Peripheral Blood Leukocyte Function Research Articles

Separation and phagocytosis analysis of peripheral blood leukocytes from Qihe crucian carp Carassius auratus

The phagocytic function of peripheral blood leukocytes (PBL) in teleost fish is the bridge between cellular-mediated and humoral immunity and plays a central role in the immune system of the teleost fish. However, the phagocytic ability of PBL from Qihe crucian carp Carassius auratus remains unclear. This study aimed to build a convenient separation method to obtain high purity PBL from Qihe crucian carp and to explore their phagocytic characteristics. The results showed that the PBL of Qihe crucian carp (QPBL) could be obtained effectively by 1.085 g/ml percoll separation liquid. Most isolated QPBL were round or elliptical, with horseshoe-shaped, reniform, or irregular nucleus. Finger-like or filamentous protrusions were present on the cell surface, and some cells had a long pseudopod. According to the results of scanning electron microscopy (SEM), transmission electron microscopy (TEM), and fluorescence microscope, fluoresbrite carboxylate YG microspheres (1 μm) could be effectively phagocytosed by QPBL, and various phagocytic stages could be observed. The phagocytic cells in QPBL have morphology similar to those of “professional” phagocytes (monocyte and granulocyte). Notably, the percentage of cells, which can engulf microspheres, increased to approximately 22% after incubation with microspheres for 90 min, and it showed no obvious changes until 180 min. Furthermore, two important phagocytosis regulatory protein genes, Rab5 and Rab7, were seriously up-regulated during the phagocytosis in QPBL. Taken together, an efficient and convenient separation method for isolating QPBL was established, and the isolated QPBL had a high phagocytic activity towards the external particles. The results could provide the basic foundation for further study of the immune function of PBL in Qihe crucian carp and other teleost fish.

Plasma exosomes impact on paracrine secretion of peripheral blood mononuclear cells in patients with chronic heart failure

The study of the exosomal effect on the paracrine secretion of peripheral blood mononuclear cells of patients with chronic heart failure (CHF) in vitro demonstrated gender-associated changes in the synthesis and secretion of the key factors of endothelial inflammation by leukocytes.Under the exosomesinfluence, the synthesis and accumulation of the cytokine monocyte chemoattractant protein-1 (MCP-1) in the leukocytes of healthy donorsincreased by 26%; while in women by 18%, in men by 38% was observed. Incubation of patients’cells with suspension of exosomes increased intracellular synthesis of MCP-1 in women by 16%, in men by 19%.Interestingly, in activated cells of control women group the synthesis of MCP-1 under the exosomes influence was 2.7 times higher than in the group of female patients and in leukocytes of male donors 4 times higher than in the CHFmale group. Thus, under the exosomes influence the synthesis of the cytokine MCP-1 in cells increaseswhereasits paracrine secretion decreases, which may be considered as one of the potential physiological mechanisms of “restraining” the progress of inflammation. Our findings suggest that the level of cytokinetumor necrosis factor-alpha (TNFα) synthesis in activated leukocytes of CHF and control groups was almost the same, while in CHF group the TNFα level increases only in the culture medium. Incubation with exosomes did not cause significant changes in cytokine production, but in the CHF group there was a tendency to decrease the “medium/cell” index by more than 1.3 times. Subsequent future studies of the exosomal corrective effects would enable to establish algorithms for potential therapeutic solutions on initiative mechanisms of endothelial inflammation and development of acute and chronic ischemia. Our primary experimental work demonstrates the need for further study of the exosome’s influence on peripheral blood leukocyte function both in vitro and in vivo experimental models.

Crotalus neutralising factor and its role in human leukocyte modulation.

Crotalus neutralising factor (CNF) is an endogenous γ-type phospholipase A2 (PLA2) inhibitor that inhibits the toxic action of crotoxin, a neurotoxin present in Crotalus durissus terrificus venom. However, its effects on the activation and modulation of immune cells, which play a major role in the development of inflammation, is not known. The objective of the present study was to assess the effects of CNF on human leukocyte modulation in vitro by analysing the following parameters: cell viability, phagocytic capacity, lipid droplet formation, reactive oxygen species production, nitric oxide production, p38 MAPK activation, and cytosolic PLA2 (cPLA2) gene expression. Neutrophils and peripheral blood mononuclear cells from healthy donors were isolated via the density gradient method, resuspended in RPMI medium, and incubated with RPMI (negative control), LPS, or PMA (positive control) or CNF (sample test) at a concentration of 50 μg/mL. Results showed that CNF was not toxic to human neutrophils after 48 and 72 h of incubation. CNF treatment induced an increase in PBMCs and neutrophil phagocytic capacity, as well as the formation of lipid droplets within these cells after 1 h of incubation. However, CNF did not induce the formation of reactive oxygen and nitric oxide species. Moreover, CNF induced p38 MAPK protein phosphorylation and cPLA2 gene expression in neutrophils. The data obtained herein showed that CNF action modulates human leukocytes, CNF activates important signalling pathways for human leukocytes, and it is pro-inflammatory. These findings also complement previous studies on CNF action on human peripheral blood leukocyte function.

Cytokine expression and lymphocyte proliferative capacity in diseased harbor porpoises (Phocoena phocoena) – Biomarkers for health assessment in wildlife cetaceans

Harbor porpoises (Phocoena phocoena) in the North and Baltic Seas are exposed to anthropogenic influences including acoustic stress and environmental contaminants. In order to evaluate immune responses in healthy and diseased harbor porpoise cells, cytokine expression analyses and lymphocyte proliferation assays, together with toxicological analyses were performed in stranded and bycaught animals as well as in animals kept in permanent human care. Severely diseased harbor porpoises showed a reduced proliferative capacity of peripheral blood lymphocytes together with diminished transcription of transforming growth factor-β and tumor necrosis factor-α compared to healthy controls. Toxicological analyses revealed accumulation of polychlorinated biphenyls (PCBs), dichlorodiphenyldichloroethylene (DDE), and dichlorodiphenyltrichloroethane (DDT) in harbor porpoise blood samples. Correlation analyses between blood organochlorine levels and immune parameters revealed no direct effects of xenobiotics upon lymphocyte proliferation or cytokine transcription, respectively. Results reveal an impaired function of peripheral blood leukocytes in severely diseased harbor porpoises, indicating immune exhaustion and increased disease susceptibility.

Circulating immune complexes of calves with bronchopneumonia modulate the function of peripheral blood leukocytes: In vitro evaluation

In this work we studied if circulating immune complexes (CIC) of calves with bronchopneumonia have the capacity to modulate function of peripheral blood leukocytes of healthy cattle.CIC of three month old calves (6 healthy and 6 diseased) were isolated by PEG precipitation. Peripheral blood mononuclear cells (MNCs) and granulocytes from healthy calves and cows were the CIC responder cells in in vitro tests.The most remarkable increase of adhesiveness to polystyrene and ROS synthesis (assessed by NBT test) was detected in cows' granulocytes stimulated with CIC of diseased calves. Results of MTT test showed that CIC of both healthy and diseased calves reduced granulocytes' viability. The strongest effect of inhibition of cows' granulocytes resulted from CIC of diseased calves. CIC only moderately reduced spontaneous viability of calves' MNCs. Again, the strongest effect of CIC isolated from diseased calves was observed. In contrast to the low impact of CIC on non-stimulated cells, their inhibitory effect on viability of mitogen stimulated MNCs was very strong. With CFSE assay we showed that both types of CIC stimulated spontaneous, but inhibited mitogen induced proliferation of calves' MNCs. Propidium iodide staining reviled that CIC increased apoptosis/necrosis of both non-stimulated and mitogen stimulated MNCs.CIC of both healthy and diseased calves modulated the function of peripheral blood MNCs and granulocytes, but a stronger effect of CIC of diseased calves was shown. The age of the donors (calves or cows) of the responder cells, and the activation state of these cells, were also of influence.

Abstract 3516: Evaluation of whole body hyperthermia system: changes of core body temperature and their effects on immune system

Abstract We have recently introduced a whole-body hyperthermia (WBH) system (Heckel ht3000) which is a specific device for WBH consisting of four water-filtered infrared-A (wIRA) irradiators and synthetic leather tent for heat-retention. We are now studying how this system should be used in combination with cancer immune-cell therapy such as DC-based vaccination and adoptive T cell therapy. The aim of this study is to examine the effects of increase in core body temperature on the number and function of peripheral blood leukocytes. Heat treatment was applied to healthy volunteers with this system; rectal temperature which was used as a representative of core body temperature, and axillary temperature were monitored simultaneously. During the irradiation phase, the volunteers were exposed to wIRA until their rectal temperature reached at 38.5°C, and they were then wrapped in synthetic leather tent for 60 min (heat retention phase) in order to maintain rectal temperature within fever range (38.5-40.5°C). Peripheral blood was taken from the each volunteer 3 times; before irradiation, at the time when rectal temperature reached at 38.5°C, and when elevated rectal temperature returned to 38.5°C after the completion of heat retention. Blood samples were analyzed by using flow cytometry to examine the number of leukocytes, lymphocytes subsets and NK cells-killing activities. During the irradiation phase, rectal temperature of every volunteer was gradually elevated, and it reached at 38.5°C after 60-90 min irradiation. Moderate but continuous elevation of rectal temperatures was observed even after irradiation, and they were kept at 39.0-39.5°C during the heat retention phase. After the volunteers got out of the heat retention tent, rectal temperatures were gradually decreased, and 30-60 min later, it returned to below 38.5°C. Regarding the number of peripheral leukocytes, NK cells showed the most striking change. In fact, they showed rapid increase in number at the time when rectal temperature reached at 38.5°C, on the other hand, the number of NK cells sharply decreased to approximately 50-60% of the one in pre-irradiation phase when the elevated rectal temperature retuned to 38.5°C. We also examined how NK cells changed their functions and phenotypes in some volunteers, and found that killing activity significantly enhanced after heat-treatment in fever range. Regarding the number of other types of cells, monocytes and granulocytes showed gradual increase during and after heat-treatment. By contrast, the number of the T cells, particularly the number of CD62L positive T cells, was moderately decreased after heat-treatment, which is presumably due to their homing to secondary lymphoid tissues. In conclusion, WBH has various impacts on human immune system so that the combination of immune-cell therapy and WBH could be a more powerful therapy of enhancing immune system. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3516. doi:1538-7445.AM2012-3516

Oral Antibiotics Attenuate Bowel Segment Reversal–Induced Alterations in Subpopulation and Function of Peripheral Blood Leukocytes, Thymocytes, and Splenocytes in Massive Bowel‐Resected Rats

The authors previously demonstrated that oral antibiotics significantly attenuated inflammatory response, improved intestinal bacterial overgrowth, and augmented anabolic response in massive bowel-resected rats with bowel-segment reversal. Herein, the effects of oral antibiotics on immune functions were investigated. Male Wistar rats were subjected to a sham operation or a 70% small bowel resection with or without a 3-cm small bowel segment reversal. Thereafter, half numbers of animals with bowel resection and reversal were orally administered clindamycin plus amoxicillin (50 plus 50 mg/kg/day) for 3 weeks. Age-matched nonsurgical rats were included as references. Peripheral blood, spleen, and thymus were collected for analyzing immunocyte subpopulations and function. Bowel resection significantly decreased weight gain, thymic weight, and splenic helper-T, suppressor-T, and mature-B cells but significantly increased splenic macrophage distribution and concanvavalin A-stimulated splenocytic proliferation and cytokine production, such as tumor-necrosis factor (TNF)-alpha, interferon-gamma, and interleukin (IL)-2 (1-way ANOVA, P<.05). Bowel segment reversal further decreased circulating suppressor-T cells but increased circulating natural killer cells and spontaneous splenocytic TNF-alpha production. Segment reversal-induced elevations in bacterial numbers of gut content, circulating white blood cell, nitric oxide, IL-6, splenocytic interferon-gamma and IL-2 production, reductions in T-thymocytic distribution, and alterations in thymocytic interferon-gamma and IL-2 production were attenuated by oral antibiotics. Moreover, oral antibiotics significantly increased splenocytic granulocytes and spontaneous thymocytic proliferation. Oral antibiotics might augment the beneficial effects of bowel segment reversal on anabolism via attenuating bacterial overgrowth and inflammatory response, and restore subpopulation and function of splenocytes and thymocytes in massive bowel-resected rats.

Heterogeneous medically unexplained symptoms and immune function

It has been suggested that dysregulation of immune-to-brain communication plays a role in the biopsychological process underlying medically unexplained symptoms (MUS). Immune and non-immune stressors can both be involved in the activation of the central sickness-behavioural-system leading to complaints like malaise, pain and fatigue. We hypothesized increased pro-inflammatory and/or reduced anti-inflammatory cytokine activity to exist in MUS patients. Twenty-seven participants (4 male; 23 female) with heterogeneous MUS were compared with 27 healthy controls (6 male; 21 females). Blood samples were analysed for leukocyte subset cell counts, in vitro T-cell mitogen-stimulated cytokine production (IL-2, IL-4, IL-5, IL-6, IL-10, TNF-α and IFN-γ) and in vitro monocyte cytokine release (IL-1β, IL-6, IL-8, IL-10 and TNF-α) in response to increasing concentrations of LPS. No significant group differences were found for any of the cytokines measured. One unexpected exception was an elevation in the number of circulating B and NK-cells in participants high on MUS. Nonetheless, no support was found for the hypothesized immunological dysregulation in peripheral blood leukocyte function of MUS patients.

Immunomodulatory effects of granulocyte and monocyte adsorption apheresis as a treatment for patients with ulcerative colitis.

Our aim was to understand the mechanism of immunological changes associated with the use of an adsorptive-type extracorporeal device (Adacolumn) that has been developed for selective adsorption of granulocytes and monocytes/macrophages from peripheral blood of patients with active ulcerative colitis. The column is filled with carriers (G-1 beads) that have a diameter of 2 mm and are made of cellulose diacetate. In peripheral blood treated with the G-1 beads or peripheral blood from patients with active ulcerative colitis following granulocyte and monocyte adsorption apheresis, a significant suppression of proinflammatory cytokines (tissue necrosis factor-alpha, interleukin-1beta, interleukin-6, and interleukin-8) production by leukocytes, neutrophil chemotaxis, down-regulation of leukocyte adhesion molecule (L-selectin) and neutrophil adhesion to interleukin-1beta-activated endothelial cells were observed. Furthermore, after granulocyte adsorption therapy, the number of CD10-negative premature granulocytes increased, indicating increased turnover of these cells in the circulation. Our observations suggest that selective granulocyte and monocyte adsorption is associated with modified peripheral blood leukocyte function favorable to patients with ulcerative colitis and possibly other autoimmune disorders which reflect leukocyte hyperactivity.

A New Lactate-Based, Plasticizer-Free, Neutral Peritoneal Dialysis Fluid Provided in a Two-Compartment System: Effect on Peripheral Leukocyte Function

Background: A new neutral peritoneal dialysis fluid (PDF; Balance^®) provided in a two-compartment bag (pH 7.4, no plasticizers, minimal glucose degradation products – GDP) was investigated in comparison with a neutral control (Hanks’ balanced salt solution with gelatin 0.1%) and other PDFs with standard properties and plasticizers (Andy plus^®, pH 5.2, GDP), plasticizer free (stay safe^®, pH 5.2, GDP), and in addition plasticizer free after sterile filtration instead of heat sterilization (pH 5.2) regarding the function of peripheral blood leukocytes. Methods: Blood was drawn from 12 volunteers, and blood monocytes (MN) and polymorphonuclear leukocytes (PMNL) were collected. The cells were incubated for 30 min in control medium and the PDFs: glucose 1.5% (83 mmol/l) and 4.25% (238 mmol/l). Respiratory burst of cells was evaluated by chemiluminescence and superoxide (SO) generation after stimulation with phorbol myristate acetate. Results: In comparison with the control medium, incubation of MN in the two-compartment PDF showed preservation of respiratory burst. In contrast, the incubation of MN in standard PDF and plasticizer-free PDF showed impaired functions. The same was found for PMNL. SO anion measurement in MN and PMNL after incubation in the new two-compartment PDF also showed preservation of cell function in comparison with the control medium. The incubation of PMNL in standard PDF and plasticizer-free PDF with a high glucose content showed depressed SO anion generation. Conclusions: These in vitro data demonstrate a better preservation of in vitro phagocyte function with adaptation of pH and reduction of glucose, GDP, and plasticizers in PDFs. The best results are achieved with the two-compartment, lactate-based neutral PDF.

Immune cell functions in industrial workers after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin: dissociation of antigen-specific T-cell responses in cultures of diluted whole blood and of isolated peripheral blood mononuclear cells.

A comparative analysis was performed of the phenotype and function of peripheral blood leukocytes of two age-matched cohorts of industrial workers in chemical plants, one of which was exposed occupationally to high concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Median actual TCDD burdens were 116 ng/kg and 4 ng/kg, respectively. The phenotype analysis of peripheral blood mononuclear cells (PBMC) revealed no significant differences in the proportions of CD3, CD4, or CD8+ T lymphocytes, of CD16+ natural killer cells, and of CD19+ B lymphocytes. However, in PBMC of the TCDD-exposed workers; the proportion of CD8+ memory T cells (CD45R0+) was significantly higher, and that of lymphocytes with naive phenotype (CD45RA+) was significantly lower than in PBMC of the control group. Polyclonal and antigen-specific T-cell activation was assessed in parallel in isolated PBMC as well as in diluted whole blood cultures. In both culture systems the polyclonally stimulated cytokine release did not differ significantly between the two cohorts; however, we found a significantly reduced interferon gamma release in diluted whole blood cultures but not in isolated PBMC cultures of the TCDD-exposed cohort when we performed an antigen-specific T-cell stimulation with tetanus-toxoid. Therefore, we propose that exposure of individuals to high doses of TCDD can partially impair in the "blood milieu" those T-cell/monocyte interactions that are essential for antigen-specific T-cell responses, whereas isolated PBMC of the same donors appear functionally less affected.

Quantitation of endopeptidase 24.11 and endopeptidase 24.15 in human blood leukocytes.

Endopeptidase 24.11 (EP 24.11; also called neutral endopeptidase, enkephalinase, CALLA, or CD10) and endopeptidase 24.15 (EP 24.15) are widely distributed neutral metalloendopeptidases that degrade a number of bioactive peptides including substance P, bradykinin, neurotensin, and chemotactic peptides. In this study we used sensitive substrates and specific inhibitors to quantitate the levels of these enzymes in purified peripheral human blood leukocytes obtained from healthy blood donors. We found that neutrophils did not contain detectable amounts of EP 24.15. In contrast, T lymphocytes, B lymphocytes, and monocytes contained significant amounts of the enzyme (446 +/- 248,314 +/- 183, and 484 +/- 212 nmol/mg protein/h, respectively). Neutrophils contained significant amounts of EP 24.11 (266 +/- 130 nmol/mg protein/h). Significantly lower levels of the enzyme were found in T lymphocytes, B lymphocytes, and monocytes (94 +/- 31, 87 +/- 38, and 20 +/- 13 nmol/mg protein/h, respectively). These findings suggest that the effects of some bioactive peptides on peripheral blood leukocyte function may be modulated by these enzymes.

Luminol-dependent chemiluminescence microassay for phagocytic function

A luminol-dependent chemiluminescence (CL) microassay was developed to measure phagocytic function of peripheral blood leukocytes. Buffy coats, obtained by centrifugation of only 100 μl of whole blood, provided an enriched population of polymorphonuclear leukocytes (PMNs). The total reaction mixture, consisting of leukocytes-luminol-inducer (opsonized zymosan), was 450 μl. Peak CL activity was seen 5 min after addition of inducer at 37°C with cells tested within 60 min after collection. Tests to determine precision and reproducibility of the microassay gave a coefficient of variation of 8.5% and 11%, respectively. There was no significant difference between the mean peak CL values for 20 healthy adult donors compared to 14 premature neonates, however, the newborns' CL activity declined more rapidly; CL activity was severely depressed in cells obtained from a patient with chronic granulomatous disease. Results suggest that this microassay provides a simple, rapid, and reliable test of phagocytic function in cases where the amount of blood available for testing is limited.

Leukocyte function in granuloma annulare.

Leukocytes from patients with granuloma annulare were studied in vitro in order to detect functional abnormalities. Random migration and chemotaxis of both polymorphonuclear leukocytes and monocytes were similar in patients and controls. Preincubation of normal cells with patients' plasma did not enhance or inhibit locomotion. Phagocytosis and oxidative burst as reflected by chemiluminescence were also normal. Peripheral blood lymphocytes incorporated uridine normally with and without stimulation by the non-specific mitogen phytohaemagglutinin. These studies suggest that peripheral blood leukocyte function in patients with granuloma annulare is normal, but do not exclude a functional abnormality in tissue.

Engulfment and bactericidal capabilities of peripheral blood leukocytes in chronic leukemias.

The phagocytic function of peripheral blood leukocytes from 7 patients with chronic myelocytic leukemia (CML; 10 studies) and 7 patients with chronic lymphocytic leukemia (CLL; 11 studies) was assessed using noncapsulated Diplococcus pneumoniae. Evaluation was based on the simultaneous use of normal controls, the exact equilibration of the phagocytic potential of suspensions of patients' and controls' phagocytes in each other's, as well as autologous serum, and the maintenance of reasonable and constant ratios of bacteria to phagocytes. Neither CML nor CLL phagocytes displayed a consistent depression of either engulfment or of bactericidal functions, compared with phagocytes from normal controls.