Opsonization of yeast cells with equine iC3b, C3b, and IgG

The main opsonins in serum are antibodies and complement factor C3. The opsonization mechanisms including complement activation and deposition are important in studies of phagocytosis and of mechanisms of microbial immune evasion. The objective of the present study was to monitor the deposition of complement C3 and IgG from equine serum on yeast cells (Saccharomyces cerevisiae) using a flow cytometric immunoassay. Correlations were made between the opsonic coating and phagocytic capacity using equine blood neutrophils. In addition, the bound C3 fragments were characterized by SDS–PAGE and Western blot analyses.

Opsonic coating of yeast with equine C3 and IgG occurred rapidly with detectable levels with as little as 0.75% serum. C3 deposition was a result of complement activation and no passive adsorption was observed. When complement was inactivated, the fluorescence indicating IgG deposition increased 3–6-fold, indicating spatial competition between C3 and IgG at binding.

Opsonization with 1.5% serum led to suboptimal equine neutrophil phagocytosis of yeast cells which was dependent on complement activation by the classical pathway. With ≥6.25% serum, IgG contributed to opsonization and phagocytosis. With 50% serum and more, C3 was deposited also by the alternative pathway. Phagocytosis rates became optimal with 3% serum, and did not increase further with higher serum concentrations. The main form of C3 on the yeast cells was iC3b and the rest was C3b without any detectable breakdown products (C3c or C3dg). The equine complement components are similar in size to the human equivalents.

It may be concluded that opsonization of yeast particles leading to phagocytosis, occurs at very low serum concentrations (1.5%) and that it is dependent on activation of the classical complement pathway at this low opsonic level. This is an important finding for efficient host defense, e.g. extravascular phagocytosis at infection sites.     

Relative importance of Salmonella-specific antibody isotypes in phagocytosis of Salmonella typhimurium by ovine mammary neutrophils

The comparative opsonic efficiency of ovine salmonella-specific antibody isotypes was determined by measurement of specific phagocytic uptake of opsonised virulent Salmonella typhimurium by ovine mammary neutrophils. An in vitro phagocytosis assay revealed that IgM was superior to IgG2 in promoting the phagocytosis of opsonised virulent organisms. IgG1, on the other hand, was non-opsonic. Superiority of the IgM isotype over IgG2 as an opsonin was also evident in studies on the viability of opsonised S typhimurium upon phagocytosis. It was revealed that the percentage of organisms killed was appreciably greater when opsonisation was carried out with the IgM than with the IgG2 isotype, although after ingestion by neutrophils there was essentially no difference in the efficiency with which the ingested organisms were killed.     

Influence of age and plasma treatment on neutrophil phagocytosis and CD18 expression in foals

The aim of this study was to evaluate the influence of age and plasma treatment on neutrophil phagocytosis, CD18 expression and serum opsonic capacity in foals in field settings. Microbial infections constitute a large threat in young foals and neutrophil functions are crucial for the defense. Blood samples were obtained from 13 foals at seven time points between the ages of 2 and 56 days and once from 16 adult horses. Six of the foals were treated with adult plasma at the age of 1 week. Neutrophil phagocytosis of yeast after various opsonizations and the expression of complement adhesion receptor CD18 were analysed by flow cytometry. Autologous serum opsonization resulted in 52+/-6.1% phagocytic neutrophils in 2-day-old foals (n = 12), a significantly lower rate than in adult horses (mean 84+/-3.1%; n = 16). In foals, yeast ingestion per neutrophil was also lower than in adults. Opsonic capacity increased with age (p < 0.05), reaching adult levels at 3-4 weeks. An increase in serum opsonic capacity followed plasma treatment (p < 0.05). The phagocytic capacity of foal neutrophils at the time-points studied was equal to or higher than that in the adults, when pooled adult horse serum or anti-yeast IgG was used as opsonin. In foals, serum IgG concentration was negatively correlated to serum opsonic capacity. CD18 receptor expression was higher in neutrophils from foals (<21 days old) than in those from adult horses (p < 0.05). The results indicate that foals are transiently deficient in serum opsonic capacity, which negatively affects their capacity for neutrophil phagocytosis. These changes in serum opsonins, unrelated to IgG, may be important factors in susceptibility to infections in foals.     

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.     

The evaluation of selected parameters of cellular nonspecific immunity in normal and allergic horses

The main aim of this study was to compare selected nonspecific immunity parameters in 14 allergic and 12 healthy horses. Each animal was assessed according to the following parameters: in vitro functional capacity of phagocytic cells using the nitro blue tetrazolium chloride reduction test, both spontaneous (NBT) and zymozan stimulated (NBTs), and ingestion capacity of phagocytic cells using a phagocytic index test (IF) and percentage of phagocytosing neutrophils activity (%KF). Differences were demonstrated between the group of allergic horses, especially with severe allergy symptoms, and healthy horses in NBTs values, with higher values in healthy horses. The values of the phagocytic index were significantly higher in horses with allergy.     

Phagocytosis and destruction of Staphylococcus aureus

Summary

A review is presented of the phagocytosis and intracellular killing of Staphylococcus aureus by polymorphonuclear and mononuclear leukocytes. Recruitment of adequate numbers of leukocytes to the site of infection occurs through the process of chemotaxis. Recognition of invading staphylococci by the phagocytic cells is mediated through bacterial opsonization. Both processess depend upon the activation of the heat‐labile complement system which generates the majority of chemotactic (C5a) and opsonic (C3b) molecules for S. aureus phagocytosis. The key role of peptidoglycan in the cell wall of staphylococci in these events is stressed. Attachment and ingestion of opsonized staphylococci occurs via poorly‐defined receptors for opsonins in the membrane of the leukocyte. The greater phagocytic capacity of neutrophils as compared to monocytes is not reflected in differences in their membrane receptors for staphylococcal opsonins. Once ingested, staphylococci are rapidly destroyed by oxygen‐dependent and oxygen‐independent bactericidal mechanisms of the phagocytes. Small numbers of S. aureus may survive within the leukocyte. Special attention is focused on the numerous ways S. aureus is able to hinder, evade, and directly damage the phagocytic defense mechanisms of the host.     

Heterogeneity in phagocytic and nitroblue tetrazolium reductive properties of neutrophils from cows.

Phagocytic and nitroblue tetrazolium (NBT) reductive activities of blood neutrophils from 19 Holstein heifers were measured by light microscopic and spectrophotometric methods, respectively. These functional properties of neutrophils correlated well (r = 0.64) and varied significantly (P less than 0.05) among animals studied. Variations in phagocytosis and NBT reductive activities attributable to the source of sera were determined in experiments in which cells from the same cows and zymogen particles opsonized with heat-inactivated autologous or homologous sera were used. Variations attributable to the source of cells were determined in experiments in which cells from different cows and particles opsonized with pooled sera from all the cows were used. Most of the variation in phagocytic properties and NBT reductive activities was attributable to the source of cells (ie, each cow). The source of sera contributed slightly to the variation in NBT reductive activities, but not to the phagocytic properties. These results support the concept of functional heterogeneity of neutrophils among cows.     

A study of cells in the mammary secretions of sows

The morphology and some of the in vitro functional properties of the cells in the mammary secretions of sows have been examined. A mean cell yield of 1 × 10⁷ cells/ml was obtained from sow colostrum but during the first week post-partum the yield decreased approximately 10 fold. The polymorphonuclear leucocyte was the predominant cell type in colostrum and milk and was associated with varying proportions of lymphocytes, macrophages and epithelial cells. The phagocytes of sow milk ingested heat-killed yeast, although the phagocytic index for milk macrophages was low compared with autologous neutrophils and alveolar macrophages. Milk whey provided an effective opsonising medium for yeast ingestion. Intra-mammary immunisation of sows with ovalbumin induced antigen-reactive lymphocytes in both peripheral blood and milk.     

Modulation of phagocytic cell function

Phagocytosis is an important factor in the defense of the host against all kinds of microorganisms. The process of phagocytosis of microorganisms by phagocytes can be separated into distinct but interrelated phases: adherence, chemotaxis, opsonization, attachment, ingestion, degranulation and killing. Phagocytosis is accompanied by an increase in oxygen metabolism in which H2O2 and activated oxygen species are generated. Modulation of phagocytic cell function can be brought about by a variety of substances. Microorganisms produce and contain components which influence the process of phagocytosis. Surrounding tissue cells and the phagocytes themselves produce biologically active molecules that modulate phagocytosis.     

Modulation of phagocytic cell function

Phagocytosis is an important factor in the defense of the host against all kinds of microorganisms. The process of phagocytosis of microorganisms by phagocytes can be separated into distinct but interrelated phases: adherence, chemotaxis, opsonization, attachment, ingestion, degranulation and killing. Phagocytosis is accompanied by an increase in oxygen metabolism in which H₂O₂ and activated oxygen species are generated. Modulation of phagocytic cell function can be brought about by a variety of substances. Microorganisms produce and contain components which influence the process of phagocytosis. Surrounding tissue cells and the phagocytes themselves produce biologically active molecules that modulate phagocytosis.     

Secretory virulence factors produced by Staphylococcus aureus isolates obtained from mastitic bovine milk--effect on bovine polymorphonuclear neutrophils

The aim of the research was to test whether exogenic virulence factors secreted by Staphylococcus aureus isolates are involved in mechanisms that allow the bacteria to modulate and evade phagocytosis by bovine polymorphonuclear neutrophils. The research was based on the comparison of the effects of supernatants, prepared from cultures of 30 S. aureus isolates, on the functional properties of bovine neutrophils in vitro. S. aureus isolates were collected from milk samples from cows with clinical mastitis. Supernatants, which were used to treat leukocytes, were prepared from 18 h S. aureus cultures. Exogenic virulence factors secreted by S. aureus isolates significantly influenced the phagocytosis parameters evaluated. Depending on their leukotoxic or superantigenic properties, supernatants could affect the ingestion process, and also showed an influence on the digestion efficiency and phagocytosis carried out by bovine polymorphonuclear neutrophils in vitro.     

Evaluation of treatment of colostrum-deprived kittens with equine IgG

OBJECTIVE: To evaluate equine IgG as a treatment for kittens with failure of passive transfer of immunity (FPT). ANIMALS: 13 specific pathogen-free queens and their 77 kittens. PROCEDURE: Kittens were randomized at birth into 9 treatment groups. One group contained colostrum-fed (nursing) kittens; the other groups contained colostrum-deprived kittens that were administered supplemental feline or equine IgG PO or SC during the first 12 hours after birth. Blood samples were collected at serial time points from birth to 56 days of age for determination of serum IgG concentrations. The capacity of equine IgG to opsonize bacteria for phagocytosis by feline neutrophils was determined via flow cytometry. RESULTS: Kittens that received feline or equine IgG SC had significantly higher serum IgG concentrations than those of kittens that received the supplements PO. In kittens that were administered supplemental IgG SC, serum IgG concentrations were considered adequate for protection against infection. The half-life of IgG in kittens treated with equine IgG was shorter than that in kittens treated with feline IgG. Feline IgG significantly enhanced the phagocytosis of bacteria by feline neutrophils, but equine IgG did not. CONCLUSIONS AND CLINICAL RELEVANCE: Serum concentrations of equine IgG that are considered protective against infection are easily attained in kittens, but the failure of these antibodies to promote bacterial phagocytosis in vitro suggests that equine IgG may be an inappropriate treatment for FPT in kittens.     

Phagocytosis of opsonized fluorescent microspheres by equine polymorphonuclear leukocytes

Equine blood polymorphonuclear leukocytes (PMN) were isolated by buffy coat and hypotonic lysis of residual erythrocytes. A highly reproducible method is described for measuring the uptake of opsonized latex microspheres by equine PMN using flowcytometry. The use of cytochalasin D allowed for differentiation of ingested from attached particles. The kinetics of phagocytosis in vitro is shown for different experimental conditions. We developed an assay for evaluation of phagocytic capacity of PMN which allows the assessment of drugs for their influence on phagocytosis in vivo as well as in vitro.     

Effects of midazolam on equine innate immune response: a flow cytometric study

Benzodiazepines (BDZ) are among the most frequently used class of psychotropic drugs employed in veterinary medicine in Brazil and worldwide due to their anxiolytic, muscle relaxant and anticonvulsant effects [J. Clin. Pharmacol. 33 (1993) 124]. Peripheral benzodiazepine receptor (PBR) sites were described in peripheral organs, endocrine steroidogenic tissues and immune organs and cells. Midazolam is a mixed-type agonist of PBRs. The present study is focused on the effects of midazolam on equine peripheral blood neutrophils, peritoneal macrophages and cortisol levels in plasma. Adult horses were treated with a single dose of midazolam (0.06 or 0.1 mg/kg) or with 0.9% NaCl. Immune cells were collected 24 h after treatment for flow cytometry analysis of Staphylococcus aureus-induced phagocytosis and oxidative burst. Plasma cortisol concentration was measured 30, 90, 180 and 360 min after midazolam treatment. Midazolam induced a dose-dependent reduction on: (1) peripheral blood neutrophil and peritoneal macrophage oxidative burst; (2) the capacity of both peripheral blood neutrophils and peritoneal macrophages to phagocyte S. aureus. Increments on plasma cortisol concentration were not found after 0.06 and 0.1 mg/kg of midazolam. The effects on oxidative burst of neutrophils and macrophages from horses treated with midazolam were interpreted as a consequence of an impairment of S. aureus-induced phagocytosis. The present data suggest that midazolam, most probably acting on PBRs present on equine macrophage and neutrophil membranes, might have changed some mechanisms related to both phagocytosis and oxidative burst. These results support the use of flow cytometry to identify functional properties and dysfunction of equine immune cells. They also confirm the notion that changes in the functional capacity of the immune system may represent an important hazard of exposure to drugs or chemicals.     

Quantitative characterization of yeast cell incorporation in phagocytes: microscopic methods for differentiation between adherent and ingested particles

A light- and a fluorescence-microscopic method for quantitative assessment of yeast cell incorporation in phagocytes were developed. The light-microscopic method offers methylene blue prestained yeast cells as phagocytosis particles and counterstains nonincorporated yeasts with eosine. The fluorescence-microscopic method works by acridine orange staining of phagocytosis assays. Fluorescence of nonincorporated yeast cells is suppressed by addition of methylene blue. Different ways of evaluating the results of microscopic quantitation of phagocytosis are discussed.     

Phagocytosis induced by yeast cells in canine granulocytes: a methodological study

A phagocytic function assay of canine granulocytes was established. This method allows the proportion of active granulocytes to be estimated as well as the number of adhered and ingested yeast cells. The influence of different factors on phagocytosis was studied. Temperature variation within the interval 36-41 degrees C did not affect phagocytosis. The incubation time for optimal phagocytosis of yeast cells was 35 min. The opsonization procedure giving the optimal phagocytosis was purified IgG and serum together.     

Effects of antibiotics on phagocyte recruitment, function, and morphology in the bovine mammary gland during the early nonlactating period

The effects of 2 antibiotic preparations administered intramammarily on phagocyte recruitment, function, and morphology were evaluated at the beginning of the nonlactating period. Twelve cows with no clinical or microbiologic evidence of mastitis were assigned to 1 of 2 treatment groups. At the end of lactation, 1 of the antibiotic preparations was infused in a fore- and hind quarter of each cow; the remaining quarters were untreated controls. One group was given benzathine cephapirin; the second group was given sodium novobiocin. Secretion samples were collected from 1 treated and 1 control quarter at 16 hours, and from the remaining 2 quarters at 64 hours after treatment. Total and differential somatic cell counts were determined, and morphology of mammary polymorphonuclear neutrophils (PMN) and macrophages was observed by transmission electron microscopy. In vitro ingestion and killing of Staphylococcus aureus by mammary PMN and macrophages were assessed by fluorescent microscopy, using acridine orange stain. Cells resident in a fixed volume of secretion were incubated with a known concentration of S aureus. Total cell and PMN concentrations were higher in treated than in control quarters. Neutrophils were the predominant cell type in both treated and control quarters over the sampling period. As measured in this study, in vitro ingestion and killing of S aureus by individual PMN from treated quarters was reduced. Antibiotic treatment also increased the proportion of morphologically abnormal phagocytes. There were significant correlations among PMN ingestion, killing, and morphology. However, increased PMN concentrations tended to compensate for the reduced phagocytic function of individual cells. Therefore, efficacy of antibiotic treatment of nonlactating cows may depend, at least in part, on increased PMN concentration, which may tend to compensate for reduced phagocytic function. Compared with PMN, macrophages appeared to have only a minor role in phagocytosis of bacteria.     

Flow cytometric characterization of bovine blood neutrophil phagocytosis of fluorescent bacteria and zymosan particles

A Flow Cytometric method for the evaluation of the phagocytic capacity of bovine blood neutrophils is described. The neutrophils were isolated from bovine blood by a one step discontinuous gradient of Percoll. By this technique of isolation, 90 ± 2.8 % (mean ± s) of the granulocytes in the whole blood were recovered.Isolated neutrophils were incubated with FITC labeled S. aureus or zymosan particles in a ratio of 1:20 and 1:10, respectively, and a final serum concentration of 10 %. Phagocytosis was terminated after 15 min and the number of extracellular bacteria or zymosan particles and the percentage of phagocytic granulocytes were registered by Flow Cytometry (FCM). FCM and microscopic studies revealed that eosinophils play a minor role in the phagocytosis of bacteria. The neutrophils were the main population of the granulocytes which were actively phagocytic. Variation among cows in the ability of their blood neutrophils to phagocytize bacteria was evident.     

Comparison of phagocytosis and chemiluminescence by blood and mammary gland neutrophils from multiparous and nulliparous cows

Neutrophils were isolated from the blood and mammary gland of 3 multiparous lactating cows and 3 nulliparous heifers. Neutrophil function was evaluated by phagocytosis and luminol-dependent chemiluminescence. Peroxidase activity was detected by use of transmission electron microscopy. Compared with that for blood neutrophils, percentage of phagocytosis was 9.6% lower for neutrophils isolated from the mammary gland of lactating cows, but this difference was not observed between neutrophils isolated from the mammary gland and from the blood heifers. Similarly, after subtraction of chemiluminescence values in the absence of zymosan, phagocytosing neutrophils from the mammary gland of lactating cows had lower chemiluminescence than did those from the blood of such cows. For heifers, however, chemiluminescent activity by phagocytosing neutrophils obtained from the mammary gland was similar to that of blood neutrophils. Chemiluminescent activity of resting neutrophils from the mammary gland of lactating cows pretreated with cytochalasin B was not inhibited, compared with that of nontreated resting neutrophils (controls). This was attributed to xanthine oxidase activity. Transmission electron microscopy of mammary gland neutrophils from lactating cows revealed peroxidase-positive material associated with milk-fat globule membranes and with phagosomes containing zymosan. Results indicated that ingestion of fat and casein by neutrophils isolated from milk caused a decrease in phagocytic and chemiluminescent activity. Also, luminol-dependent chemiluminescence was not a reliable measure of milk neutrophil function, because of interference by xanthine oxidase.     

Ultrastructural observations on peritoneal exudate cells from the striped bass

An ultrastructural study was conducted of the principal cells recovered from the peritoneal exudate of striped bass (Morone saxatilis) injected with killed Bacillus cereus. The most frequently observed cells were identified as macrophages, eosinophils, and neutrophils on the basis of comparing their structure with that reported for other fish species and higher vertebrates. All three of the types of cells were phagocytic; however, the numbers of bacteria engulfed were limited. Like other studies of this type in fish, the peritoneum of the striped bass proved to be a good source of cells for morphological investigations of phagocytosis.