Calcium↗

PubMed1990

The respiratory burst response to Histoplasma capsulatum by human neutrophils. Evidence for intracellular trapping of superoxide anion.

Human neutrophils (PMN) have received little attention as to the role they play in host defense against Histoplasma capsulatum (Hc). We have characterized the binding and phagocytosis of Hc yeasts by human PMN and quantified the PMN respiratory burst in response to this organism. mAb specific for CD11a, CD11b, and CD11c all partially blocked the attachment of unopsonized yeasts to PMN; a mAb to CD18 inhibited attachment by greater than 90%. Thus, human PMN recognize and bind Hc yeasts via CD18 adhesion receptors as has been found for human cultured macrophages and alveolar macrophages. Unopsonized yeasts were phagocytosed by PMN, but phagocytosis was increased markedly by heat-labile and heat-stable serum opsonins. These opsonins promoted enhanced phagocytosis of yeasts by increasing the attachment of Hc yeasts to the PMN membrane. Phagocytosis of viable or heat-killed Hc yeasts by PMN did not induce the secretion of superoxide anion (O2-) as quantified by the reduction of cytochrome c. O2- was not detected when yeasts were opsonized in normal serum or immune serum, or at a ratio of yeasts to PMN of up to a 100:1. However, phagocytosis of opsonized yeasts by PMN did not prevent them from subsequently releasing O2- after further incubation with opsonized zymosan or PMA. Opsonized Hc yeasts clearly stimulated the PMN respiratory burst as quantified by intracellular reduction of nitroblue tetrazolium, reduction of cytochrome c in the presence of cytochalasin D, oxygen consumption, luminol-enhanced and nonenhanced chemiluminescence, and H2O2 production. These data suggest that phagocytosis of Hc yeasts by PMN is associated with intracellular entrapment of O2- that is not detectable by reduction of extracellular cytochrome c.

CD18 Antigens↗

PubMed1990

Peritoneal defence mechanisms and Staphylococcus aureus in patients treated with continuous ambulatory peritoneal dialysis (CAPD).

Peritonitis in continuous ambulatory peritoneal dialysis (CAPD) patients due to S. aureus is associated with an adverse clinical outcome, suggesting impaired clearance of this organism by the host. The ability of peritoneal macrophages (PM0) derived from CAPD patients to take up S. aureus and mount a respiratory burst was investigated. Whilst significant activity was observed in the absence of opsonin, both parameters of phagocytosis were augmented by addition of 20% pooled human serum (PHS), complement-depleted PHS, and fibronectin. When used as sole opsonin, fibronectin resulted in a dose-related increase in chemiluminescent response by both blood neutrophils and PM0. The opsonic activity of dialysis effluent, as judged by neutrophil chemiluminescence, correlated with IgG and fibronectin content, but not with complement as assessed by C3 levels. The addition of urokinase to dialysate improved its opsonic properties whilst having no effect on the activity of PHS-20%; this would suggest that the formation of fibrin in dialysate, promoted by S. aureus, interferes with phagocytosis. This and the low IgG, complement and fibronectin levels in dialysate may explain in part the relatively poor clearance of this organism from the peritoneum.

Dialysis Solutions↗

PubMed1989

Opsonic activity of a new intravenous immunoglobulin preparation: Pentaglobin compared with sandoglobulin.

Standard preparations of immunoglobulin for intravenous use consist predominantly of IgG (greater than 95%). We have compared the ability of a standard preparation (Sandoglobulin) with that of a new preparation (Pentaglobin, containing 12% IgM and 12% IgA) to improve the opsonic activity of antibody-deficient human sera in vitro. Panhypogammaglobulinaemic sera were poorly opsonic for five of six organisms tested, particularly Pseudomonas aeruginosa, Escherichia coli and Streptococcus pneumoniae, but opsonized Staphylococcus aureus almost normally. Both immunoglobulin preparations significantly improved the opsonic activity of the antibody-deficient sera for most organisms. The major difference between the two preparations was the ability of Pentaglobin to supply opsonins for P. aeruginosa, E. coli and Klebsiella pneumoniae, while Sandogloblin was significantly more potent in opsonins for Haemophilus influenzae. Pentaglobin demonstrates significant in vitro opsonic activity, particularly for enterobacteria (coliforms) and P. aeruginosa. Its content of IgM antibodies appears to confer special properties on Pentaglobin not seen with standard preparations of immunoglobulin for intravenous use. Its place in clinical practice remains to be determined but it may have a possible role in augmenting host defence mechanisms in Gram-negative septicaemia.

Agammaglobulinemia↗

PubMed1989

Functional importance of cystic fibrosis immunoglobulin G fragments generated by Pseudomonas aeruginosa elastase.

We examined the functional importance of immunoglobulin polypeptide fragments generated by Pseudomonas aeruginosa elastase (Pseudomonas elastase). The purpose of this study was to determine whether the elastase produced by Pseudomonas aeruginosa cleaves human IgG into immune fragments that functionally inhibit opsonophagocytosis. Our results confirm that IgG isolated from patients with cystic fibrosis (CF) incubated with purified pseudomonas elastase results in the generation of two major polypeptide fragments and that, furthermore, these fragments significantly inhibit bacterial uptake by human neutrophils. After 75 minutes bacterial uptake was six times greater when intact IgG was used as an opsonin (uptake 90.2% +/- 18.6% SEM) compared with a IgG was used as an opsonin (uptake 90.2% +/- 18.6% SEM) compared with a mixture of pseudomonas-lipopolysaccharide-reactive Fab and F(ab')2 fragments generated by pseudomonas elastase (uptake 15.4% +/- 0.8% SEM, p less than 0.001). Hydrolyzed CF IgG antibodies consistently resulted in a level of bacterial uptake less than that of normal saline negative controls (NS): (at 10 minutes, NS 26.6% vs CF 16.8%, p less than 0.05; at 75 minutes, NS 28.2% vs CF 15.4%, p less than 0.01. This suggests that the immune polypeptides are active inhibitors of the essential neutrophil phagocyte-bacterial cell interaction. Intact immune IgG reversed the defect in opsonophagocytosis. When intact IgG was mixed with IgG fragments the phagocytic rates increased directly with increasing amounts of intact IgG. We conclude that the elastase exoproduct secreted by Pseudomonas aeruginosa is capable of cleaving IgG into functionally important fragments that inhibit bacterial uptake. Furthermore, this inhibition can be overcome by increasing amounts of a commercially available preparation of intact immune IgG.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Bacterial↗

PubMed1985

Opsonization of group B Streptococcus type III: studies using clinical strains and maternal sera.

Luminol-dependent phagocytic chemiluminescence was used to measure opsonins to group B Streptococcus type III in serum samples from pregnant women. Mean levels were similar amongst patients colonized with this organism and those who were not. Values remained fairly constant for individual women during pregnancy apart from a small, but consistant fall in cord blood samples. Again using luminol-dependent chemiluminescence, 54 clinical isolates of group B Streptococcus type III were evaluated for susceptibility to serum opsonization. Six were found to be resistant and these originated from both colonized babies and babies with systemic GBS infections. Further studies demonstrated strain-to-strain variation in the degree of dependence on both heat-labile and heat-stable opsonins.

Antibodies, Bacterial↗

PubMed1983

Neutrophil iodination micro-method as an index of neutrophil and opsonic function.

A neutrophil iodination micro-method has been developed for assessment of neutrophil and opsonic function in any serum/micro-organism combination. The test is performed in micro-titre trays and thus requires relatively small amounts of reagents and numbers of cells. The variables of the test have been explored and the effect of immunization with different organisms on opsonin levels defined. The versatility of the test has been demonstrated in its ability to measure both heat labile and heat stable opsonins in normal as well as immune serum, for a number of different micro-organisms. The method is simple, rapid and a large number of tests can be performed at the one time.

Immunologic Techniques↗

PubMed1983

Evaluation of cellular and humoral mechanisms of carbaryl-induced reticuloendothelial phagocytic depression.

The simultaneous injection of carbaryl and colloidal carbon phagocytized by the reticuloendothelial cells results in competition between the two substances in favor of the carbon particles. Experiments with opsonized carbaryl suggest that the decrease in carbaryl blood clearance by the colloid is mediated by a depletion of serum opsonins. Following blockade, the liver carbaryl uptake was depressed in the control group (17%), while it was increased in the opsonized group (12%). With all preparations of carbaryl, opsonized or non-opsonized, colloidal carbon produced a slight and variable increase in carbaryl uptake by the spleen and lungs. These results indicate that, besides the uptake of carbaryl by the hepatocytes, other clearance sites must also be considered such as the Kupffer cells and other liver sinusoidal cells. Moreover our results show that intravenous administration of carbaryl induces a state of phagocytic depression as indicated by impaired intravascular phagocytosis and depressed hepatic uptake of the reticuloendothelial (RE)-test colloidal suspension. The results obtained from injection of opsonized colloidal particles during carbaryl-induced RE-depression, and the fact that carbaryl and carbon are both opsonized by the same serum factor, suggest that the mechanisms of RE-blockade involve selective hepatic and splenic macrophage failure and depletion of serum opsonins. According to our enzymatic investigation, this failure of the RE system to incorporate colloids during carbaryl--RE-blockade could be due to a defect in the activity of macrophage membrane-bound serine esterase.

Animals↗

PubMed1981

Serum opsonic activity in acute protein-energy malnutrition.

Phagocytic host defence mechanisms require both normally functioning cells and humoral factors. For example, activated complement components and/or specific immunoglobulin are essential for effective ingestion and killing of bacteria by neutrophils, and complement is especially important early in infection, before specific antibody has been produced. Abnormalities of serum complement have previously been reported in malnutrition, and the present study investigated the levels of serum opsonins in children with protein-energy malnutrition (PEM).Opsonic activity for Escherichia coli and Staphylococcus aureus was depressed in acute PEM patients, but recovered to higher levels with treatment. This depression was detected only when low concentrations of serum (10-20 ml/litre) were used. Marked and persistent opsonin deficiencies were associated with poor clinical response. Reduced opsonic activity may adversely affect host defence mechanisms and contribute to morbidity and mortality from pyogenic infections in PEM. Replacement therapy with fresh or fresh frozen plasma might restore opsonic activity in these patients and reduce the risk of septicaemia and its attendant high mortality.

Child↗

PubMed1982

Opsonization of encapsulated Staphylococcus aureus: the role of specific antibody and complement.

Previous studies of encapsulated Staphylococcus aureus have shown that the opsonins of normal, nonimmune human serum (complement factor C3 and IgG) bind beneath the capsule, i.e., on the cell wall, and when bound at this site these opsonins are not effective in promoting phagocytosis of the bacteria by polymorphonuclear leukocytes (PMN). In this investigation immune antibody was added to human serum to effect opsonization of encapsulated S. aureus. Opsonization was assessed by quantitating the uptake of 3H-labeled staphylococci by human PMN, and the amount of C3 fixation to bacteria was measured in a quantitative fluorescent immunoassay. Low levels of immune antibody (IgG) effectively opsonized encapsulated S. aureus when added to fresh but not to heated serum; phagocytosis of the staphylococci was mediated via pronase-sensitive membrane receptors (presumably C3b receptors) of PMN. Experiments with C2-, C3-, or C5-deficient human sera revealed that C3 was required for opsonization and that activation of C3 was mediated via the alternative complement pathway. Encapsulated S. aureus bound significantly less C3 than unencapsulated strains in diluted normal serum; addition of immune antibody, however, increased C3 fixation 4.7-fold (p less than 0.005). Immunoelectron microscopy localized C3 throughout the capsule as well as on the staphylococcal cell wall when bacteria had been opsonized in human serum with immune antibody. Without immune antibody, C3 binding was restricted to the cell wall. At approximately 10-fold higher levels of immune antibody, opsonization and phagocytosis of encapsulated S. aureus was independent of complement and pronase-sensitive receptors on PMN. These studies show that, in addition to immune antibody, the alternative pathway of complement plays an important role in the opsonization of encapsulated S. aureus strains and suggest that complement may be crucial to the in vivo clearance of these organisms.

Antibodies, Bacterial↗

PubMedDate not supplied

[The manifestation of functional heterogeneity of the peptidoglycans in opsonophagocytic reactions].

The peptidoglycan activity of Staphylococcus aureus CCM 885 and Bifidobacterium bifidum 1 was studied in the opsonophagocytic system on the basis of the luminol-dependent chemiluminescence of human neutrophils. B. bifidum peptidoglycan was more active in direct interaction with neutrophils. After treatment with normal human serum S. aureus peptidoglycan showed more pronounced neutrophil-stimulating properties. This phenomenon was in accord with a higher content of IgG antibodies to S. aureus peptidoglycan, as well as with its capacity for the classical direct activation of the complement. Moreover, in reactions with S. aureus peptidoglycan "minor" opsonins, independent of antibodies and complement, were detected. B. bifidum peptidoglycan did not activate the complement, could not be opsonized with the serum having no specific antibodies and did not react with "minor" opsonins.

Bifidobacterium↗

PubMed1977

Mediation of CRP-dependent phagocytosis through mouse macrophage Fc-receptors.

The purified acute phase serum constituent, C-reactive protein (CRP), was examined for its ability to interact with mouse macrophage Fc-receptors when serving as an opsonin for C-polysaccharide (CPS)-coated erythrocytes (E.CPS). The ingestion of E.CPS-CRP by macrophage monolayers was dependent on the presence of CRP on the erythrocyte surface and on the treatment of the CRP-sensitized cells with human complement (C). The ingestion of E.CPS-CRP-C was inhibited by exposing the macrophages to either 2-deoxyglucose, a selective inhibitor of Fc-receptor activity, or to aggregated gamma-globulin. Inhibition was also brought about by plating macrophages on glass-bound immune complexes of BSA/anti-BSA (IgG) but not on complexes formed with the F(ab')2 fragment of the anti-BSA. Substrate-bound complexes of CPS-CRP selectively inhibited the uptake of IgG-coated E, EA(IgG); the inhibition was proportional to the concentration of CRP used to form the complex. The opsonin-independent ingestion of latex particles was not altered by these Fc-receptor blocking procedures. These findings coupled with the recent demonstration of a sequence homology between CRP and the CH3 domain of human IgG lead us to propose shared opsonic functions for antibody and CRP.

Animals↗

PubMed1997

Increased phagocyte Fc gammaRI expression and improved Fc gamma-receptor-mediated phagocytosis after in vivo recombinant human interferon-gamma treatment of normal human subjects.

Recombinant human interferon-gamma (rhIFN-gamma) decreases the frequency of serious infections in patients with chronic granulomatous disease (CGD) through an unknown mechanism. To test the hypothesis that it exerts a beneficial effect by enhancing clearance of microbes from the bloodstream and tissues, normal human subjects were treated in vivo with rhIFN-gamma. Phagocyte opsonic receptor expression, serum opsonin levels, and phagocytosis of bacteria were then measured. A 4.7-fold increase in neutrophil expression of the high-affinity Fc gamma-receptor (Fc gammaRI) was observed that peaked 48 hours after the initiation of rhIFN-gamma treatment (P < .05). Monocyte expression of Fc gammaRI, Fc gammaRII, Fc gammaRIII, CD11a, CD11b, CD18, and HLA-DR also significantly increased with peak expression at 48 hours. Phagocytosis by neutrophils of killed Staphylococcus aureus opsonized with heat-inactivated pooled human serum significantly improved after rhIFN-gamma treatment (P < .05) and correlated with Fc gammaRI expression by neutrophils (r = .8, P < .001). This increase in ingestion could be inhibited by anti-Fc gammaRI monoclonal antibodies. Levels of the serum opsonin lipopolysaccharide-binding protein also significantly increased after in vivo rhIFN-gamma (P < .05). These results suggest that the protective effect of rhIFN-gamma in patients with CGD may involve improved microbial clearance. Moreover, improved phagocyte trafficking may occur secondary to increased expression of monocyte beta2-integrins. Because these IFN-gamma-related improvements in host defense were seen in normal hosts, rhIFN-gamma may have broader applications in the treatment of various disorders of immunity in addition to its demonstrated efficacy in CGD.

Acute-Phase Proteins↗

PubMedDate not supplied

Measurement of phagocyte chemiluminescence using a microtitre plate luminometer.

The use of chemiluminescence techniques to study the interaction between bacteria and phagocytes has been useful for examining the extent to which serum factors, such as opsonins, are important in internalization of the organisms and the response of the cell to phagocytosed bacteria. However, such methods have been limited by the number of experiments which can be performed at one time using most commercial luminometers. However, the recent introduction of the Amerlite microtitre plate luminometer allows the measurement of chemiluminescence responses in 96-well microtitre plates. Using this instrument, lucigenin-enhanced chemiluminescence can be detected from as few as 5000 cells (polymorphonuclear leukocytes or monocytes) per well with a 1:10 ratio of cells to zymosan particles opsonized with 10% serum. The opsonic capacity of up to 100 sera can be measured in triplicate wells in a single experiment using four microtitre plates and polymorphonuclear leukocytes prepared from less than 40 ml freshly obtained venous blood. We are currently using this technique to investigate the effect of serum opsonins on the interaction between normal human polymorphonuclear leukocytes and monocytes with mycobacteria of three species (Mycobacterium leprae, M. tuberculosis, and M. avium-intracellulare). Other possible applications of this method are discussed.

Humans↗

PubMed2003

Mannose-binding lectin engagement with late apoptotic and necrotic cells.

The serum opsonin mannose-binding lectin (MBL) has been shown to be involved in the handling of apoptotic cells. However, at what stage in the process this happens and whether this mediates activation of complement is unknown. Cells rendered apoptotic or necrotic were incubated with purified MBL/MBL-associated serine protease (MASP) complexes and assessed by flow cytometry and fluorescence microscopy. MBL bound specifically to late apoptotic cells, as well as to apoptotic blebs and to necrotic cells, but not to early apoptotic cells. Binding of MBL could be inhibited by EDTA as well as with an antibody against the CRD region. Addition of C1q, another serum opsonin involved in the handling of apoptotic cells, prior to MBL partly inhibited MBL binding to apoptotic cells and vice versa. MBL/MASP could initiate deposition of purified complement C4 on the target cells. However, addition of MBL/MASP to whole serum deficient for both C1q and MBL did not enhance deposition of C4, but MBL enhanced phagocytosis of apoptotic cells by macrophages. These results demonstrate that MBL interacts with structures exposed on cells rendered late apoptotic or necrotic and facilitates uptake by macrophages. Thus, MBL may promote non-inflammatory sequestration of dying host cells.

Apoptosis↗