Flow cytometric assay for combined measurement of phagocytosis and intracellular killing of Candida albicans

A rapid quantitative flow cytometric (FCM) assay for the combined kinetic measurement of phagocytosis and intracellular killing of fluorescein-isothiocyanate (FITC) labelled Candida albicans is described. The fraction of phagocytosing leukocytes and the numbers of attached or internalized Candida albicans per phagocytosing leukocyte were quantified by FCM, using trypan blue and a fluorescence quenching technique. Results obtained by microscopy agreed with the FCM estimates of phagocytosis. Dead, but not live, Candida albicans stained by propidium iodide (PI). Thus, both viable and intracellularly killed fungi could be discriminated and measured by FCM. Phagocyte killing determined by the FCM assay correlated with killing measured by a standard microbiological test and by methylene blue staining. The method allows rapid and accurate testing of opsonic and phagocytic functions under both experimental and clinical conditions.     

Intracellular killing of Candida albicans by human polymorphonuclear leucocytes: comparison of three methods of assessment

Three different methods, [3H]uridine uptake, viable count and 51Cr-release were used to assess the intracellular survival of a strain of Candida albicans, 19321, which was lethal for mice injected intravenously. Intracellular survival 1 h after ingestion ranged from 50 to 80% depending on the method employed and the detergent used to lyse the phagocytes. Inhibition of uridine uptake by detergents used to lyse the phagocytes led to difficulty in assessment of intracellular killing by this method.     

A simple and rapid flow cytometric method for routine assessment of baker's yeast uptake by human polymorphonuclear leukocytes

A new method for measuring uptake of baker's yeast (BY) by human polymorphonuclear leukocytes (PMN) using flow cytometry is described. The method correlates excellently with the visual method, is reproducible and provides a means for investigating the early phases of the phagocytic process as well as the phagocytic capacity of PMN. This quick and accurate method allows the counting of large numbers of cells, and monitoring of the process of particle uptake and has a considerable potential in the routine assessment of polymorph function in various clinical situations.     

Simultaneous flow cytometric measurement of antigen attachment to phagocytes and phagocytosis

The current available assays cannot differentiate the stages of phagocytosis. We, therefore, established methods for concurrent detection of antigen attachment and engulfment by phagocyte using latex beads coated with lipopolysaccharide, rabbit IgG, and carboxyfluorescein diacetate succinimidyl ester. The generated beads were incubated with whole blood at 37°C for 1 hr and stained with PE-Cy5.5 anti-rabbit IgG antibody. By flow cytometry, attachment and phagocytic processes could be detected, simultaneously. The established method is a valuable tool for diagnosis of phagocytic disorder and study of molecules involved in phagocytosis.     

Interaction and intracellular killing of Candida albicans blastospores by human polymorphonuclear leucocytes, monocytes and monocyte-derived macrophages in aerobic and anaerobic conditions.

Polymorphonuclear leucocytes (PMN), monocytes and monocyte-derived macrophages were capable of interacting with opsonized C. albicans in both aerobic and anaerobic conditions. Superoxide anion release by these cells was inhibited in anaerobic conditions while lysozyme release and phagocytosis were equally efficient in both aerobic and anaerobic conditions. All cell types tested were capable of intracellular killing of C. albicans and this appeared to be maximum at 6 h for monocytes and macrophages and 24 h for PMN. Monocytes killed the lowest number of organisms, 1 x 10(6), and the killing was similar for aerobic and anaerobic conditions. In contrast, PMN and macrophages demonstrated greater killing of C. albicans in aerobic conditions compared with anaerobic conditions; PMN killed 1.9 x 10(6) organisms and macrophages 3 x 10(6) when incubated anaerobically. Inhibitors of oxygen metabolism decreased intracellular killing of C. albicans by macrophages and PMN in aerobic but not anaerobic conditions. The oxygen reaction products involved in the killing of C. albicans appeared to be different however: macrophage killing was decreased by superoxide anion and hydrogen peroxide inhibitors. PMN killing was decreased by superoxide anion, hydrogen peroxide, hypochlorous acid and hydroxyl radical inhibitors. The present study shows that although monocytes, macrophages and PMN function similarly in their interaction with C. albicans, they appear to use different oxygen reactive products for the intracellular killing of C. albicans.     

In vitro phagocytosis of opsonized latex beads by HD11 cells as a method to assess the general opsonization potential of chicken serum

Opsonins, an important arm of the innate immune system, are various soluble proteins, which play a critical role in destruction of invading pathogens directly or via engulfment of pathogens through the intermediate of phagocytosis. The diversity of opsonin profiles is under genetic influence and may be associated with variation in disease resistance. The aim of this study was to set up an assay to determine serum opsonophagocytic potential (OPp) for chicken sera by flow cytometry and to evaluate the assay using samples from different chicken lines. Two chicken lines selected for high and low concentrations of mannose-binding lectin, a known opsonin, in serum were used to establish the method. Furthermore, the presumed “robust” Hellevad chickens and two other commercial chicken lines (Hisex and Bovans) were tested to evaluate OPp as a parameter reflecting general immune competence. The results showed that Hellevad and Bovans chickens had higher OPp than Hisex chickens. There were no correlations between concentrations of total IgY or mannose-binding lectin and OPp. However, a strong positive correlation was observed between vaccine-induced infectious bronchitis virus titres and OPp. Moreover, inverse relationships were observed between concentrations of total serum IgM as well as natural antibody levels, and OPp. In conclusion, in vitro opsonophagocytosis assessment and determination of OPp may be of relevance when addressing general innate immunocompetence.

RESEARCH HIGHLIGHTS

• A flow cytometry method was developed to assess poultry serum opsonophagocytosis potential.

• This method is based on serum-opsonin-coated polystyrene beads and HD11 cell phagocytosis.

• Serum samples from different commercial chicken lines were compared.

• Opsonophagocytic potential may be included in assay panels for general immune competence of poultry.

     

A flow cytometric method for the detection of adenosine deaminase in mononuclear cells

The purpose of this study was to develop a flow cytometric method for the detection of adenosine deaminase (ADA) in a single cell suspension of mononuclear cells. Anti-human ADA antibody was purified by affinity chromatography on a column of Sepharose 4B to which calf ADA was covalently linked. This antibody was used for indirect immunofluorescent staining of cells fixed in 4% paraformaldehyde. The specificity of staining was proved by substitution of anti-human ADA with normal rabbit IgG and by absorption experiments. The fluorescence profile of the cells was then analyzed by flow cytometry. Two groups of cells were studied: (a) thymocytes, tonsil cells and peripheral blood mononuclear cells (PBMC), (b) ADA-positive and ADA-deficient cell lines. In each of these populations of cells a peak of specific immunofluorescence staining for the enzyme could be easily distinguished from weak background staining of control preparations. Within each group, the cell population with higher ADA activity also displayed a greater intensity of cell fluorescence. Flow cytometry provides a means for quantitation of ADA in individual mononuclear cells.     

A rapid evaluation of phagocytosis and killing of Candida albicans by CD13+ leukocytes

Flow cytometry can be adopted for routine monitoring of the immune functions of human polymorphonuclear leukocytes (PMNs) in several disease states. We recently developed a rapid and reproducible assay for the evaluation of the phagocytosis and killing of Candida albicans blastospores by human PMNs. Whole blood leukocytes were incubated with opsonized fluorescein isothiocyanate-labeled (FITC-labeled) blastospores for phagocytosis and killing assays. To discriminate between ingested, membrane-bound and free C. albicans blastospores, ethidium bromide (EtBr) was added to the samples prior to the flow cytometric analysis. EtBr induces a loss of green fluorescence in non-phagocytized C. albicans blastospores. Phagocytosis is determined by gating the phagocytes and calculating the percentage of phagocyte-associated green fluorescent cells. Intracellular killing is determined by first lysing phagocytes by hypotonic shock and then adding propidium iodide (PI) in order to identify red dead blastospores. Killing is measured in terms of the percentage of double-marked blastospore cells. We suggest that this method is a reliable and inexpensive technique to evaluate the immune reactivity of PMNs and peripheral blood monocytes (PBMs) in cases of immunosuppression.     

A new multiparameter flow cytometric method for human semen analysis

BACKGROUND: The objectives of this study were (i) to evaluate whether the combined use of Syto 16 and 7-amino-actinomycin-D (7-AAD) allows the detection of sperm apoptosis and (ii) to describe a new multiparameter flow cytometric method to assess simultaneously sperm concentration (SC), viability and apoptosis as well as leukocyte concentration. METHODS: Semen samples from 68 patients were evaluated according to World Health Organization (WHO) criteria (normal, n=26; abnormal, n=42). The detection of activated caspases before and after betulinic acid (BA) incubation was carried out in 13 semen samples by flow cytometry using fluorescein-labelled inhibitors of caspases (FLICA). A multiparameter flow cytometric analysis was performed in 55 semen samples. Fluorescent microspheres were used to assess SC. Sperm apoptosis was detected by staining sperm with Syto 16 and 7-AAD. Leukocytes were counted using monoclonal anti-CD45. RESULTS: A significant correlation between the percentage of the spermatozoa with low Syto 16 fluorescence and the percentage of spermatozoa containing activated caspases was found (r=0.68, P=0.0106; n=13). After incubation with BA, an increase of the percentage of apoptotic cells was observed in all samples, using both the Syto 16/7-AAD and the caspase activation methods. There was a good correlation between flow cytometry and optical microscopy for sperm (r=0.98, P < 0.0001) and leukocyte counting (r=0.64, P <0.0001). The percentage of apoptotic sperm was inversely correlated with both SC (r=-0.303, P=0.0246) and morphology (r=-0.384, P=0.0050) but not with motility. CONCLUSIONS: The combination of Syto 16/7-AAD provides a sensitive assay to detect sperm apoptosis. The multiparameter flow cytometric method described offers the possibility of a simultaneous, simple, rapid and accurate assessment of several semen parameters.     

Effect of immunoglobulin therapy on phagocytosis by polymorphonuclear leucocytes in whole blood of neonates

There has been some disagreement as to the clinical effect of intravenous immunoglobulin (IVIG) therapy on neonatal bacterial infections. We therefore evaluated the effect of IVIG therapy on neonatal polymorphonuclear leucocyte (PMN) functions by monitoring phagocytosis and hydrogen peroxide (H₂O₂) production. Subjects were 10 mature neonates who had normal plasma levels (i.e. equal to adult plasma levels) of IgG and nine premature neonates who had lower plasma levels of IgG. Phagocytosis by PMN was measured using flow cytometric analysis of whole blood. Addition of γ-globulin to the whole blood of mature neonates increased phagocytosis, but not significantly. Higher doses of added γ-globulin (> 2.0 mg/ml, the concentration was expressed as that in the final reaction volume) decreased phagocytosis to under baseline level. In premature neonates addition of γ-globulin increased phagocytosis and the significant maximum effect was observed with 0.5 mg/ml of the additional γ-globulin. Higher doses of additional γ-globulin (2.5 mg/ml) decreased phagocytosis to baseline level. Phagocytosis in four mature and four premature neonates was compared before and after 1 g/kg of IVIG therapy. Phagocytosis in mature neonates after IVIG therapy did not change compared with the pretreatment level. On the other hand, phagocytosis in premature neonates after IVIG therapy significantly increased compared with its pretreatment level. In both mature and premature neonates H₂O₂ production following phagocytosis varied in parallel with changes of phagocytosis. The patterns of H₂O₂ production following phagocytosis were essentially similar to those observed with phagocytosis. The above results are expected to form the basis for a rational indication for IVIG therapy against bacterial infections in neonates with low plasma IgG levels.     

Simultaneous cytofluorometric measurement of phagocytosis, burst production and killing of human phagocytes using Candida albicans and Staphylococcus aureus as target organisms

Objective   Polymorphonuclear leukocytes (PMN) play a central role in the elimination of most extracellular pathogens, and an impairment of their functions predisposes an individual towards local and systemic bacterial and fungal infections. Here we describe a rapid and easy-to-perform cytofluorometric assay for investigation of PMN activity using Candida albicans and Staphylococcus aureus as target organisms.
Methods   Phagocytes were stained with anti-CD13-RPE antibody, and microorganisms were stained with calcein-AM. Oxidative burst production was measured by oxidation of dihydroethidium. The percentage of killed target organisms after ingestion was determined by staining with ethidium-homodimer-1 after lysis of human cells. The dyes and procedures used in this method were chosen after comparison of different stains and cell preparation techniques described in previous assays.
Results   Concerning phagocytosis, the percentages of active phagocytes and of ingested microorganisms were determined. Furthermore, the method allowed measurement of the resulting percentage of PMNs producing respiratory burst, and of the percentage of killed microorganisms. We minimized artifactual changes, which might have been the reason for the difficulties and conflicting results of other cytofluorometric methods.
Conclusions   The described method provides a new whole blood cytofluorometric assay, which combines rapid and simple handling with high reproducibility of results obtained by investigation of PMN activity using Candida albicans and Staphylococcus aureus as target organisms.     

A flow cytometric technique for quantitation of B-cell activation

A flow cytometric method for enumeration of intracytoplasmic antibody-containing cells was developed. Flow cytometry was found to yield results comparable to traditional fluorescence microscopy in cells stimulated to produce intracytoplasmic antibody by either pokeweed mitogen or Epstein-Barr virus. This technique offers several advantages over traditional fluorescence microscopy, including objective measurement of fluorescence intensity and rapid analysis of large numbers of cells.     

A novel flow cytometric assay for rapid detection of extended-spectrum beta-lactamases

The rapid detection of extended-spectrum beta-lactamases (ESBLs) is a challenge for most clinical microbiology laboratories because inaccurate identification of ESBL producers has important clinical implications for both antibiotic treatment and infection control. The aim of our study was to develop a rapid detection assay of ESBL producers based upon flow cytometric analysis. Antimicrobial susceptibility testing followed by molecular characterization of bla_TEM, bla_SHV or bla_CTX-M genes was performed on clinical isolates (41 ESBL positive and 20 ESBL negative) and isolates expressing well-characterized beta-lactamases, including ESBLs (n = 13), plasmid AmpCs (n = 3), oxacillinases (n = 5) and carbapenemases (n = 3). Additionally, two ATCC strains recommended by CLSI for susceptibility testing were used as controls. The flow cytometry analysis protocol involved an incubation of bacterial cells with different concentrations of ceftazidime (1, 2 and 4 mg/L) and cefotaxime (4, 8 and 16 mg/L) for 1 and 2 hours, in the presence and absence of clavulanic acid; subsequently, cells were stained with the fluorescent dye Bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)], a lipophilic anion able to diffuse across depolarized membranes. Additionally, CFU counts were performed. Susceptible isolates displayed increased fluorescence after 1 hour of incubation; conversely, the increase of the depolarized population was only observed after incubation with clavulanic acid associated with ceftazidime or cefotaxime in ESBL producers. An excellent correlation was obtained between the number of non-depolarized bacteria quantified by flow cytometry and by conventional CFU assays. A novel, accurate and fast flow cytometric assay is available to detect the presence of ESBLs.     

A quantitative fluorescent method for measurement of bacterial adherence and phagocytosis

We have developed a new two-color fluorescent method for the quantitative measurement of adherence and ingestion of Streptococcus pneumoniae by human monocytes. The method employs a fluorescent naphthalimide, Lucifer Yellow VS, that has been covalently linked to the bacterial cell wall. Bacteria were opsonized and allowed to adhere to monocytes. Lucifer Yellow did not alter bacterial interaction with complement in serum or with the phagocytic cell. The ability of monocytes to ingest the adherent bacteria was tested under a variety of conditions. Rabbit antibody to Lucifer Yellow derivatized with Texas Red was used to detect monocyte-bound, but uningested bacteria. Dual laser flow cytometry simultaneously quantitated the total number of monocyte-associated S. pneumoniae and the number that remained surface adherent. This method allows separate analysis of the opsonins and receptors involved in bacterial adherence to phagocytes and in the ingestion process.     

Detection of allergen-induced basophil activation by expression of CD63 antigen using a tricolour flow cytometric method

In the field of allergy diagnosis, most in vitro functional tests are focused on basophils. Nevertheless, the very small number of circulating basophils limits these experiments and their clinical benefit remains controversial. As flow cytometry is a valuable tool for identifying cell populations, even at low concentrations, we developed a tricolour flow cytometric method for the study of allergen-induced basophil activation. Identification of cells was based both on CD45 expression and on the presence of IgE on the cell surface, since basophils express high-affinity receptors for IgE (Fc epsilon RI). Cell activation upon allergen challenge was assessed by the expression of CD63 antigen on the plasma membrane. Basophil isolation and activation (with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine) were validated in 32 non-allergic patients. In 12 allergic patients, basophil stimulation by a relevant allergen was in most cases positive (10/12). Furthermore a concentration-dependent hook effect was observed. Of the allergic and non-allergic patients, none showed non-specific activation with an irrelevant allergen (specificity 100%). Overall, our preliminary results, even in a small population, suggest that this is a reliable and valuable method for the diagnosis of allergies complementing specific allergen IgE and skin test results. Obviously, additional clinical studies are needed to validate these first results.     

A microcytotoxicity assay for thyroid-specific cytotoxic antibody, antibody-dependent cell-mediated cytotoxicity and direct lymphocyte cytotoxicity using human thyroid cells

A solid-phase enzyme immunoassay for the detection of antibodies, specific for hemoglobin (Hb) is described. The application of glutaraldehyde resulted in a sensitive assay and allowed the use of urea, which is an important advantage if polypeptides not soluble in aqueous buffers are to be used. Mutation-carrying Hb chains can be purified, solubilized in urea and used in the immunoassay to monitor the purification and selection of antibodies specific for these variants. Specific antibodies are the main tools for the development of a hemoglobin-locus mutation system for detection of potentially mutagenic environmental agents. With erythrocytes as target cells, this system permits in vivo monitoring of subjects under exposure. Conventional antibody production, however, frequently turns out to be unsuccessful. The production of monoclonal antibodies has several advantages over conventional antibody production, but a sensitive antibody screening system is essential. Because of the sensitivity and the ease with which a large panel of antibody fractions against a vast panel of Hb antigens can be examined, the described immunoassay has potential value for the screening of hybridoma cultures.     

In vivo expression of rubella antigens on human leucocytes: detection by flow cytometry

Flow cytometry has been used to detect in vivo expression of rubella antigens on human leucocytes. Sequential samples of peripheral blood were obtained from four volunteers with naturally acquired rubella and five persons immunised with RA27/3 rubella vaccine. Leucocytes were stained for rubella antigens using a pool of rubella monoclonal antibodies. Rubella antigens were detected on the leucocytes of all four volunteers with naturally acquired rubella between 1 and 13 days after onset of illness. Viral antigens were expressed more frequently on the monocyte (9-51%) than the lymphocyte (less than 1-4%) and granulocyte (less than 1-3%) populations. Among the vaccines, rubella antigens were detected on the leucocytes of four of the five volunteers between 5 and 12 days after immunisation. The expression of viral antigens was more transient and the proportion of cells exhibiting rubella-specific fluorescence considerably lower following vaccination (1-12%) than natural infection (9-51%). Our results demonstrate that flow cytometry provides a rapid and sensitive analytical technique for detecting viral antigens on leucocytes from infected persons. Leucocytes may play an important role in the pathogenesis of rubella infection.     

Effect of extracellular ionic calcium and magnesium on opsonic and non-opsonic phagocytosis of escherichia coli bovine blood polymorphonuclear leucocytes

The effects of extracellular Ca²⁺ and Mg²⁺ concentrations on opsonic and non-opsonic phagocytosis ofEscherichia coli by bovine polymorphonuclear leucocytes (PMN) isolated from blood were evaluated by flow cytometry. Eight cows were used as blood donors. The green fluorescence of blood PMN selectively gated in the forward scatter (FS) - side scatter (SS) dot plot after incubation with fluorescein isothio-cyanate (FITC) - labelledE. coli was used to characterise phagocytosis. Parameters for phagocytosis were percentage fluorescent PMN (% phagocytosis) and mean fluorescence intensity (MFI). The fluorescence of adherent bacteria was quenched with trypan blue to distinguish between adherence and ingestion. Nonopsonic and opsonic phagocytosis were decreased in the absence of extracellular ionic Ca²⁺ and Mg²⁺ compared to physiological levels. Addition of 10 him EGTA to the incubation medium was necessary to block all extracellular Ca²⁺ and resulted in a significant decrease of opsonic phagocytosis, with only 5% phagocytic PMN after quenching. Increasing Ca²⁺ concentrations resulted in a gradual increase in percentage opsonic and non-opsonic phagocytosis and in MFI for opsonic phagocytosis. Ionic calcium plays an important role in phagocytosis (attachment as well as ingestion) by bovine blood PMN in the presence of opsonins, whereas non-opsonic phagocytosis appeared to be less dependent on Ca²⁺. However, reduced serum or milk calcium levels in cows are unlikely to cause a substantial reduction of PMN phagocytosis in vivo.     

Three-colour flow cytometric method to measure antibody-dependent tumour cell killing by cytotoxicity and phagocytosis

We have developed a three-colour flow cytometric method to assay the contributions of cytotoxicity and phagocytosis to antibody-dependent cell-mediated tumour cell killing. In this assay, tumour target cells are pre-labelled with CFSE, and mixed with effector cells and a tumour antigen-specific monoclonal antibody. After incubation of the cells with the antibody, effector cells are labelled with PE and dead cells with PI. Using flow cytometry, dead and phagocytosed tumour cells can be quickly and easily counted and the numbers summed to determine the total number of killed cells. One can thereby measure the phagocytic aspect of antibody-dependent cell-mediated tumour cell killing, otherwise only revealed by microscopic examination. The failure to detect phagocytosed, in addition to live and dead target cells, by standard assays may result in an underestimation of tumour cell killing and hence the potential of an antibody for immunotherapy of cancer. We illustrate the new method by analysing human monocyte-mediated cytotoxic and phagocytic cell killing of IGROV1 ovarian tumour cells by the ovarian tumour antigen-specific anti-folate binding protein monoclonal antibody, MOv18 IgE.