Polymorphonuclear Phagocytosis Research Articles

Oral vitamin C treatment increases polymorphonuclear cell functions in type 2 diabetes mellitus patients with poor glycemic control

This study investigated the effect of vitamin C on polymorphonuclear (PMN) cell functions in type 2 diabetes mellitus patients with poor glycemic control. We hypothesized that oral vitamin C treatment improves PMN cell functions. Patients (14) received either a vitamin C (1000 mg/d) or placebo (anhydrous calcium hydrogen phosphate) tablet for 6 weeks and were subjected to a 6-week washout period followed by a 6-week treatment crossover period. Blood samples were collected at pretreatment and posttreatment for PMN cell functions (by flow cytometry) and plasma vitamin C concentration. Phagocytosis was examined by incubating whole blood samples with fluorescein isothiocyanate–labeled Staphylococcus aureus, and oxidative burst was simultaneously evaluated by adding hydroethidine. In comparison with placebo, vitamin C increased both PMN cell phagocytosis (pretreatment: placebo, 17.8% ± 1.6% and vitamin C, 19.0% ± 3.4%, P = .70; posttreatment: placebo, 16.6% ± 1.7% and vitamin C, 27.1% ± 2.9%, P = .005) and oxidative burst (pretreatment: placebo, 6.4% ± 0.8% and vitamin C, 7.1% ± 1.2%, P = .60; posttreatment: placebo, 6.9% ± 1.3% and vitamin C, 12.1% ± 1.6%, P = .02). The plasma vitamin C concentration was elevated after vitamin C treatment as compared with that before treatment (P < .001) and was higher than that observed in the placebo treatment group (P < .01). Plasma vitamin C concentration and PMN cell functions were not significantly different before both treatments. We conclude that the 6-week 1000-mg/d vitamin C increased PMN phagocytosis and oxidative burst in type 2 diabetes mellitus patients with poor glycemic control.

Enhancement of vitamin C on PMN function in healthy individuals

Abstract Vitamin C (ascorbic acid, ascorbate) promotes immune cell activities by supporting various cellular functions especially, in neutrophils or polymorphonuclear (PMN) leukocytes, which play an important role in bacterial infection and acute inflammation. Little data have been reported in impact of vitamin C on PMN function during bacterial phagocytosis in healthy individuals. Here, we have elucidated the effect of vitamin C on PMN phagocytosis and oxidative burst to Staphylococcus aureus by whole blood assay and flow cytometry after pre-incubation without and with vitamin C in vary concentrations. The results showed that vitamin C concentration at 20 mM induced a significant increase phagocytosis (p-value=0.03) and had trend to significant increase oxidative burst (p-value=0.06) while vitamin C concentration of 30 mM showed significant increase both phagocytosis and oxidative burst (p-value=0.04 and 0.02, respectively) at only 15 min of exposure to the bacteria. As a minimal concentration that can increase phagocytosis, we selected vitamin C at 20 mM as optimal concentration to investigate more data by enrollment of healthy volunteers (n=17). The results revealed significant difference both phagocytosis and oxidative burst after 20 mM vitamin C pre-incubation (p&amp;lt;0.0001). In conclusion, we showed that vitamin C at 20 mM can boost phagocytosis and oxidative burst of neutrophils in healthy adults within 15 min after bacterial exposure. This rapid activation may be applied to fight bacterial infection. Nevertheless, further research in clinical trial and underlying mechanisms await further studies.

Enhancement of Natural Killer Cell Activity and Phagocytosis in Healthy Subjects by Immulina, a Spirulina Extract Enriched for Braun Type Lipoproteins

Immulina is a commercial extract of Spirulina (Arthrospira) platensis that is standardized by biological activity. We previously reported that this extract is a potent activator of THP-1 monocytes in vitro and that oral consumption enhanced several immunological functions in mice [1]. In this study we further characterized Immulina by determining that Braun type lipoproteins are responsible for a major portion of the in vitro monocyte activation exhibited by this material. In order to understand the effect of Immulina on the human immune system, a pilot study was conducted on ten healthy individuals who supplemented their diet with Immulina (400 mg/day) for seven days. Blood was drawn from the participating individuals at two time points: before and after seven days of Immulina supplementation. Changes in mononuclear and polymorphonuclear phagocytosis were determined in heparinized whole blood as well as the cytotoxicity exhibited by natural killer (NK) and lymphokine activated killer cells. We observed statistically significant increases both in tumor cell killing by NK cells (p = 0.0019) and in phagocytosis by blood mononuclear cells (p = 0.0124) after Immulina supplementation. Acknowledgements: This research was partly funded by a USDA, Agricultural Research Service Specific Cooperative Agreement No. 58-6408-012. Immulina capsules were supplied by Scandinavian Clinical Nutrition Denmark A/S, Greve, Denmark.

Measurement of phagocytosis and oxidative burst of canine neutrophils: high variation in healthy dogs.

Quantitative analysis of phagocytosis and oxidative burst in canine polymorphonuclear (PMN) cells was performed by flow cytometry techniques. Different concentrations of phorbol myristate acetate (PMA) were used to modulate PMN phagocytosis. A low concentration of PMA (3 nmol) resulted in increased phagocytic activity of canine PMN, which could not be enhanced by higher dosages. Experiments with a reference cell population showed high losses of PMN, most probably by adherence to plastic material. It was possible to avoid this loss by layering all ingredients on cushions of Histopaque. However, Histopaque had a negative influence on the phagocytic activity of canine PMN. The use of PMA led to a dosage-dependent increase in the oxidative burst measured by the production of reactive oxygen species (ROS). Cushions of Histopaque were used to avoid cell loss. There was no negative influence of Histopaque on ROS formation. Storage of canine PMN for 24 h at room temperature had no negative influence on phagocytosis or oxidative burst measurements. Variations in the ROS assays conducted by two different examiners could be eliminated by use of a Histopaque-cushion.

Measurement of phagocytosis and oxidative burst of canine neutrophils: high variation in healthy dogs

Quantitative analysis of phagocytosis and oxidative burst in canine polymorphonuclear (PMN) cells was performed by flow cytometry techniques. Different concentrations of phorbol myristate acetate (PMA) were used to modulate PMN phagocytosis. A low concentration of PMA (3 nmol) resulted in increased phagocytic activity of canine PMN, which could not be enhanced by higher dosages. Experiments with a reference cell population showed high losses of PMN, most probably by adherence to plastic material. It was possible to avoid this loss by layering all ingredients on cushions of Histopaque®. However, Histopaque® had a negative influence on the phagocytic activity of canine PMN. The use of PMA led to a dosage-dependent increase in the oxidative burst measured by the production of reactive oxygen species (ROS). Cushions of Histopaque® were used to avoid cell loss. There was no negative influence of Histopaque® on ROS formation. Storage of canine PMN for 24 h at room temperature had no negative influence on phagocytosis or oxidative burst measurements. Variations in the ROS assays conducted by two different examiners could be eliminated by use of a Histopaque®-cushion.

Effects of taurine on polymorphonuclear phagocytosis activity in burned patients.

This study determines the effects of taurine (Tau) on phagocytosis of polymorphonuclear neutrophils (PMN) isolated from normal subjects (n = 41) and severely burned patients (n = 20). Phagocytosis was measured by nitroblue of tetrazolium (NBT) reduction in samples with and without latex bead stimulation. Taurine was added at doses of 0.2, 0.4, 0.8 and 1.6 mM to stimulated samples. In control cells there were statistically significant increases in phagocytosis after addition of Tau 0.8 mM and 1.6 mM to as compared to samples without Tau addition (295 +/- 23% and 330 +/- 35% vs. 248 +/- 18%; mean +/- S.E.; p < 0.05). A statistically significant increase in phagocytosis was observed in cells from the burned population after addition of Tau 1.6 mM (288 +/- 38% vs. 198 +/- 13%; mean +/- S.E.; p < 0.05). No changes in phagocytosis were found in cells from a subgroup of burn patients (n = 13) followed over 7, 15 and 21 days. These results indicate that taurine supplementation in vitro at doses of 0.8 to 1.6 mM improves the phagocytic capacity of neutrophils in healthy subjects and in patients with severe burn injury, mainly when neutrophil function is unaltered.

Parenteral lipid emulsions and phagocytic systems.

Lipid emulsions (LE) for parenteral use are complex emulsions containing fatty acids, glycerol, phospholipids and tocopherol in variable amounts and concentrations. In clinical practice, LE have been employed for more than 30 years. Fatty acids may have different impacts on phagocytic cells according to their structure. Experimental and clinical studies have consistently shown that LE modify monocyte/macrophage and polymorphonuclear phagocytosis. The inhibitory effect of LE on the functional activity of the phagocytic system, although still clinically controversial, may have a harmful impact because total parenteral nutrition with lipids may be recommended in hypercatabolic conditions where inflammation and infection are present. LE based on triglycerides containing long chain fatty acids (termed long chain triglycerides or LCT) are the main parenteral fat source and are typically rich in n-6 polyunsaturated fatty acids. They may have adverse effects on the immune system, especially when given in high doses over a short period of time. However when administered properly they can be used safely. LE containing medium chain triglycerides (MCT) may have some advantages because of their positive effects on polymorphonuclear cells, macrophages, and cytokine production, particularly in critically ill or immunocompromised patients. New parenteral LE containing n-3 polyunsaturated fatty acids or monounsaturated olive oil are already available in Europe. Judicious use of these new LE is mandatory especially relating on their potential impact on the immune system. New experimental and clinical studies are required to further establish the role of LE in clinical nutrition.

Immunostimulating and protective effects of an oral polybacterial immunomodulator ‘Dentavax’ in a rabbit experimental model

The immunostimulating and protective effects of an oral polybacterial immunomodulator, Dentavax (D), composed of killed cells from Klebsiella pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Candida albicans and Lactobacillus acidophilus and their lysates, have been investigated on an experimental rabbit model. In this model, mixed suspensions of the above bacterial wild strains have been injected in six sides of oral mucosa. A long-lasting inflammation with the development of infiltrates and confluating abscesses has been observed. The influence of orally given Dentavax on the course of the model infection as well as on the dynamics of the immune response has been studied. A two-fold decrease in the duration and severity of inflammatory reaction, confirmed by the histological findings, has been registered. In immunised animals, an activation of polymorphonuclear phagocytosis, together with stimulation of humoral systemic and mucosal immunity with synthesis of specific serum (predominantly, IgG) and coproantibodies (predominantly, S-IgA) determined by ELISA, has been found. The results obtained proved the strong immunostimulating and protective effects of the preparation D, which is meant for the prophylaxis and treatment of inflammatory periodontal diseases.

In vitro Effects of Meropenem and Imipenem/Cilastatin on Some Functions of Human Natural Effector Cells

Meropenem, a new carbapenem antibiotic, was assessed to evaluate its effects on some functional parameters of human polymorphonuclear (PMN) and natural killer (NK) cells in comparison with imipenem/cilastatin. Both drugs significantly inhibited PMN phagocytosis and chemotaxis at concentrations of 2,000 and 4,000 μg/ml. They affected PMN microbicidal activity, evaluated against Candida albicans, only at 4,000 μg/ml. A study of the effects of both drugs on peripheral NK populations and the human NK line (NK-92) showed that even at 4,000 μg/ml there was no effect on antitumor activity. These data indicate that meropenem can reduce some PMN antimicrobial functions only at very high concentrations like imipenem/cilastatin, whereas no concentration influenced NK activity.

The Role of Chemotherapy in Polymorphonuclear Phagocytosis in Pneumonia

The efficacy of the host defense system is a determining factor for the outcome of antimicrobial events in infected patients. The neutrophil granulocyte plays a key role in the lung’s defense against bacterial invasion and in the absence of a sufficient attraction of functionally intact neutrophils in the lung, following bacterial challenge, severe pulmonary infection may result. The involvement of phagocytes in pneumonia is well known: infiltration of lung parenchyma by neutrophils occurs within a short time in response to infection and is followed by an influx of monocytes.We investigated the effects of antimicrobial therapy in pneumonia on polymorphonuclear neutrophil (PMN) phagocytosis.

Polymorphonuclear Phagocytosis and Killing in Workers Occupationally Exposed to Hexachlorobenzene

Phagocytosis and intracellular killing of Candida albicans and Candida pseudotropicalis by neutrophils from 66 workers exposed to chlorinated compounds were studied. Phagocytosis with both antigens was normal in all the workers studied. However, lytic activity of the neutrophils in the presence of both antigens, C. albicans and C. pseudotropicalis was impaired. These findings might be related to a derangement in the antioxidant mechanisms and to some interference with GSH levels that might lead to the abnormal functions observed in neutrophils.

A comprehensive hematologic study in calves with total artificial hearts.

Thromboembolism and infection remain potential threats for long-term circulatory assist and replacement devices. The alteration of the hemostatic system and of blood cell functions caused by device implantation may predispose the recipient to these complications. Many sensitive coagulation assays and the technology of flow cytometry would be powerful tools for this investigation. The availability of such immunologic technologies for animal species other than humans has yet to be established. In a series of in vitro tests we found that the following assays, among others, are usable in calves: TAT, TxB2, platelet surface glycoprotein IIbIIIa, and membrane aminophospholipid. F1.2, D-dimer, beta TG, PF-4, and platelet surface expression of GMP-140 and receptors for fibronectin, thrombospondin, and vWF were not measurable. A sustained mild decrease in hematocrit levels in six calves with the Cleveland Clinic-Nimbus total artificial heart for 11-120 days was attributed to an increase in circulating blood volume, but not to red blood cell damage. Whole blood platelet aggregation was suppressed only for the first 3 post operative days, with decreased GPIIbIIIa expression. Polymorphonuclear phagocytosis, chemotaxis, and superoxide anion production were not altered. Device infection and thromboembolism occurred in one of 13 cases overall.

Polymorphonuclear phagocytosis and killing in workers exposed to inorganic mercury

The ability of neutrophils to phagocytose and kill Candida species as well as the splenic phagocytic function were investigated in workers from a mercury-producing plant. In the neutrophil phagocytosis study, two species of Candida were used in since in individuals with myeloperoxidase deficiency neutorphils are unable to kill Candida albicans, while Candida pseudotropicalis can be effectively lysed. Phagocytosis of both antigens and splenic phagocytic function were normal in all the workers studied. However, following ingestion of the organisms there was considerable reduction in the ability of neutrophils from exposed workers to kill both species of Candida, and this was not explained by a mild impairment of phagocytosis. After improvement in the hygiene conditions in the factory, a new evaluation was performed, 6 months later, in the same workers and urinary mercury concentrations were determined monthly in each worker. Despite a significant reduction in urinary mercury concentrations, a greater impairment in the ability of neutrophils to kill C. albicans was observed. The killing of C. pseudotropicalis presented no further impairment when compared to the previous evaluation. These results suggest that impairment of the lytic activity of neutrophils from workers with urinary mercury concentrations within the safe level for exposed population is due, at least in part, to some interference with myeloperoxidase activity. In addition, the mercury-NADPH complex, once formed, could limit the utilization of reduced pyridine nucleotides by NADPH- dependent enzymes such as NADPH oxidase, thereby inhibiting the PMN respiratory burst.

INTRACISTERNALLY GIVEN ELASTASE/E/ENHANCES BLOOD-BRAIN BARRIER/BBB/ PERMEABILITY IN NEWBORN PIGLETS

Elastase, released during polymorphonuclear phagocytosis, is a potentially tissue damaging enzyme. We investigated its effect on BBB-permeability in newborn piglets. 11 anesthetized animals /Group 1/ were given 1 μg porcine E in 0,5 ml artificial CSF intracisternally, 7 piglets (given mock CSE) served as controls /Group 2. Through 4 hours afler E administration permeability changes of pial microvessels were followed intravitally by fluorescence photomacroscope using an open cranial window. Tracer was 1 % Na-fluorescein (NaF). Physiological parameters (HR, MABP, blood gases, acid-base status) were continuously monitored. Spolly NaF extravasation was seen in all animals in Group 1 (meantime elapsed from E administration: 78±13 min.), but the BBB remained tight in Group 2 till the end of the experiments, α1-proteinase inhibitor /α1-VI/ activity was measured in sera and in CSF at 0, 2, 4 h after E injection. In CSF of Group 1 it increased significantly (Oh: 8,9±4,1 - 2h: 173,7 ± 46,5 - 4h 416,8±132,9 μg/ml), while its activity wasn't elevated in Group 2 (Oth: 8,8 ± 4,6 2h: 12,8 ± 3,9 - 4h 6,7±2,4 μg/m]). In the sera measured paralelly, α1-PI activity remained constant in both groups. Free E wasn't detectable in all samples. White blood cell count elevated in CSF (Oh: 1 ± 1 - 4h: 63±14 μl−1) and in sera (Oh: 2453 ± 355 - 4h: 7518 ± 751 μl−1) in Group 1, but didn't change in Group 2. We conclude ihat elastase may play an additional role in BBB injury during neonatal meningitis. All values are: x, + S.E.

IL-2-based immunotherapy alters circulating neutrophil Fc receptor expression and chemotaxis.

We performed functional assays on polymorphonuclear (PMN) leukocytes from 21 patients with advanced cancers, before, during, and after IL-2 administration. Of these, 19 were treated with high dose bolus IL-2 infusions (10(5) U/kg every 8 h) and 2 patients received low dose continuous infusions of IL-2 (250 U/kg/h). Five of six patients studied after IL-2 therapy had a decrease in their PMN chemotactic response to FMLP after bolus IL-2 (mean 8 doses) or, after the 4th day of continuous infusion IL-2 (pre-IL-2 values of 82% +/- 17% to 45% +/- 1% post-IL-2, p2 less than 0.004) compared with normal control values. In 8 of 10 patients studied, PMN capacity to oxidize intracellular dichlorofluorescein dye, an indirect measurement of O2- production in response to PMA stimulation, decreased after IL-2 administration (pre-IL-2 mean dichlorofluorescein oxidation (by channel number) 243 +/- 128 vs 3-day post-IL-2 87 +/- 86, p2 less than 0.02). Furthermore, a marked decrease in Fc gamma R III (Leu-11, CD16) expression was observed in 12/13 patients' PMN studied after IL-2 therapy (mean percent of PMN population with positive FcR expression was 81.1 +/- 15.4% pre-IL-2 which decreased to 56.0 +/- 30.5% post-IL-2, p2 less than 0.001). Other PMN surface markers (My4, My7, ICAM-1, LFA1, LFA3, Mac1) did not change significantly. PMN-mediated antibody-dependent cellular cytotoxicity did not change after IL-2 therapy (only 4/15 patients demonstrated more than 50% reduction in antibody-dependent cellular cytotoxicity). PMN phagocytosis of Staphylococcus aureus was also not significantly altered by IL-2 administration in six patients studied (pre-IL-2, 99 +/- 17% vs 111 +/- 28% post-IL-2, p2 greater than 0.2). We conclude that the systemic administration of IL-2 by intermittent or continuous administration is associated with marked changes in PMN function and cell surface receptor expression. These alterations may contribute to the apparent increased susceptibility to bacterial infection observed in these patients.

Impaired polymorphonuclear leukocyte function in chronically hemodialyzed patients with iron overload.

Polymorphonuclear (PMN) leukoyte function tests and clinical data analyses were performed in 28 chronic renal failure (CRF) patients receiving regular hemodialysis. We divided them into two groups; 12 patients with normal serum ferritin were classified as group 1 and 16 patients with high serum ferritin as group 2. There was no difference in age, BUN, serum creatinine, complement (C3, C4), peripheral white blood cell count and the duration of dialysis between the two groups, but the serum iron level was higher in group 2 (129.1 ± 46.58 μg/dl) than in group 1 (74.3 ± 20.9 μg/dl) (p<0.001). The total iron binding capacity was lower in group 2 (p<0.05) and the number of transfusions was higher in group 2 (25 ± 16.1) than in group 1 (12 ± 8.7) (p<0.05). The nitroblue-tetrazolium (NBT) test showed no difference among groups 1, 2 and the healthy control group. In chemotaxis to fMLP (N-formylmethionylleucylphenylalanine), the mean number of migrated neutrophils to fMLP (10−6 M/L) per high power field was significantly decreased in group 2 (99.5 ± 37.6) compared with the healthy control group (140.1 ± 13.4) (p<0.005), but not in group 1 (155.8 ± 79.4). In the phagocytosis test using Staphylococcus aureus, the phagocytic index ratio compared to the healthy control group was significantly decreased in group 2 (0.59 ± 0.14), but not in group 1 (0.97 ± 0.18). These results suggest that iron overload due to multiple transfusions in patients receiving regular long term hemodialysis may play a part in causing susceptibility to infection by impairing PMN leukocyte functions, especially chemotaxis and phagocytosis.

Are there circadian variations of polymorphonuclear phagocytosis in man?

Eleven male subjects were investigated to detect a possible circadian rhythm of the polymorphonuclear phagocytosis. Both cell activity and serum opsonins were studied for numerical detection of granulocytes having ingested at least one particle and for the mean number of ingested particles per cell. No significant temporal differences (ANOVA and cosinor) were found.

Effect on leukocyte locomotion and superoxide production by uremic toxins and polyamines

We have isolated peak II, which is the peak of the middle molecules containing polyamines, from dialysate of uremic patients in recirculating dialysis. We investigated the effect of total dialysate, chromatographic peaks, I, II, III, IV and commercial polyamines on polymorphonuclear leukocyte chemotaxis and superoxide production (i.e. phagocytic activity) in healthy and uremic patients. Polymorphonuclear chemotaxis was inhibited by total dialysate and peak II but not by polyamines; polyamines, total dialysate and peak II had no activity on polymorphonuclear phagocytosis.

Characterization of neutrophil iodination for the assessment of phagocytic and opsonic function in septic patients

To increase our understanding of the nature of surgical infection, further studies on the host defense abilities of infected patients are required. Therefore, a more thorough investigation of the iodination method for the measurement of polymorphonuclear leukocyte function and serum opsonic activity was undertaken to characterize its application in surgical infection. A significant relationship was found between the phagocytic indices derived from different standard neutrophils or sera measured on the same day. When expressed as a value of normal phagocytic indices minus abnormal phagocytic indices, this relationship was constant from day to day despite wide variations in the absolute phagocytic index values. This finding enables direct comparisons to be made between the values obtained both from the same patient and from different patients during the course of their illness by reference to daily control values. We also found that the system was sufficiently sensitive to detect, in a dose-responsive manner, the changes induced in normal neutrophil phagocytosis and serum opsonic activity by a specific bacterial challenge with either K. pneumoniae or E. coli. In addition, zymosan, which is utilized in the iodination reaction but also has immunoadjuvant properties, was found to enhance neutrophil function but depress serum opsonic activity in the face of such bacterial challenges. We conclude that the iodination technique is a credible method for the indirect measurement of polymorphonuclear phagocytosis and serum opsonic function in the face of a bacterial challenge and can be reliably employed in studies of septic patients provided these findings are taken into account.

Impaired opsonic activity but normal phagocytosis in low-birth-weight infants.

Abstract Blood of 19 term and 19 low-birth-weight (LBW) neonates was studied for polymorphonuclear phagocytosis, intracellular killing and serum opsonin activity. Serums from 12 of 13 infants under 1925 gm had inadequate opsonins against Staphylococcus aureus 502A and Serratia marcescens when combined with either autologous or normal adult leukocytes. Serums from adults, term newborn infants and physiologically hypogammaglobulinemic infants did not exhibit this serum abnormality. With adult opsonin, cells from all 15 normal term and 13 of 14 normal LBW neonates phagocytized and killed bacteria normally. However, cells from six of nine term and LBW infants with abnormal clinical findings showed diminished phagocytic and killing rates. Levels of γG and β1c globulins were significantly higher in term than in LBW serums; γM and γA globulins were similar in the two groups. Low levels of γG globulin in LBW infants are probably the major factor responsible for their opsonic defect.