N-truncation and pyroglutaminylation enhances the opsonizing capacity of Aß-peptides and facilitates phagocytosis by macrophages and microglia.

Abnormal accumulations of amyloid-ß (Aß)-peptides are one of the pathological hallmarks of Alzheimer's disease (AD). The precursor of the Aß-peptides, the amyloid precursor protein (APP), is also found in peripheral blood cells, but its function in these cells remains elusive. We previously observed that mononuclear phagocytes release Aß-peptides during activation and phagocytosis, suggesting a physiologic role in inflammatory processes. Here, we show that supplementing the media with soluble N-terminally truncated Aß(2-40) and Aß(2-42) as well as Aß(1-42) induced the phagocytosis of polystyrene particles (PSPs) by primary human monocytes. If the PSPs were pre-incubated with Aß-peptides, phagocytosis was induced by all tested Aß-peptide species. N-terminally truncated Aß(x-42) induced the phagocytosis of PSPs significantly more effectively than did Aß(x-40). Similarly, the phagocytosis of Escherichia coli by GM-CSF- and M-CSF-elicited macrophages as well as microglia was particularly facilitated by pre-incubation with N-terminally truncated Aß(x-42). The proinflammatory polarization of monocytes was indicated by the reduced MSRI expression and IL-10 secretion after phagocytosis of PSPs coated with Aß(1-42), Aß(2-42) and Aß(3p-42). Polarization of the macrophages by GM-CSF reduced the phagocytic activity, but it did not affect the capabilities of Aß-peptides to opsonize prey. Taken together, Aß-peptides support phagocytosis as soluble factors and act as opsonins. Differential effects among the Aß-peptide variants point to distinct mechanisms of interaction among monocytes/macrophages, prey and Aß-peptides. A proinflammatory polarization induced by the phagocytosis of Aß-peptide coated particles may provide a model for the chronic inflammatory reaction and sustained plaque deposition in AD.

Human colostral phagocytes eliminate enterotoxigenic Escherichia coli opsonized by colostrum supernatant.

BACKGROUND: Several elements in colostrum and human milk, including antibodies and nonspecific factors with bactericidal and antiviral activity, may play an important anti-infectious and protective role. In developing countries, enterotoxigenic Escherichia coli (ETEC) is the main etiological agent of diarrhea in low-socioeconomic level children. In the present work, we studied the functional activity of mononuclear (MN) and polymorphonuclear (PMN) phagocytes of human colostrum against ETEC, as well as the interactions between these cells and colostral or serum opsonins. METHODS: Colostrum samples were collected from 33 clinically healthy women between 48 and 72 hours postpartum. We verified superoxide release in colostral MN and PMN using cytochrome C reduction methods, phagocytosis, and bactericidal activity using acridine orange methods and superoxide dismutase (SOD) in the colostrum supernatants. RESULTS: Colostral MN and PMN phagocytes exposed to ETEC opsonized with colostrum supernatants caused a significant increase (p<0.05) in superoxide release. Phagocytosis by colostral PMN cells increased significantly (p<0.5) when the phagocytes were incubated with both sources of opsonins (sera and colostrum). Increases in superoxide release in the presence of opsonized bacteria triggered the bactericidal activity of the phagocytes. Phagocyte treatment with SOD decreased their ability to eliminate ETEC. Colostrum supernatant had higher SOD concentrations (p<0.05) compared with normal human sera. CONCLUSIONS: These results suggest that the ability of phagocytes to eliminate ETEC depends on the activation of cellular oxidative metabolism; moreover, activation of colostral phagocytes is likely an additional breast-feeding protection mechanism against intestinal infections in infants.

Influence of dietary vitamin E on phagocytic functions of macrophages in broilers.

Vitamin E (VE) is known for its antioxidant properties and has been shown to modulate immune system functions in various species. This study examined the influence of different levels of dietary VE (alpha-tocopherol acetate) on phagocytic functions of macrophages (abdominal exudate cells) in broiler chickens at 3, 5, and 7 wk. Birds were fed commercial diets containing 16 (control), 110, or 220 mg of VE/kg of feed. Macrophages were elicited into the abdominal cavity by injecting a 3% Sephadex solution prepared in PBS (G50-50, 1 mL/100 g of BW) 42 h prior to harvest. The percentage of phagocytically active macrophages and the number of SRBC phagocytosed per macrophage for unopsonized and antibody-opsonized SRBC were determined. These aspects of macrophage function were assessed based on 900 macrophages per sample. When unopsonized SRBC were used, dietary VE supplementation above control level did not affect phagocytic function of macrophages at wk 3, 5, or 7. With antibody-opsonized SRBC, the percentage of phagocytically active macrophages and the number of SRBC phagocytosed per macrophage were higher (P = 0.08 and P = 0.01, respectively) in 3-wk-old birds fed 110 and 220 mg of VE/kg of feed compared with age-matched controls. This enhancing effect of VE supplementation on macrophage function was not observed in 5- and 7-wk-old broilers. It appears from this study that supplemental VE enhances Fc-receptor-mediated macrophage phagocytic activity at early stages of broiler growth.

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.

Optimization of nb-4 and hl-60 differentiation for use in opsonophagocytosis assays.

Production of effective vaccine formulations is dependent on the availability of assays for the measurement of protective immune responses. The development and standardization of in vitro human cell-based assays for functional opsonophagocytic antibodies require critical evaluation and optimization of the preparation of cells for the assay. We report evaluation of a number of protocols with two continuous cell lines (NB-4 and HL-60) for the provision of differentiated cells for use in functional assays. Flow cytometric analysis of CD11b antigen expression, as a marker of differentiation, indicated that all-trans-retinoic acid (ATRA) gave improved differentiation (>80% of cells differentiated at 96 h) when compared with dimethylformamide (DMF) (<60% of cells differentiated at 96 h). Morphological changes during differentiation toward a neutrophil-like phenotype were assessed by scanning electron microscopy. HL-60 and NB-4 cells treated with ATRA showed more spreading and flattening than cells treated with DMF, further evidence that they may have achieved a more differentiated phenotype. The number of cell divisions in culture appeared to be critical because cell lines maintained in exponential growth for >40 passages failed to express CD11b antigen or show morphological changes associated with differentiation after exposure to either differentiation-inducing reagent. Late-passage cells also demonstrated increased tolerance to DMF. Our results indicated that ATRA supplemented with vitamin D(3) and granulocyte colony-stimulating factor affords robust, rapid, and reproducible differentiation of both cell types.

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.

Depressed humoral immunity after weight reduction in competitive judoists.

We studied changes in serum opsonic activity (SOA) in male judoists who were engaged in active weight reduction. Serum immunoglobulins, complements and SOA, measured by neutrophil-associated chemiluminescence responses, were investigated 20 days, 7 days and 1 day before a competition and 5 days after the competition. In addition, muscle strength and anaerobic work capacity, as well as body composition, were also determined. A dietary survey was conducted daily during the observation period. Body weight decreased by 4.2 kg over 19 days. SOA significantly decreased 5 days after the competition, as well as the concentrations of serum immunoglobulins, complements and total proteins. These trends were noted in the marked weight reduction group (i.e. reduction weight of body fat/body fat weight before weight reduction > or = 25%) more than the slight reduction group (<25%). Depressed SOA was closely correlated with the decreased concentrations of immunoglobulins and complements. These results suggest that the decrease in immunoglobulins and complements following weight reduction is associated with reduced SOA, which might cause susceptibility to infections. This study demonstrated that such immunosuppression appeared in the recovery period after the competition rather than immediately before the competition.

Colostral neutrophils express Fc alpha receptors (CD89) lacking gamma chain association and mediate noninflammatory properties of secretory IgA.

Colostrum plays an important role in protecting newborn infants against acute gastrointestinal and respiratory infections. IgA antibodies have been considered the major effector component; however, the role of their receptors on colostral phagocytes, especially neutrophils, has not been studied. Here, we demonstrate that CD15+ colostrum neutrophils express IgA Fc receptors (Fc alphaR, CD89) at levels similar to those of blood neutrophils. Most colostral cells (70%) bear secretory IgA (SIgA) on their surface (and intracellularly), whereas blood cells do not. The Fc alphaR on colostral neutrophils was identified as the a.1 isoform with a similar molecular mass (55-75 kDa) as that identified for blood neutrophils. Removal of N-linked carbohydrates revealed a major protein core of 32 kDa for both cell types. In contrast, co-immunoprecipitation and immunoblot experiments using a mild detergent, digitonin, revealed a lack of gamma chain association with Fc alphaR (gamma-less) exclusively on colostral neutrophils. The functional role of these gamma-less Fc alphaR cells was evaluated by measuring superoxide release and killing of SIgA-coated enteropathogenic E. coli. No increase in superoxide release was observed in colostral cells compared with blood neutrophils, whereas optimal release was obtained with PMA stimulation. Furthermore, despite similar bacterial phagocytosis index between both cell types, IgA-mediated bacterial-killing was not detectable with colostral neutrophils, whereas killing was detectable on blood cells. These results reveal exclusive expression of gamma-less Fc alphaR on colostral neutrophils associated with receptor hyperoccupation by IgA and with low, bacterial-killing activity, which suggest that this receptor may mediate noninflammatory effects of SIgA.

Opsonic capacity of foal serum for the two neonatal pathogens Escherichia coli and Actinobacillus equuli.

Two of the most commonly isolated foal pathogens are Escherichia coli and Actinobacillus equuli. The hypothesis tested in this study was that young foals carry a lower opsonic capacity for these bacteria compared to adult horses. A flow-cytometric method for the phagocytosis of these by equine neutrophils was established. The opsonic capacity of serum from healthy foals from birth to age 6 weeks was evaluated and related to the concentrations of IgGa and IgGb. Phagocytosis of yeast was used as a control. Serum was required for phagocytosis, with higher concentrations for E. coli than for A. equuli. Ingestion of colostrum led to a significantly higher serum opsonic capacity. After that, there was no consistent age-related trend for opsonic capacity for the different microbes. Foal serum showed similar or higher opsonisation of E. coli and A. equuli compared to serum from mature individuals. During the studied period, the predominance among IgG subisotypes switched from IgGb to IgGa. Although the overall correlation between concentrations of IgG subisotypes and serum opsonic capacity was poor, sera with IgGb levels below 1.9 mg/ml induced lower opsonisation of E. coli and yeast, but not of A. equuli. Complement activation was important for opsonisation of all tested microbes. The results of this study are significant to the understanding of a key immunological facet in the pathophysiology of equine neonatal septicaemia in clinical practice.

Compensation of preliminary blood phagocyte immaturity in the newborn calf.

To estimate the functional maturity of the phagocytic defence in neonatal calves, we analyzed the characteristics of blood phagocytes from calves (n = 10) 1 h post partum (p.p.) and 4 h p.p. At 1 h p.p., all calves were colostrum-deprived, while 5 calves had received colostrum before the 4 h p.p. sampling. The results were compared to those obtained from 3-9-week-old calves (n = 10). Phagocytic and oxidative burst activity of polymorphonuclear leukocytes (PMNL) and monocytes were determined in whole blood and separately analyzed by flow cytometry. In neonates prior to colostrum ingestion (1 h p.p.), phagocytic activity of PMNL against non-preopsonized E. coli was lower when compared to PMNL of 3-9-week-old calves. Opsonization of bacteria with pooled plasma from adult animals only partially restituted this lower PMNL phagocytic activity, indicating that humoral as well as cellular aspects of PMNL phagocytosis are altered in neonatal calves. In contrast to PMNL, monocytes of neonates exhibited an enhanced phagocytic activity. The oxidative burst activity of PMNL, as well as of monocytes was higher in newborn calves. During the first 4 h of life, the activities of blood phagocytes changed. Colostrum ingestion was accompanied by an increase in the percentage of phagocytizing PMNL and monocytes. This increase was absent in colostrum-deprived calves. In contrast, the oxidative burst activity of phagocytes decreased with age. In monocytes, the decrease of oxidative burst activity was only observed in colostrum-fed calves. In conclusion, some blood phagocyte functions in calves were found to be immature at birth, but these functions are presumably compensated by high absolute PMNL numbers and by other the more active mechanisms.

Effects of growth hormone and insulin-like growth factor I on opsonin receptor expression on local and systemic phagocytes in a lethal peritonitis model.

OBJECTIVE: To investigate the effects of pretreatment with growth hormone (GH) and insulin-like growth factor I (IGF-I) on phagocyte exudation and bacterial clearance, focusing on CD11b and CD32/CD16 expression on local and systemic phagocytes, in a lethal peritonitis model. DESIGN: Prospective randomized experimental study. SETTING: Research laboratory in a university hospital. SUBJECTS: Balb/c mice (n = 21). INTERVENTIONS: Mice were challenged intraperitoneally with 1 x 10(8) Escherichia coli, after 6 days of pretreatment with saline (control), GH (4.8 mg/kg/day), or IGF-I (24 mg/kg/day). Samples were harvested at 4 hrs after the challenge. MEASUREMENTS AND MAIN RESULTS: Viable bacterial counts in peritoneal lavage fluid (PLF) and blood were determined. Peritoneal exudative cells and peripheral blood leukocytes were counted and analyzed for receptor expressions by flow cytometry. GH reduced viable bacterial counts in PLF, as compared with the saline control. GH (three-fold) and IGF-I (two-fold) increased the number of peritoneal exudative neutrophils (PENs), as compared with the saline control. The number of PENs showed an inverse correlation with PLF viable bacterial counts. By contrast, there were no differences in peripheral blood neutrophil (PN) counts among the three groups, nor was there a correlation between PN and PEN counts. CD11b expression was greater on PENs than on PNs in all three groups. CD11b expression on PNs did not differ among the three groups. However, GH increased CD11b expression on PENs, as compared with saline and IGF-I, and this expression showed a positive correlation with PEN numbers and an inverse correlation with PLF viable bacterial counts. CD11b expression on peritoneal macrophages and peripheral blood monocytes did not differ among the three groups. There were no differences in phagocyte CD32/CD16 expression among the three groups. CONCLUSIONS: GH pretreatment enhanced CD11b expression on PENs, but not PNs, possibly in association with enhanced neutrophil recruitment, phagocytosis, and bacterial elimination by PENs, without activation of PNs. GH prophylaxis may be useful for reducing the frequency rate and severity of septic complications, via modulation of CD11b expression on local and systemic neutrophils.

Opsonic activity of bovine serum and mammary secretion after Escherichia coli J5 vaccination.

Six pairs of cows were used to determine the effects of immunization with an Escherichia coli (O111:B4) J5 bacterin on in vitro opsonization of a smooth heterologous strain of E. coli. One cow in each pair was either immunized with the vaccine or sham-immunized at drying off, 30 d after drying off, and at calving. Opsonizing bacteria with serum collected from vaccinated cows 21 d after calving resulted in higher mean number of intracellular bacteria per phagocytosing neutrophil than opsonizing bacteria with serum collected from control cows. Phagocytic parameters using serum collected at drying off and calving did not differ between treatment groups. A trend for enhanced opsonic activity of colostrum from vaccinates was noted. Enhanced opsonization by serum from vaccinated cows coincided with higher serum IgM titer to E. coli J5 whole cell antigen compared with controls. Serum IgG titers to E. coli J5 did not differ between groups. Colostrum IgG titers to E. coli J5 were greater at calving in vaccinated than in control cows. Colostrum and milk collected 21 d after calving from vaccinated cows had higher IgM titers to E. coli J5 than did mammary secretions from control cows. Numbers of intracellular bacteria per phagocytizing neutrophil were correlated positively with IgM titers to E. coli J5 in both serum and colostrum.

Serum opsonic activity and neutrophil phagocytic capacity of newborn lambs before and 24-36 h after colostrum uptake.

Neutrophil (PMN) counts, immune complex (IC) uptake by PMN, and serum opsonising activity for promoting yeast uptake were used to evaluate infection clearing capacity in 16 lambs prior to colostrum feeding (two lambs fed bovine colostrum, 14 suckled lambs) and at 2 days of age. At 2 days of age lambs had more circulating PMN than they had prior to colostrum uptake (P less than 0.01). Colostrum feeding caused a significant increase in the percent of lamb PMN phagocytosing IC, although at Day 2 the percent phagocytosis was significantly lower (32.2%) than for adult controls (90%). Yeast opsonophagocytosis was greater when 24-36 h post-feeding serum was the source of opsonin than when pre-feeding serum was used (P less than 0.001). When adult serum was the opsonin, yeast opsonophagocytosis was approximately twice the phagocytosis mediated by 24-36 h post-feeding serum. The peripheral neutrocytosis and the enhancement of opsonophagocytosis generated by absorption of either ovine or bovine colostrum did not differ. The results of this study suggest that the parameters evaluated may be used for indicating the presence (or absence) of passively acquired protective immunity.

Beneficial effects of aggressive protein feeding in severely burned children.

To determine any potential benefit of feeding increased amounts of protein to hypermetbolic burned patients, 18 children with burns averaging 60% total surface area were randomized into two matched groups and studied serially for at least six weeks: the first group was given a normal diet with a balanced nutritional supplement, and the second group was supplemented with milk whey protein. The normal protein group received 87.1% of their desired caloric intake with 16.5% of calories from protein compared to 77.7% of desired caloric intake with 23.0% of calories from protein for the high protein group. Despite a higher caloric intake, the normal protein group had a worse opsonic index compared to the high protein group (0.42 +/- 0.04 vs. 0.62 +/- 0.05, p < 0.0007), lower levels of C3 (1371 +/- 55 vs. 1585 +/- 64 micrograms/ml, p < 0.01), lower levels of IgG (805 +/- 52 vs. 975 +/- 56 micrograms/ml, p < 0.03), lower levels of transferrin (200 +/- 10 vs. 283 +/- 18 mg/dl, p < 0.0001), lower levels of total serum protein (5.5 +/- 0.1 vs. 6.3 +/- 0.2 g/dl, p < 0.005), more bacteremic days (11% vs. 8%, p < 0.005) and worse survival (5/9--56% vs. 9/9--100%, p < 0.03). Patients receiving the high protein diet had significantly higher plasma levels of valine, lysine, threonine, leucine, aginine, isoleucine, proline, serine, asparagine, tryptophane, and tyrosine. Asparagine levels were significantly (p < 0.01) associated with better neutrophil function and opsonic index. Except for phenylalanine, significant associations were found for serum levels of each of the amino acids with concentrations of one or more serum proteins. These studies provide evidence that many immunologic functions are dependent upon optimal availability of specific amino acids, and that routine diets do not provide sufficient protein to satisfy the needs of seriously burned children.