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PubMed1974

Corticosteroids, serum, and phagocytosis: in vitro and in vivo studies.

The phagocytic-bactericidal capacity (PBC) of human polymorphonuclear leukocytes (PMNs) for strains of Klebsiella (K) and of Enterococcus (E) was unaffected in vitro by the presence of 100 mug of either hydrocortisone (HC) or of methylprednisolone (MP) per ml in the medium. At higher concentrations (500 to 2,000 mug/ml) both compounds impaired PBC-K and PBC-E, but the latter was less sensitive to steroid-induced inhibition. In addition to interfering with intracellular killing of both organisms by PMNs, 2,000 mug of HC per ml also inhibited ingestion of E, although not of K. Steroid-induced inhibition of PBC-K in vitro was completely abolished by increasing the concentration of serum used as opsonin. The PBC-K of human PMNs obtained 30 min after intravenous injection of 1 g of MP was unimpaired in vitro in the presence of 10 to 90% simultaneously obtained autologous serum containing 42 mug of MP per ml. These findings suggest that short-term, high-dosage administration of MP is unlikely to produce clinically significant impairment of intraleukocytic bacterial killing.

Blood↗
PubMed1990

Phagocytosis of mastitis isolates of Staphylococcus aureus and expression of type 5 capsular polysaccharide are influenced by growth in the presence of milk.

Phagocytosis by bovine polymorphonuclear granulocytes of seven capsular polysaccharide type 5 Staphylococcus aureus strains isolated from mastitis [corrected] was investigated by means of luminol-dependent chemiluminescence. Bacteria were grown on four different agar media (brain heart infusion, Columbia broth, modified staphylococcus medium 110, and skim milk) and were opsonized by normal bovine serum. When compared to growth on brain heart infusion agar, Columbia agar, and modified staphylococcus medium 110 agar, growth on skim milk agar rendered five of the strains more resistant to opsonization. The other two strains were resistant in all culture media used. Short periods of incubation in milk after growth on brain heart infusion agar did not augment resistance to phagocytosis, indicating that mere adsorption of milk components on bacteria was not responsible. The variability of the chemiluminescence response of polymorphonuclear leukocytes was pronounced among strains with each growth medium except milk. Growth on modified staphylococcus medium 110 and on milk agar favored the masking of teichoic acid, as shown by inagglutinability with rabbit antiserum. Interestingly, agglutination by a monoclonal antibody to capsular polysaccharide type 5 was optimal when bacteria were grown on skim milk agar. This suggests that capsular polysaccharide participated in the masking effect. These findings indicate that masking of the bacterial target of most of the naturally acquired opsonins present in normal bovine serum occurred when bacteria grew in the presence of milk, resulting in an increased resistance to phagocytosis by polymorphonuclear leukocytes.

Animals↗
PubMed1991

Protein X of Streptococcus agalactiae induces opsonic antibodies in cows.

Protein X of Streptococcus agalactiae is a surface protein frequently associated with strains isolated from cases of mastitis of dairy cows. By immunizing cows with purified protein X, we obtained an antibody response which was restricted to X-bearing strains of S. agalactiae in a whole-cell enzyme-linked immunosorbent assay. This response resulted in an increase in the opsonic activity of serum for strains bearing protein X, as assessed through the augmentation of the chemiluminescence response of phagocytosing polymorphonuclear cells and through an increased ingestion of bacteria, although the proportion of ingested bacteria which were killed (about 60%) remained unchanged. Protein X behaved as a target of opsonins and, as such, could be a protective antigen worth incorporating in a vaccine against S. agalactiae mastitis.

Animals↗
PubMed1984

Fibronectin as an opsonic regulator of monocyte phagocytosis.

Experiments using human monocytes and the yeast Saccharomyces cerevisiae have shown that fibronectin is a major plasma opsonin. Further studies have shown that fibronectin promotes the ingestion as well as the adherence of micro-organisms. These observations are independent of the non-physiological concentrations of heparin used in other assay systems.

Fibronectins↗
PubMed1991

Role of mesothelial cells in peritoneal antibacterial defence.

Whether phagocytosis of Staphylococcus aureus by polymorphonuclear neutrophils, monocytes, and peritoneal macrophages takes place when the staphylococci are adherent to monolayers of human mesothelial cells in the absence of opsonins was investigated. Adherence of S aureus to mesothelial monolayers increased significantly when the bacteria were opsonised with pooled human serum, but phagocytosis by polymorphonuclear neutrophils and monocytes occurred independently. Phagocytosis by peritoneal macrophages, however, was only marginal. Pretreatment of polymorphonuclear neutrophils with inhibitors of cellular metabolism and motility reduced their phagocytic capacity. These results indicate that the surface of mesothelial cells provides favourable conditions for the elimination of staphylococci in the peritoneal cavity. Phagocytic motility seems to be important in surface phagocytosis. The inability of peritoneal macrophages to phagocytise staphylococci adherent to the mesothelial cells suggests that they contribute little to the antibacterial defence of the peritoneal membrane of patients receiving peritoneal dialysis.

Cells, Cultured↗
PubMed1993

Bacteraemia due to recurrent reinfection with Staphylococcus epidermidis associated with defective opsonisation and procidin function in serum.

AIMS: To differentiate between reinfection and relapsing infection with Staphylococcus epidermidis in a middle-aged woman with defective opsonisation and procidin function in serum. METHODS: Microbiological typing was done by biotyping, phage typing, and polyacrylamide gel electrophoresis of radiolabelled bacterial proteins (radioPAGE method). Polymorphonuclear cell function was assessed in vitro by phagocytosis and killing of Candida albicans; measurement of neutrophil random locomotion and chemotaxis; reduction of nitroblue tetrazolium after stimulation by opsonised Candida and a radiometric saccharomyces opsonisation assay. The effect of plasma infusions on opsonic activity was assessed by chemiluminescence using control polymorphonuclear leucocytes with a laboratory strain of S epidermidis opsonised with either patient or control serum. RESULTS: Recurrent reinfection with different strains of Staphylococcus epidermidis rather than relapsing infection was confirmed as having occurred by typing bacterial strains. The RadioPAGE method detected all the S epidermidis strains involved in this patient's illness. The patient's serum was shown to be defective in both opsonin and procidin function. The defects were correctable in vitro by the addition of normal serum. Clinical recovery occurred after repeated infusions of normal fresh frozen plasma and prolonged antibacterial treatment; antibacterial treatment alone was insufficient. CONCLUSIONS: The radioPAGE method is useful in distinguishing recurrent reinfection with S epidermidis from relapsing infection with this organism. Elucidation of the nature of, and underlying predisposition to, infection in the patient studied allowed a rational treatment plan of plasma infusion combined with antibacterial treatment to be devised which ultimately proved successful.

Autoradiography↗
PubMed1987

New microbial and host factors in disseminated gonococcal infection: case report.

Disseminated gonococcal infection was diagnosed in an immunocompromised patient who presented with oliogoarthropathy and tenosynovitis. The gonococcal isolate was prototrophic, showed intermediate resistance to penicillin, and belonged to serogroup WII/III. An isolate from the patient's sexual contact showed similar characteristics. The patient had a Saccharomyces opsonin defect, which is associated with childhood infections and has not been reported previously in association with disseminated gonococcal infection. The pathogenetic importance of the unusual isolate and the underlying host defence defect is considered.

Adolescent↗
PubMed1993

Effects of recombinant GM-CSF and IgA opsonisation on neutrophil phagocytosis of latex beads coated with P6 outer membrane protein from Haemophilus influenzae.

BACKGROUND: IgA is the major antibody class in mucosal secretions, yet its biological functions remain poorly understood and its role as an opsonin for neutrophils has been the subject of controversy. It has been reported that treatment of neutrophils with granulocyte-macrophage colony stimulating factor (GM-CSF) induces the cells to phagocytose particles opsonised with IgA. A study was performed to investigate the effects of GM-CSF and IgA opsonisation on the ability of human neutrophils to recognise and phagocytose latex beads coated with the P6 outer membrane protein of Haemophilus influenzae. METHODS: Human neutrophils with and without preincubation with 100 pmol/l GM-CSF, were incubated with non-opsonised P6-coated latex beads or beads opsonised with IgA purified from the blood of a bronchiectatic patient with high titres of IgA anti-P6. Phagocytosis was measured by counting internalised beads during microscopic examination. RESULTS: The phagocytosis of IgA opsonised beads by untreated neutrophils (mean (SE) 2.1 (0.43) beads/cell) was significantly greater than that of non-opsonised beads (mean (SE) 1.3 (0.30) beads/cell). Treatment of neutrophils with GM-CSF resulted in increased phagocytosis of non-opsonised beads (mean (SE) 2.1 (0.39) beads/cell) but opsonisation with IgA increased this further (mean (SE) 3.4 (0.53) beads/cell). CONCLUSIONS: Human neutrophils recognise and phagocytose non-opsonised particles coated with bacterial antigen. Antibodies of the IgA isotype opsonise for neutrophil phagocytosis of particles coated with bacterial antigen but this behaviour is enhanced, in an additive fashion, by treatment of the cells with GM-CSF. The results suggest that IgA and GM-CSF are important cofactors for neutrophil recognition and elimination of bacterial pathogens.

Bacterial Outer Membrane Proteins↗
PubMed1989

Effects of selected carbohydrates and the contribution of the prophenoloxidase cascade system to the adhesion of strains of Pseudomonas aeruginosa and Proteus mirabilis to hemocytes of nonimmune larval Galleria mellonella.

The adhesion of strains of Pseudomonas aeruginosa and Proteus mirabilis to the plasmatocytes and granular cells of nonimmune larval Galleria mellonella was influenced by and varied with the type of carbohydrate. Laminarin enhanced prophenoloxidase activation and bacterial adhesion to the hemocytes whereas sucrose suppressed both activities. For all other sugars there was no correlation between bacterial adhesion to the hemocytes and phenoloxidase activity. It is proposed that bacterial adhesion to the hemocytes may be mediated by both lectinlike binding and components of the prophenoloxidase activating system acting like opsonins.

Animals↗
PubMed1977

Electroimmunoassay of alpha-2-opsonic protein during reticuloendothelial blockade.

Physiological regulation of reticuloendothelial (RE) phagocytic activity by a plasma opsonic factor has been documented. In the recent study, serum levels of this alpha-2-opsonic protein in rats during colloid-induced RE blockade were measured utilizing an electroimmunoassay (Rocket immunoelectrophoresis) with monospecific antiserum to the purified alpha-2-glycoprotein. RE blockade was produced by the intravenous injection of the gelatinized "RE-test-lipid emulsion" at a dose of 50 mg/100 g body wt. The opsonic activity of serum at various intervals during colloid-induced RE blockade as measured by tissue slice bioassay manifested a high correlation (r = 0.98) with the serum opsonic protein concentration as measured by the electroimmunoassay. During RE blockade (30 min), there was a rapid depletion of the opsonic alpha-2-glycoprotein to 20% of the initial preinjection levels. Serum concentration of this glycoprotein remained low for at least 2-3 h after which time its concentration progressively increased with approximation of normal values by 6 h postblockade. Opsonic protein concentration at 24 h postinjection were significantly (P less than 0.05) elevated above controls. Thus, colloid-induced RE blockade is associated with the removal of this glycoprotein from the serum and recovery from RE blockade is accompanied by a restoration of opsonin levels. The electroimmunoassay can provide a sensitive technique to monitor this humoral factor known to exert a physiological control on the RE system.

Alpha-Globulins↗
PubMed1994

Bactericidal/permeability-increasing protein ameliorates acute lung injury in porcine endotoxemia.

Bactericidal/permeability-increasing protein (BPI), a cationic protein isolated from human neutrophils, binds lipopolysaccharide (LPS), kills gram-negative bacteria, and neutralizes many of the effects of LPS in vitro and in vivo. We hypothesized that a recombinant 23-kDa NH2-terminal fragment of BPI (BPI23) would reduce acute lung injury in endotoxemic pigs. At -18 h, pigs received an intravenous priming dose of LPS (20 micrograms/kg). Anesthetized ventilated swine were randomized to receive 1) no further treatment (n = 4); 2) LPS (250 micrograms/kg over 50 min) and BPI23 (3-mg/kg bolus and 3 mg/kg over 60 min) (n = 6); or 3) LPS and thaumatin, a cationic protein devoid of LPS neutralizing activity that has a molecular mass and isoelectric point that are similar to that of BPI23 (n = 7). BPI23 treatment significantly ameliorated LPS-induced hypoxemia, functional upregulation of opsonin receptors on circulating phagocytes, and alveolitis but had no effect on the elaboration of tumor necrosis factor-alpha or thromboxane A2. The salutory effects of BPI23 on acute lung injury in endotoxemic pigs may be mediated, at least in part, by inhibition of direct activation of phagocytes by LPS.

Animals↗
PubMed1977

Evaluation of nonspecific (alternative pathway) opsonic activity by neutrophil chemiluminescence.

The role of alternative complement pathway activation in protection against infection is not well understood. We have investigated nonspecific opsonic activity in human adult, term neonatal and premature serum using the technique of neutrophil chemiluminescence (cl) to measure particle uptake. Following phagocytosis, neutrophils become metabolically active, produce excited molecular oxygen and emit a burst of light which can be detected and quantitated in a liquid scintillation counter. In the present study, zymosan particles were preopsonized in human serum and added to control neutrophils. Particles opsonized in adult serum produced a marked peak in CL. Serial dilutions of the serum prior to opsonization yielded proportionally lower peaks in CL. Opsonic activity as measured by the CL procedure was completely blocked by the addition of chelating agents which remove calcium and magnesium ions and block both classic and alternative complement pathway activation. Addition of an excess of magnesium ions (needed for alternative pathway activation) to the reaction mixture partially restored the opsonic activity. Opsonic activity as measured by the CL procedure was significantly depressed in approximately two-thirds of term and premature infant sera tested. Deficient nonspecific opsonic activity was closely correlated with serum levels of C3PA. These studies suggest that the CL procedure may be of value in the investigation of nonspecific opsonins in human serum.

Adult↗
PubMed1991

Human neonatal monocyte-derived macrophages and neutrophils exhibit normal nonopsonic and opsonic receptor-mediated phagocytosis and superoxide anion production.

Human neonates are at risk for invasive infections with fungi and gram-negative bacteria, microorganisms which are often susceptible to phagocytosis in either the presence or the absence of opsonins. Although opsonic phagocytosis has been investigated, to date there have been no systematic studies of nonopsonic phagocytosis in neonates. We investigated receptor-mediated phagocytosis by neonatal neutrophils and monocyte-derived macrophages in order to determine if a functional defect exists in any of the classes of nonopsonic or opsonic receptors. Ingestion or binding by mannosyl/fucosyl, Fc and complement receptors and the receptor(s) for unopsonized Pseudomonas aeruginosa were normal in neutrophils and macrophages from the cord blood of 26 healthy full-term neonates as compared to simultaneous normal adult controls. Superoxide anion production by neonatal neutrophils in response to both soluble and particulate stimuli was also normal. We conclude that neonatal nonopsonic and opsonic receptor-mediated phagocytosis are normal.

Adult↗
PubMed1996

Alveolar macrophage uptake of the environmental particulate titanium dioxide: role of surfactant components.

Pulmonary surfactant components can modulate uptake of microorganisms and viruses by alveolar macrophages (AMs), but little is known about their role in the uptake and clearance of inert environmental particulates. We tested the hypotheses that surfactant components [e.g., surfactant protein A (SpA) and the artificial bovine surfactant Survanta] modulate phagocytosis of inert environmental particulates by acting as particle opsonins, or by direct activation of AMs. AM uptake of a model inert particulate [titanium dioxide (TiO2)] was measured using flow cytometry to quantitate increased right angle scatter caused by particle uptake (e.g., fold increase in right angle scatter versus control: 2.6 +/- 0.3; and 5.0 +/- 0.2 for AMs plus TiO2, 20 and 80 micrograms/ml TiO2, respectively). Opsonization of TiO2 with surfactant components resulted in a modest increase in AM uptake compared with that of unopsonized TiO2 [e.g., fold increase, uptake of TiO2 (50 micrograms/ml), opsonized with SpA, Survanta, and rat immunoglobulin G, respectively: 1.6 +/- 0.1; 1.3 +/- 0.01; 1.5 + 0.02, n = 3-4]. Uptake of inert latex beads was similarly enhanced after opsonizing with SpA and Survanta (beads per cell: unopsonized, 3.2 +/- 0.40; SpA, 5.0 +/- 0.55; Survanta, 6.0 +/- 0.12; n = 3-6). Pretreating AMs with surfactant components and measuring the subsequent uptake of unopsonized TiO2 resulted in approximately the same magnitude of enhancement. The data indicate that surfactant components can increase AM phagocytosis of environmental particulates in vitro, but only slightly relative to the already avid AM uptake of unopsonized particles.

Animals↗
PubMed1977

Influence of a deficiency of the second component of complement on the bactericidal activity of neutrophils in vitro.

Serum from three patients with a complete, selective deficiency of the second component of complement (C2) did not promote optimal killing of Staphylococcus aureus, 502A by neutrophilic polymorphonuclear leukocytes (PMN) in vitro. The addition of C2 reagent or the presence of heat-stable opsonin in the C2-deficient serum corrected the defective killing of S. aureus that was observed with patient or control PMN. PMN from the patients or control subjects killed bacteria with equal efficiency under conditions of optimal opsonization (normal pooled serum). However, twice-washed control PMN were better than patient PMN in killing S. aureus under circumstances of suboptimal opsonization (C2-deficient serum, heated C2-deficient serum, heated normal pooled serum, or no replacement of serum). The latter finding was due to residual C2 on the surface of twice-washed control cells. As repeated washing of control PMN progressively removed cell-associated C2, the staphylocidal effectiveness of the control RMN decreased to the level of patient PMN. In contrast to the findings with S. aureus, triply-washed PMN from patients or controls killed normal numbers of Escherichia coli, ON2, in C2-deficient serum.

Animals↗
PubMed1981

Role of cell-generated hydrogen peroxide in granulocyte-mediated killing of schistosomula of Schistosoma mansoni in vitro.

Human as well as murine granulocytes have been shown to kill the larval stages of helminth parasites; the mechanism of this cell-mediated cytotoxicity is, however, poorly understood. The present study was designed to assess the role of peroxidative processes in killing of schistosomula of Schistosoma mansoni by human granulocytes in vitro. The rate of H(2)O(2) production by human neutrophils, eosinophils, and basophils was measured upon incubation with schistosomula alone or in the presence of specific antibody or complement. Opsonized parasites (antibody and/or complement) increased the rate of H(2)O(2) production by neutrophils, eosinophils, and basophils by respective percentages of 500, 500, and 371. The rate of H(2)O(2) release was directly related to the number of granulocytes and to the proportion of cells attached to the surface of the schistosomula. Increased hydrogen peroxide release occurred by 10 min of incubation and was demonstrable up to 16 h after addition of leukocytes to schistosomula. The primary source of this oxygen product was found to be the granulocytes adherent to the schistosomula and not those that remained unattached. Hydrogen peroxide production by neutrophils and eosinophils was quantitatively similar (schistosomula coated with antibody plus complement stimulated 5 x 10(6) neutrophils and eosinophils to release H(2)O(2) at respective rates of 0.35 and 0.40 nmol/min). Granulocyte-mediated parasite killing correlated with rate of H(2)O(2) generation; both processes were inhibited by catalase. To define further the role of oxidative metabolites, neutrophils and eosinophils of two subjects with chronic granulomatous disease were used; marked reduction of granulocyte-mediated parasite mortality was observed. Peroxidase was required for H(2)O(2)-mediated killing. Addition of the peroxidase inhibitors azide (1 mM), cyanide (1 mM), or aminotriazole (1 cM) to neutrophilschistosomula mixtures significantly reduced parasite cytotoxicity (P < 0.01); similar reduction was observed when eosinophils were used (P < 0.01). Fixation of halide (iodide) to trichloroacetic acid-precipitable protein (2.4-6.0 nmol/h per 10(7) neutrophils) was demonstrated in the presence of granulocytes, opsonins, and parasites; this process was completely inhibited by 1 mM azide. These data indicate that contact between the surfaces of human granulocytes and schistosomula results in release of cellular hydrogen peroxide and iodination. The generation of H(2)O(2) and its interaction with peroxidase appear to be crucial in effecting in vitro granulocyte-mediated parasite cytotoxicity.

Animals↗
PubMed2006

Bypassing complement: evolutionary lessons and future implications.

Lectins like mannan-binding protein are part of the innate immune system. They circulate in association with serine proteases. Upon binding oligosaccharides, they activate the complement cascade analogous to the more familiar but evolutionarily more recent classical pathway, which is triggered by antibody binding to antigen. In this issue of the JCI, Selander et al. developed a sensitive and specific ELISA employing Salmonella-specific sugars to assess the activity of the lectin pathway of complement activation (see the related article beginning on page 1425). This more physiologic assay system allowed the investigators to rigorously define the requirements for lectin pathway activation. Furthermore, they uncovered an unsuspected means for this pathway to reach the desired critical step of activation of the opsonin C3. These types of functional assays will eventually replace the more laborious, less physiologic, and less informative approaches currently in use to monitor complement activation.

Animals↗
PubMed1991

Activation of complement by Treponema denticola.

Oral spirochetes have been shown to be associated with periodontal diseases and are present in increased numbers in lesions of greater severity. In this study, the interaction of Treponema denticola with human complement, a major antibacterial defense system, was examined. For each of two strains of T. denticola, it was found that both the classical and alternative pathways of human complement were activated in human serum upon incubation at 37 degrees C. C3 fragments were deposited on the surface of this organism following complement activation; the fragments bound included both of the major C3-derived opsonic fragments C3b and iC3b. Under incubation conditions identical to those carried out for complement activation in serum, T. denticola failed to degrade purified, hemolytically-active C3, although it readily degraded inactivated C3. Thus, despite the documented proteolytic activity of this organism, complement activation and deposition of complement-derived opsonins may be important defense mechanisms in the control of infections with T. denticola.

Complement C3↗