Neutrophil Function Assays Research Articles

268. Human Mesenchymal Stem Cells Augment Neutrophil Function to the Human Fungal Pathogen, Candida albicans

Abstract Background Neutrophils are critical innate immune cells that play a crucial role in protection against invading pathogens. Mesenchymal stem cells (MSC) are multipotent progenitors that can differentiate into osteoblasts, chondrocytes, and adipocytes and possess immunomodulatory effects. MSC are the focus of clinical investigation in various comorbidities, including diabetes, cancer, and invasive infections. Methods Human MSC were derived from bone marrow and have been characterized to meet all criteria of MSC (ISCT guideline). Cells were seeded at 100,000 cells per well in fibronectin (50ug/mL)-coated 24-well plate and incubated for 24 hours at 37°C, 5% CO2. The next day, 0.5 mL of whole blood from three healthy donors was added for another 24 hours, after which the neutrophils were isolated, viability measured, and a high throughput multiparametric neutrophil function assay was performed, compared to non-MSC exposed neutrophils (control neutrophils). Functions tested included ROS (dihydrorhodamine 123 (DHR123), phagocytosis of iRFP-expressing Candida albicans, ectodomain shedding (loss of CD62L), and degranulation (increased expression of CD66b). Multiparametric assays were measured on a BD FACS Celesta 4 laser flow cytometer and data was analyzed on FlowJo software. Results Neutrophils co-cultured with MSC had an average viability of 96.3% compared to control neutrophils at 92.15%. Using the multiparametric neutrophil functional assay, neutrophils exposed to MSC ex vivo had increased functional responses to the fungal pathogen Candida albicans. MSC-conditioned neutrophils exhibited a 33.2% increase in degranulation, a 13.4% increase in ROS generation, and 16.7% additional phagocytosis compared to non-MSC neutrophil (control). Compared to fresh neutrophils isolated the day of testing, MSC-conditioned neutrophils had an average increase in phagocytosis, degranulation, shedding, and ROS generation of 36.9%, 62.7%, 50.3%, and 37.8%, respectively. Conclusion MSC increase the functional effector activity of neutrophils in healthy controls, specifically ROS generation, phagocytosis, ectodomain shedding, and degranulation in response to C. albicans. These data raise the possibility of MSC as a novel therapeutic intervention for patients with invasive infections. Disclosures Michael Mansour, MD, PhD, Thermofisher: Grant/Research Support

Seborrheic Keratosis Caused by Human Papillomavirus Type 20 Ameliorated by Zinc Oxide Ointment

A 91-year-old woman visited our department with scattered small nodule lesions and multiple pules or plaques with a stuck-on appearance. The lesions were intractable and resistant to several treatments. Immunodeficiency was excluded by examinations including a CT scan, white blood cell (WBC) counts, natural killer and neutrophil function assays, and IgG titers against human papillomavirus (HPV) 20. HPV20 was identified using the PCR method. The finding of the skin biopsy showed an irritated type of feature of seborrheic keratosis. Additionally, immunohistochemical staining of the lesion revealed that both TNF-α and IFN-ɤ were produced at the skin lesions. The patient's serum zinc level was slightly low. We noticed that zinc deficiency has been reported to decrease the cytotoxic activity of natural killer cells, which play an important role in eliminating virus-infected cells and tumor cells. Finally, zinc oxide ointment was found to improve the lesions dramatically. HPV20 causes tumors only in immunodeficient patients or in patients with epidermodysplasia verruciformis (EV). In EV, EVER1- or EVER2-encoding membrane proteins, of which are related to zinc transport protein-1 expressed on the membrane of the endoplasmic reticulum, were mutated, leading to increased susceptibility to various viral and bacterial infections due to the decreased intracellular zinc concentration. We speculated that the reduction in local zinc concentration was ameliorated by using zinc oxide ointment, resulting in the recovery from HPV20 infection.

Association of metabolic markers with neutrophil function in healthy postpartum dairy cows

The objective of this study was to evaluate the associations of serum markers for systemic inflammation, liver, mineral, and energy status, and blood neutrophil counts with the function of circulating neutrophils in postpartum dairy cows. Blood samples were collected from 21 healthy Holstein cows at 5, 10, 14, and 21 d postpartum. Serum samples were used to measure concentrations of total calcium, phosphorus, magnesium, total protein, albumin, globulin, cholesterol, urea, glucose, gamma-glutamyl transferase, aspartate aminotransferase, glutamate dehydrogenase, haptoglobin (Hp), β-hydroxybutyrate, non-esterified fatty acids (NEFA), and insulin-like growth factor-1. The shift of percentage of activated neutrophils for phagocytosis (PPC) and oxidative burst (POB) and the median fluorescence intensity (MFI) for PC (MFIPC), OB (MFIOB), and endocytic and proteolytic degradation measured via DQ-ovalbumin (MFIDQ) were evaluated using flow cytometry. Mixed linear regression models were used to assess the associations of serum concentrations of metabolites and blood neutrophil counts with each neutrophil function assay outcome accounting for d postpartum, parity, and body condition score. Pearson correlation tests (r) were used to describe the relationships of metabolites and neutrophil counts with neutrophil function assays that were significant in the regression models. Greater serum concentrations of Hp were associated with lesser OB function (POB and MFIOB; r = −0.2 for both), but greater PPC and MFIDQ (r = 0.3 for both). Greater serum NEFA and cholesterol were associated with lesser (r = −0.3) and greater (r = 0.2) POB function, respectively. Blood neutrophil counts were associated with PPC (r = −0.2) and MFIDQ (r = −0.3). At 5 d postpartum, greater serum total protein was associated with greater OB function (POB and MFIOB; r = 0.4 for both). Other markers were not associated with neutrophil function. Some metabolic and inflammatory markers in healthy postpartum dairy cows were associated with circulating neutrophil function. However, these associations only explained a small proportion of the variance in neutrophil function. Serum Hp concentration was most associated with neutrophil function changes but had opposite directions of association with OB- and PC-related functions. Future studies should focus on understanding the mechanisms by which Hp and other metabolic indicators affect neutrophil function in healthy and diseased postpartum dairy cows.

Polycarbonate and polystyrene nanoplastic particles act as stressors to the innate immune system of fathead minnow (Pimephales promelas).

Water pollution with large-scale and small-scale plastic litter is an area of growing concern. Macro-plastic litter is a well-known threat to aquatic wildlife; however, the effects of micro-sized and nano-sized plastic particles on the health of organisms are not well understood. Small-scale plastic particles can easily be ingested by various aquatic organisms and potentially interfere with their immune system; therefore, the authors used a freshwater fish species as a model organism for nanoplastic exposure. Characterization of polystyrene (41.0 nm) and polycarbonate (158.7 nm) nanoplastic particles (PSNPs and PCNPs, respectively) in plasma was performed, and the effects of PSNPs and PCNPs on the innate immune system of fathead minnow were investigated. In vitro effects of PSNPs and PCNPs on neutrophil function were determined using a battery of neutrophil function assays. Exposure of neutrophils to PSNPs or PCNPs caused significant increases in degranulation of primary granules and neutrophil extracellular trap release compared to a nontreated control, whereas oxidative burst was less affected. The present study outlines the stress response of the cellular component of fish innate immune system to polystyrene and polycarbonate nanoparticles/aggregates and indicates their potential to interfere with disease resistance in fish populations. Environ Toxicol Chem 2016;35:3093-3100. © 2016 SETAC.

P-123 Chronic Granulomatous Disease in a Female Presenting with Granulomatous Ileo-Colitis

There are numerous mimickers of inflammatory bowel disease (IBD) which can lead to delays in diagnosis as well as inappropriate use of immunosuppressive therapy. We present a case of Chronic Granulomatous Disease (CGD) manifesting with acute ileo- colitis in a young girl. Case Report: 2 year old female born in the US to Ecuadorean parents presented with 6 weeks of rectal bleeding, abdominal pain, frequent stooling, and anorexia. Although there was no recent travel the parents reported that a friend had given her “dog soup” for 5 days before becoming ill. They denied any rashes, joint pains or aphthous stomatitis. Fecal assays revealed a negative stool culture, C. diff, and stool for ova and parasites. Laboratory studies revealed a Hemoglobin = 10.0, ESR = 70, CRP = 3.3, and Albumin of 3.8. The patient underwent an Endoscopy and Colonoscopy which revealed a pan-colitis as well ileitis with non-caseating granulomas in every biopsy. She was given a 6 week course of steroids to which she responded nicely. However, 3 weeks after stopping she developed a recurrence of bleeding. At that time she was started on mesalamine with no further bleeding. The past medical history was remarkable for a right cervical adenitis which was PCR positive for Mycobacterium abscessus, recurrent draining otitis media which was positive for MRSA, pneumonia requiring hospitalization and a history of incomplete Kawasaki's Disease. Extensive immune work up revealed an abnormal neutrophil function assay and genetic testing is pending. CGD is a phagocytic immunodeficiency syndrome, affecting the NADPH oxidase complex which is responsible for generating the respiratory burst. Mutations in the 5 genes (gp91phox, p47phox, p22phox, p67phox, and p40phox) that make up the complex account for all of the known cases of CGD. There is 1 X-linked and 4 autosomal recessive forms of CGD. The X- linked form, primarily seen in males, occurs in 65–70% of all CGD patients. CGD has an incidence between 1/200,000 and 1/250,000 live births. Infections are caused by catalase positive organisms specifically: Staphylococcus Aureus, Aspergillus, Burkholderia Cepacia, Serratia Marcescens Nocardia, Chromobacter Violaceum, Atypical Mycobacteria, Klebsiella and Granulibacter Bethesdensis. The most common infections are pneumonia, skin abscesses, lymphadenitis, liver abscesses and osteomyelitis. Noninfectious complications include gastric outlet obstructions, urinary tract obstruction, autoimmune diseases and colitis/enteritis which has been reported in 43% of X-linked and 11% of AR patients. The X-linked phenotype typically presents earlier with a more severe clinical course. Diagnosing CGD is through ascertaining the respiratory burst via DHR (preferred) or nitroblue tetrazolium (NBT). This is followed by genetic mutational analysis. Treatments strategies for the colitis/enteritis have included steroids, mesalamine, anti-TNF, surgery, and GM-CSF. None of have been consistently successful and immunosuppressive therapies have resulted in fatal opportunistic infections. Although these therapies appear to be beneficial initially overtime they become less effective. Infectious treatments include antibacterial, antifungal prophylaxis and Interferon Gamma. Stem cell transplantation, which resolves the colitis should be considered for patients with minimal respiratory burst and recurrent severe infections despite prophylaxis. It is imperative that CGD be considered in every IBD patient prior to initiating immunosuppression.

Genetically Determined Severity of Anti-Myeloperoxidase Glomerulonephritis

Myeloperoxidase (MPO) is a target antigen for antineutrophil cytoplasmic autoantibodies (ANCA). There is evidence that MPO-ANCA cause necrotizing and crescentic glomerulonephritis (NCGN) and vasculitis. NCGN severity varies among patients with ANCA disease, and genetic factors influence disease severity. The role of genetics in MPO-ANCA NCGN severity was investigated using 13 inbred mouse strains, F1 and F2 hybrids, bone marrow chimeras, and neutrophil function assays. Mouse strains include founders of the Collaborative Cross. Intravenous injection of anti-MPO IgG induced glomerular crescents in >60% of glomeruli in 129S6/SvEv and CAST/EiJ mice, but <1% in A/J, DBA/1J, DBA/2J, NOD/LtJ, and PWK/PhJ mice. C57BL6J, 129S1/SvImJ, LP/J, WSB/EiJ, NZO/HILtJ, and C3H mice had intermediate severity. High-density genotypes at 542,190 single nucleotide polymorphisms were used to identify candidate loci for disease severity by identifying genomic regions that are different between 129S6/SvEv and 129S1/SvImJ mice, which are genetically similar but phenotypically distinct. C57BL/6 × 129S6 F2 mice were genotyped at 76 SNPs to capture quantitative trait loci for disease severity. The absence of a dominant quantitative trait locus suggests that differences in severity are the result of multiple gene interactions. Invivo studies using bone marrow chimeric mice and invitro studies of neutrophil activation by anti-MPO IgG indicated that severity of NCGN is mediated by genetically determined differences in the function of neutrophils.

Primary immunodeficiency

Primary immunodeficiency disorder (PID) refers to a heterogeneous group of over 130 disorders that result from defects in immune system development and/or function. PIDs are broadly classified as disorders of adaptive immunity (i.e., T-cell, B-cell or combined immunodeficiencies) or of innate immunity (e.g., phagocyte and complement disorders). Although the clinical manifestations of PIDs are highly variable, most disorders involve at least an increased susceptibility to infection. Early diagnosis and treatment are imperative for preventing significant disease-associated morbidity and, therefore, consultation with a clinical immunologist is essential. PIDs should be suspected in patients with: recurrent sinus or ear infections or pneumonias within a 1 year period; failure to thrive; poor response to prolonged use of antibiotics; persistent thrush or skin abscesses; or a family history of PID. Patients with multiple autoimmune diseases should also be evaluated. Diagnostic testing often involves lymphocyte proliferation assays, flow cytometry, measurement of serum immunoglobulin (Ig) levels, assessment of serum specific antibody titers in response to vaccine antigens, neutrophil function assays, stimulation assays for cytokine responses, and complement studies. The treatment of PIDs is complex and generally requires both supportive and definitive strategies. Ig replacement therapy is the mainstay of therapy for B-cell disorders, and is also an important supportive treatment for many patients with combined immunodeficiency disorders. The heterogeneous group of disorders involving the T-cell arm of the adaptive system, such as severe combined immunodeficiency (SCID), require immune reconstitution as soon as possible. The treatment of innate immunodeficiency disorders varies depending on the type of defect, but may involve antifungal and antibiotic prophylaxis, cytokine replacement, vaccinations and bone marrow transplantation. This article provides a detailed overview of the major categories of PIDs and strategies for the appropriate diagnosis and management of these rare disorders.

Use of rapid cytochemical staining to characterize fish blood granulocytes in species of special concern and determine potential for function testing

Studies of innate immunity in fish species of special concern are essential for better understanding of their health status during hatchery rearing conditions. The cytochemical and morphological characterizations of blood granulocytes have been used to provide information about phylogenetic differences and determine the potential use of neutrophil function assays. Rapid, simple, cytochemical staining kits used routinely for staining mammalian granulocytes have been used to characterize granulocytes from blood of four fish species: Arctic grayling, cutthroat trout, June sucker, and shovelnose sturgeon. Blood smears were stained with Peroxidase 391 (myeloperoxidase, MPO), alkaline phosphatase (AP), Periodic Acid Schiff (PAS) and Diff-quick stain; examined using bright field and differential interference contrast microscopy. Granulocytes on blood smears were evaluated based on the cell morphology, and presence or absence of the specific chromogen. Presence of lymphocytes, monocytes, platelets/thrombocytes and granulocytes was determined in all fish species. Arctic grayling, June sucker, and cutthroat trout had MPO positive granulocytes, while shovelnose sturgeon heterophils had positive reaction for leukocyte AP, but not MPO. Presence of MPO indicated potential to measure oxidative burst and degranulation of neutrophil primary granules in Arctic grayling, cutthroat trout and June sucker. Absence of MPO in shovelnose sturgeon suggested use of different enzyme marker (AP) in degranulation assay for this species. Standardization of cytochemical techniques allowed for rapid screening of leukocyte types, reducing the number of fish, time and effort to select adequate neutrophil function assays to be used in studies of health status in species of special concern.

158 DEVELOPMENT OF FIELD ASSAY FOR EVALUATION OF WHITE RHINOCEROS NEUTROPHIL FUNCTION AS A STRESS MARKER

Kruger National Park (KNP) is the primary source of translocated white rhinoceroses (Ceratotherium simum) in South Africa. Capture and transport of the rhinoceroses is a highly successful procedure. However, some relocated rhinoceroses present with fertility problems in the 1–2 years post capture. Novel research has shown that one can assess stress using respiratory neutrophil burst levels in several species (Huber et al. 2006 Protoplasma 229, 221–224; Weyts et al. 1998 Dev. Comp. Immunol. 22, 563–572). The hypothesis is that some rhinoceroses respond poorly to stress. The response to stress might be measurable by a neutrophil function (NF) assay, and NF depression might identify adversely stressed animals. The aim was to develop an in-the-bush test for NF and evaluate the changes in neutrophil activity during capture and loading into transportation crates. Neutrophil function was assayed using a portable luminometer (3M Clean-Trace™, 3M, St. Paul, MN, USA) while driving from one capture to the next. For the reaction mixture, 500 μL of PBS was incubated with 10 μL of fresh heparinized blood at 37.6°C. The incubator unit was power by a 230 V convertor in the vehicle. After 5 min of incubation, 100 μL of luminol was added. This sample was read in the luminometer as a blank and then to the sample, 100 μL of 12-O-tetradecanoylphorbol-13-acetate (TPA), also commonly known as phorbol 12-myristate 13-acetate (PMA), a diester of phorbol, was added. The luminescence readings or relative light units (RLU) were read at 2.5 min and then 5 min after PMA addition. All samples were prepared in triplicate. The readings were then taken every 5 min for 65 min. Two samples of blood were evaluated for each rhinoceros: the first sample at capture (anaesthetic induction) and then a second sample after loading of the rhinoceros into the transport crate, 20 to 30 min later. Two response curves were produced for each rhinoceros using the means of the triplicate readings. The curves were then transformed with a trend line and the area under the curve (AUC) was calculated. The 2 AUC for rhinoceros were then compared statistically using Wilcoxon rank regression, with P &lt; 0.05 considered statistically different. Three distinct response curve patters were seen. Sixty-two percent (16/26) of the rhinoceroses had no statistical (P &lt; 0.05) difference between capture and loading samples. In 5 of 26 (19%), the loading AUC was statistically greater than the capture sample, and in 5 of 26 (19%), the capture AUC was statistically greater than the loading sample. In conclusion, NF can be assayed in the field using the blood of wild caught rhinoceroses. The assay is repeatable and can distinguish 3 populations of wild caught rhinoceroses. The hope is that future research will allow us to identify rhinoceroses that have negative stress reactions and to change the capture conditions to make the process less stressful.

Hydroxylated fullerenes inhibit neutrophil function in fathead minnow ( Pimephales promelas Rafinesque, 1820)

Hydroxylated fullerenes act as potent inhibitors of cytochrome P450-dependent monooxygenases, and are reported to be very strong antioxidants quenching reactive oxygen species (ROS) production. Effects of nanosized hydroxylated fullerenes on fish neutrophil function and immune gene transcription was investigated using fathead minnow ( Pimephales pr omelas). Neutrophil function assays were used to determine the effects of fullerene exposure in vitro and in vivo on oxidative burst, degranulation and extracellular trap (NETs) release, and the innate immune gene trascription was determined with quantitative PCR (qPCR). Application of fullerenes (0.2–200 μg mL −1 in vitro) caused concentration dependent inhibition of oxidative burst and suppressed the release of NETs and degranulation of primary granules (up to 70, 40, and 50% reduction in activity compared to non-treated control, respectively). Transcription of interleukin 11 and myeloperoxidase genes was significantly increased and transcription of elastase 2 gene was significantly decreased in fish exposed to hydroxylated fullerenes for 48 h in vivo (12 and 3 fold increase, and 5 fold decrease, respectively). Observed changes in gene transcription and neutrophil function indicate potential for hydroxylated fullerenes to interfere with the evolutionary conserved innate immune system responses and encourages the use of fish models in studies of nanoparticle immunotoxicity.

Immunotoxicity assessment for the novel Spleen tyrosine kinase inhibitor R406

Spleen tyrosine kinase (Syk) is a novel pharmaceutical target for treatment of allergic, autoimmune, and neoplastic disorders. Previous studies have indicated that Syk signaling plays critical roles in regulating the lymphohematopoietic system. These observations prompted us to investigate whether inhibition of Syk would promote immunotoxicity. In a series of studies, rats were treated orally with R406, at dose levels up to and including 100 mg/kg/day (or its prodrug R788 at dose levels up to and including 100 mg/kg/day, reduced to 50 mg/kg/day for females as MTD was exceeded), a potent Syk inhibitor, twice daily for 28 days. In addition to standard toxicological assessments, immunophenotyping by flow cytometric analysis, and a study of humoral immune response measuring anti-KLH IgM and IgG levels, were undertaken. Other immunotoxicity studies included three host resistance models in female Balb/c mice to further ascertain effects of R406 on innate and acquired immunity. Following R406 treatment, expected immunomodulating effects (e.g., decreased thymic and spleen weight, hypocellularity of bone marrow, and reduced lymphocyte counts, including T and B cells) were observed in the rat studies. These changes essentially resolved during a 14-day treatment-free recovery period. A KLH challenge in rats demonstrated no adverse effects on IgG or IgM response. R788/406, administered orally at dose levels up to and including 80 mg/kg/day for 28 days, did not affect bacterial or viral clearance in the Listeria, Streptococcal, or Influenza host resistance mouse models, respectively. This correlated with previous in vitro macrophage and neutrophil function assays (assessing migration, phagocytosis, oxidative burst and microbicidal activity), which revealed that R406 did not adversely affect macrophage or neutrophil function in innate immune responses. Collectively, these results demonstrate that R406 has minimal functional immunotoxicity notwithstanding its lymphocytopenic effect, suggesting that inhibition of Syk might not lead to unacceptable mechanism-based adverse effects.

Lymphocyte subpopulations and neutrophil function in calves during the first 6 months of life

Fifteen clinically healthy calves were sampled every week during the first 5 weeks of life and thereafter every month until the age of 6 months. The percentages and absolute values of CD4+, CD8+ γδ TCR+ and WC1+ T cells, CD21+ B cells and NKp46+ NK cells were determined by flow cytometry, and the expression of the interleukin-2 receptor α chain (CD25) was measured to assess the level of activation of the lymphocyte subpopulations. Neutrophil phagocytosis, respiratory burst and bactericidal activity were measured in five different neutrophil function assays. Most of the parameters examined reached a stable level during the first 6 months of life. The proportions of CD4+ and CD8+ lymphocytes remained relatively stable during the study period, while there was a moderate decrease in the relative percentage of γδ T cells from birth to approximately 5 months of age. However, the absolute numbers of γδ T cells per millilitre of blood remained stable throughout the study period and did not display significant variation with age. The percentage of cells expressing the B-cell maturation marker CD21 increased significantly over the first 5 months of life. The proportion of NK cells showed substantial variation during the study. Marked differences in the relative proportions of the lymphocyte subpopulations were noted between the individual calves, and the individual ranking of the animals was largely maintained over time. CD25 expression was detected on a mean of 6.6% of the CD4+ cells, while a lower percentage of the other lymphocyte subpopulations expressed this receptor. Phagocytic activity was demonstrated in approximately 90% of the neutrophils, and this proportion remained stable during the entire study period, while respiratory burst activity showed a moderate decrease during the first 2 months of life. The present study shows that the T-cell subpopulations are present in peripheral blood of calves at levels comparable with adult values, while the B-cell population increases significantly with age. The decrease in the relative percentage of γδ T cells appears to be attributable to an increase in the absolute numbers of CD4+ and CD21+ cells, rather than a change in absolute γδ T-cell numbers. Furthermore, the results indicate that the neutrophilic granulocytes are functional and able to mount an effective response in young calves from the first week of life.

The effects of danofloxacin and tilmicosin on neutrophil function and lung consolidation in beef heifer calves with induced Pasteurella (Mannheimia) haemolytica pneumonia.

Pneumonia caused by Pasteurella (Mannheimia) haemolytica was induced in weaned beef heifer calves, approximately 6 months of age. Calves were treated at 20 h after challenge with therapeutic doses of danofloxacin or tilmicosin. Peripheral blood neutrophils were collected at 3, 24 and 48 h after treatment. The ex vivo effects on neutrophil function, neutrophil apoptosis, and hematological parameters were examined, as was the effect on percentage lung consolidation. Neutrophil function assays included random migration under agarose, cytochrome C reduction, iodination, Staphylococcus aureus ingestion, chemotaxis, and antibody-dependent and antibody-independent cell-mediated cytotoxicity. Apoptosis was determined using a cell death detection kit. Killing was performed at 72 h after treatment. Statistical comparisons were made among the three groups of challenged-treated animals: saline, danofloxacin, and tilmicosin. Comparisons were also made between nonchallenged nontreated animals (NCH) and challenged saline-treated animals. There were no significant differences for any of the neutrophil function assays or neutrophil apoptosis among the challenged-treated groups. This suggests that danofloxacin and tilmicosin have no clinically significant effects on neutrophil function or apoptosis. There were also no significant differences in percentage lung consolidation among the challenged-treated groups. Significant differences were found between the NCH calves and the challenged saline-treated calves in several neutrophil assays, which were attributed to effects of P. haemolytica infection.

Variability of neutrophil and pulmonary alveolar macrophage function in swine

Neutrophils and alveolar macrophages are essential defence mechanisms against bacterial infection of the lung. The purpose of this study was to evaluate the variability of a panel of neutrophil and alveolar macrophage function assays in swine, and to determine if the function of these leukocytes differed at various stages of production. Measured neutrophil functions included chemotaxis, phagocytosis, oxidative burst, and degranulation. Phagocytosis and oxidative burst were measured in alveolar macrophages isolated from bronchoalveolar lavage fluid (BALF). Both neutrophil and alveolar macrophage functions were highly variable from day-to-day and between pigs. Individual pigs did not have consistently high or low neutrophil and macrophage responses over time when compared to their cohorts. Older grower–finisher pigs had significantly greater neutrophil oxidative burst responses than younger suckling and weaner pigs ( P<0.001). Similarly, alveolar macrophages from suckling and early weaner pigs less than 40 days of age had significantly lower oxidative burst responses than those from older pigs ( P=0.02). Age-related variation in phagocytosis, chemotaxis, or granule secretion were not detected. These results establish baseline data for individual and age-related variation in swine leukocyte function, and form a basis for further evaluation of the contribution of non-infectious factors to development of the porcine respiratory disease complex.

Inhibition of neutrophil responses by cyclosporin A. An insight into molecular mechanisms.

Cyclosporin A (CsA) is an effective agent in rheumatoid arthritis (RA), slowing joint damage progression. Its therapeutic effect on T lymphocytes has been studied extensively, but there is little information available about neutrophils, the cells responsible for a substantial proportion of inflammation. A study was performed to investigate the in vitro effects of CsA on neutrophil functions triggered by several agonists and determine whether the drug could counteract the binding of formyl-methionyl-leucyl-phenylalanine (fMLP) to its receptor and/or modulate changes in the intracellular Ca(2+) concentration ([Ca(2+)]i). CsA was added to neutrophils 5-50 min before the incubation steps for neutrophil function assays (chemotaxis, superoxide anion production, lysozyme release), calcium measurements and receptor binding experiments. CsA appeared to be particularly effective in lowering chemotaxis, superoxide anion production and lysozyme release induced by different agonists. However, it did not significantly affect either basal or agonist-stimulated neutrophil [Ca(2+)]i and the interaction between fMLP and its receptor. Because of its in vitro inhibition of neutrophil functions, CsA appears to have considerable potential as an anti-inflammatory drug. Moreover, as it is also a potent immunosuppressive agent, it may reduce the progression of joint damage in RA. More work remains to be done to clarify the molecular mechanism of CsA action on neutrophils.

Function of neutrophils and chemoattractant properties of fetal placental tissue during the last month of pregnancy in cows

SUMMARY Neutrophils were isolated from the blood of pregnant cows on days 255, 265, and 275 of pregnancy, and on the day of parturition (n = 5/group), and in addition, simultaneously from 4 ovariectomized healthy cows (control animals). Neutrophils were subjected to neutrophil function assays (chemotaxis against zymosan-activated serum, random migration, ingestion of 125I-iododeoxyuridine [IdUR]-labeled Staphylococcus aureus, iodination of proteins, cytochrome C reduction, antibody-independent and antibody-dependent cell-mediated cytotoxicity). Results were expressed as percentage of control animals. Fetal placental tissue (cotyledon), uterine wall tissue, and skeletal muscle were obtained from the principal animals on the aforementioned days via laparotomy, and tissue suspensions were prepared. Chemotaxis of neutrophils was tested against tissue supernatants. Compared with day 255, there was an increase in ingestion of 125I-IdUR-S aureus at parturition, whereas iodination of proteins and cytochrome C reduction were reduced on the day of calving. The other neutrophil functions tested did not change over time of gestation. Fetal placental and uterine wall tissue attracted neutrophils with uterine wall tissue having a tendency to be more potent than cotyledonary tissue. Skeletal muscle tissue did not attract neutrophils. There was no change in chemotaxis response of neutrophils evoked by intrauterine and uterine tissues over time of gestation. It was concluded that at parturition, neutrophil function is impaired with respect to their bactericidal effects, which may render the animal more susceptible to bacterial infections, and that the chemoattractant properties of fetal placental and uterine wall tissues are tissue-specific, at least when compared with skeletal muscle. The presence of neutrophils in the placenta and other parts of the uterus may be necessary for placental separation and prevention from postpartum intrauterine bacterial infections, respectively.

Effect of two anticoagulants on leukocyte yield and function, and on lysosomal enzyme activity.

We compared acid citrate-dextrose (ACD-B) and heparin to determine which anticoagulant better preserves leukocytes for lysosomal enzyme assays if processing was done immediately or delayed for 24 h or more. Twenty normal subjects had blood drawn into tubes containing either ACD-B or heparin. The leukocytes were isolated by sedimentation in dextran (50 g/L) less than 2, 24, 48, and 72 h later. The most apparent difference was that cell counts indicated a 30% reduction in the number of leukocytes for ACD-B and a 95% reduction for heparin-treated cells at 48 h. The neutrophil function assay indicated that leukocyte processing must be done in less than 24 h regardless of the anticoagulant used, and that heparin is to be preferred. A comparison of heparin and ACD-B for maintenance of the activity of arylsulfatase A (EC 3.2.6.1) and hexosaminidase (EC 3.2.1.50) indicates that there is no effect of anticoagulant. However, at 48 h after venipuncture, there is an 80% reduction in the number of heparin-treated samples that are suitable for use in the assay. Those laboratories doing lysosomal enzyme tests on mailed specimens, which are most often greater than 24 h old, should use ACD-B as anticoagulant.