Chemiluminescence Response Of Whole Blood Research Articles

VALIDATION AND NOVEL APPLICATIONS OF THE WHOLE-BLOOD CHEMILUMINESCENCE ASSAY OF INNATE IMMUNE FUNCTION IN WILD VERTEBRATES AND DOMESTIC CHICKENS

The whole-blood chemiluminescence (WBCL) assay is a simple and rapid method of measuring production of reactive oxygen species by circulating leukocytes, particularly heterophils (birds) and neutrophils (other vertebrates). In the interest of substantiating a broadly applicable measure of innate immunity, we investigated the microplate WBCL method for several wildlife species as well as domestic broiler chickens. Lucigenin as a light enhancer was used for all avian blood, wild and domestic, while luminol was used in bear and frog blood. Use of ethylenedinitrilotetraacetic acid (EDTA) as the anticoagulant caused hemolysis of frog blood and decreased WBCL responses in all animals tested. Heparin, even in high concentrations, caused modest to no decrease of WBCL responses. The WBCL response correlated highly with heterophil or neutrophil numbers in all species tested. The WBCL response was tested in freshly collected blood as well as in blood one to five days postcollection in order to determine the utility of this assay for field studies when immediate access to laboratory facilities is not possible. One to three days of delay in performing the test after blood collection caused no, or only a slight, decay of the chemiluminescence response in most animals. Using domestic chickens, we tested the sensitivity of the WBCL method to detect differences between treatment groups and looked for loss of chemiluminescence response over several days using the original blood samples. Significant differences in the WBCL response between experimental groups of broiler chicken were detectable in freshly collected blood, as well as one- to four-day-old blood. Our results show that the innate immune response of populations of wild animals may be successfully compared using this assay, even when blood cannot be tested until a few days after collection.

Chemiluminescence response of whole blood in patients undergoing urological operations.

Polymorphonuclear leukocytes (PMNs) are one of the most important components of the defence mechanisms against bacterial infection. The functions of PMNs are believed to be impaired in patients during the perioperative period. Bactericidal function of PMNs was investigated together with the luminol-dependent chemiluminescence (CL) reaction of whole blood in 23 patients, 12 undergoing open surgery and 11 undergoing endoscopic surgery. Blood samples were collected one day before surgery (day -1) and 2 hours (day 0), 24 hours (day 1) and 7 days (day 7) after surgery. Counts of whole white blood cells (WBCs), PMNs and lymphocytes were not different between the two surgery groups. CL responses in the open surgery group were increased on days 0, 1 and 7. In the endoscopic surgery group, CL response was increased on day 1, but not on day 0 or day 7. These results suggest that the PMN function during the perioperative period was not impaired, but increased just after surgery, mainly due to an increasing number of WBC caused by the surgical intervention.

Effect of Chitosan Implantation on Activation of Canine Macrophages and Polymorphonuclear Cells after Surgical Stress.

The effect of cotton type chitosan implantation under the skin on the immunological response mediated by macrophages and whole blood was evaluated by chemiluminescence (CL) in dogs. The number of white blood cells was significantly decreased until 120 hr after operation in the control group (p < 0.05), while chitosan implantation increased the number of white blood cells, particularly neutrophils, from 24 to 96 hr after implantation (p < 0.05). The CL response of whole blood in the control group seemed to be reduced at 48 and 96 hr after operation (p < 0.05), but in the chitosan groups it maintained higher activity until 120 hr after implantation (p < 0.05). The macrophage activity measured by CL assay in the control group was markedly decreased from 24 to 120 hr after operation, and that for the 5 mg/kg chitosan group was also decreased at 24 and 48 hr after implantation (p < 0.05), although high activity was observed from 72 to 120 hr after implantation (p < 0.05). Neither 5 mg/kg nor 10 mg/kg chitosan groups showed any reduction in CL response of macrophages after operation, and the 20 mg/kg chitosan group retained high CL response of macrophages until 120 hr after operation (p < 0.05). These findings suggest that chitosan may be an efficacious and useful immunopotentiator for preventation of immunosuppression after surgery.

Chemiluminescence response of whole blood and separated blood cells in cases of experimentally induced pancreatitis and MDTQ-DA—Trasylol—ascorbic acid therapy

The formation of reactive O 2 species in the pancreas of pigs after induction of necrotizing or oedematous pancreatitis was studied by means of luminol- and lucigenim-sensitized chemiluminescence. The effect of 2,2-dimethyl-4-methanesulphonic acid-1,2-dihydroquinoline in combination with Trasylol and ascorbic acid was studied in vivo. This combined therapy leads to a reaction in the chemiluminescence response by 50–70% with prevention of pancreatogenic shock and multiple organ failure by improvement of the glutathione status. The combination of radical traps, kallikrein inhibitors and natural antioxidative substances is an efficient alternative therapy in cases of acute pancreatitis.

Study on Nonspecific Immunity in Pregnant Women: II. Effect of Hormones on Chemiluminescence Response of Peripheral Blood Phagocytes

To analyze the mechanisms of increased nonspecific immunity in pregnant women, the effect of various hormones on the phagocytic activity was estimated by a luminol-dependent chemiluminescence (CL) response during phagocytosing opsonized zymosan. The CL response of whole blood supplemented with exogenous human chorionic gonadotropin (hCG) increased significantly in all the male and female subjects and pregnant women. An approximate two- to fourfold increase was observed in comparison with the unsupplemented control in each subject at concentrations ranging from 1 to 1,000 IU/ml after 48 h of incubation (P less than 0.05). Progesterone slightly stimulated the CL response in female subjects only, but had no effect on male and pregnant women. Estradiol (E2) did not stimulate the CL response in any subject. The expression of Fc and C3b receptors on the surface of polymorphonuclear leucocytes (PMNL) in pregnant women was also investigated by measuring the immunofluorescence stained with monoclonal antibody to Fc and C3b receptors, respectively. The relative numbers of Fc receptors increased significantly in the third trimester compared to those of female control (P less than 0.05). Those of C3b receptor also increased in the second and third trimester (P less than 0.005). These results suggested that the nonspecific immunity represented by phagocytic activity in pregnant women increased with both oxidative metabolic responsiveness and the expression of membrane receptors. Besides, the increased phagocytic activity of the maternal host is probably due to the stimulatory effect of both endogenous and exogenous hCG on their peripheral blood phagocytes.

Stimulating effect of E.coli L-form cytoplasmic membranes on the macrophage functions of influenza virus infected mice

To evaluate their defense level against bacterial infection of patients with liver cirrhosis, we compared the luminol-dependent chemiluminescence (CL) response of peripheral blood from 40 patients with that from 40 healthy volunteers. Small quantities of heparinized whole blood (100 μl; final dilution, 1:10) were used for phagocytes, and CL was measured on addition of nonopsonized zymosan or Escherichia coli without special opsonization. Whole blood CL in cirrhotic patients was significantly lower than that in the healthy controls. The incidence of lower CL response in patients increased as disease stage advanced. Polymorphonuclear leukocytes (PMN) from cirrhotic patients exibited a slightly lower CL response than those from controls, but this was not statistically significant. In contrast, the CL response of monocytes in patients was significantly lower than that of controls. The opsonizing capacity of the patients’ sera and ascitic fluid was also decreased. In fact, the levels of opsonins such as complement in the patients’ sera and both immunoglobulins and complement in the ascitic fluids were found to be lower in cirrhotic patients. On the basis of these findings, defect of opsonophagocytic function seems to participate in the increased susceptibility to infection in cirrhotic patients. Furthermore, whole blood CL induced by nonopsonized zymosan at the onset of relatively severe bacterial infections such as sepsis, pneumonia, or spontaneous bacterial infection was less augmented in the blood of cirrhotic patients than that in noncirrhotic patients. To clarify the reason why whole blood exhibits a lower CL response in the acute phase of bacterial infections, we investigated the priming effects of lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α), well-known CL activators, on the CL response of whole blood obtained from cirrhotic patients in comparison with that from healthy persons. The priming effects were significantly decreased in patients’ blood when compared with that of healthy persons. These low responses of patients’ blood to LPS or TNF-α support our finding that phagocytes are not fully activated when gram-negative bacterial infections occur.

In vitro and in vivo effect of mercury, lead and cadmium on the generation of chemiluminescence by human whole blood

A method based on the use of the luminogenic substance, luminol, was developed for measuring the respiratory burst induced by phorbolmyristate acetate in 4 μl of human whole blood. In vitro, the three cations, Hg 2+, Pb 2+ and Cd 2+ inhibit the peak luminescence response at concentrations higher than those usually found in blood of subjects exposed to these metals. Cadmium metallothionein, however, in a concentration range similar to that found in whole blood of workers exposed to cadmium strikingly reduces the respiratory oxidative burst. In workers exposed to inorganic lead or mercury vapour, no reduction of the chemiluminescence response of whole blood was observed while a slight but statistically significant reduction was found in whole blood from cadmium exposed workers. If a similar effect occurs in lung macrophages, it might decrease the respiratory tract resistance to infection.

透析患者における食細胞の活性酸素放出能の検討

In order to elucidate the vulnerability to infection in patients on chronic hemodialysis, as one of the host defense mechanisms, the production of oxygen free radicals by phagocytes was studied in patients by luminol- or lucigenine-enhanced chemiluminescence (CL). Whole blood CL of the patients, in both luminol- or lucigenine-enhanced was significantly higher than that in healthy adults after stimulation by zymosan, PMA, and Staphylococcus aureus (S. aureus) 209 P. However, the CL response of the patients' polymorphonuclear neutrophils (PMNs) with the same stimuli was slightly lower than that in healthy adults. There were no differences in the levels of opsonins, such as complements and immunoglobulins, between the patients and healthy adults. It appears that any factor in the patients' serum enhances CL response, because of the PMN CL response after addition of patients' serum was higher than that after addition of healthy controls' serum, and the PMN CL response after the addition of patients' serum obtained after hemodialysis was higher than that before hemodialysis. The addition of erythrocytes to PMNs from healthy adults caused a reduction in the PMN CL response, but the addition of urea and creatinine had no effect. The CL response induced by microsphere-bound luminol (lumisphere), which makes possible the direct measurement of highly reactive oxygen within phagosomes, was studied in the patients and controls. The CL response in the patients was slightly lower than those in controls, but not significant. These results suggest that not only CL response of phagocytes but also other defense mechanisms should be studied further to make clear the vulnerability to infection in these patients. In addition, the effect of three antibiotics, cefbuperazone, cefminox and latamoxef on luminol-enhanced CL of whole blood was studied in healthy adults and the patients. After 3-hour exposure to those drugs at subinhibitory concentration (1/4 MIC), Klebsiella pneumoniae (K. pneumoniae) 163 treated by drugs induced higher CL response of whole blood than that by untreated bacteria in both healthy adults and the patients, and the peak time of the CL response induced by the drug-treated bacteria was shorter than that by untreated bacteria. This study suggests that the three drugs at sub-MIC work in partnership with host defense against infection due to K. pneumoniae even in patients on chronic hemodialysis.

The use of luminol-dependent chemiluminescence assay for monitoring the phagocytic and opsonic activity in bovine whole blood

Luminol-dependent chemiluminescence (CL) assay using whole bovine blood was developed to evaluate its phagocytic function and opsonic activity. The optimal assay system was as follows. A whole blood sample (100 μl) was mixed with 400 μl of Hanks balanced salt solution containing 25 m m Hepes, luminol (1 × 10 −4 m: final concentration) and zymosan (925·9 μg/ml: final concentration) were added and the mixture was measured continuously in a chemiluminometer. Final concentration of plasma in a reaction vial was 18·5%. Results were expressed as peak CL (cpm), CL index and the time (peak time) taken to show the peak CL. There was a significant correlation between the peak CL and the number of neutrophils in blood ( r = 0·722, P < 0·01, n = 25). The peak CL and peak time were closely related to the plasma concentration. The addition of sodium azide completely abolished CL response and Superoxide dismutase significantly inhibited CL response by 70% of the control. In the presence of sodium benzoate, catalase and histidine, a slight decrease of CL response was observed. CL response of whole blood was decreased considerably depending on the storage conditions and the relative percentages of the CL response under three different conditions were as follows: 83·3% at 5·7 h for 10 °C, 71·0% at 5 h for 30 °C and 60·5% at 4·3 h for 0 °C, as compared with the value which obtained from freshly drawn blood (10 min after blood collection). Whole blood CL assay was simple, rapid and is a useful tool for monitoring the phagocytic function and opsonic activities. It is potentially applicable to other animal systems.

Study on nonspecific immunity in pregnant women: increased chemiluminescence response of peripheral blood phagocytes.

The total number of circulating leucocytes in the peripheral blood of pregnant women progressively increased with advance in gestation because of neutrophilia. When the phagocytic activity, representing nonspecific immunity, was estimated by a luminol-dependent chemiluminescence (CL) response during phagocytosing opsonized zymosan, we observed that the CL response of whole blood and Ficoll-Paque separated polymorphonuclear leucocytes (PMNL) significantly increased throughout the pregnancy (P less than 0.01). The CL responses of mononuclear leucocytes (MN) and monocytes also increased and reached peak levels in the third trimester (P less than 0.05). These findings suggest that the phagocytic activity in pregnant women increases, not only with regard to the number of phagocytes but also with regard to individual cell function, from a relatively early stage of the pregnancy, and that this increased nonspecific immunity may compensate in part for a weakened specific immunity of the maternal host. Attention should be directed to effects of human chorionic gonadotropin (hCG) relative to the increased CL response during pregnancy.

Production of luminol-reactive oxygen radicals during Plasmodium vinckei infection.

We tested the ability of whole blood and enriched fractions of peripheral blood polymorphonuclear leukocytes obtained from mice during the course of infection with Plasmodium vinckei to produce luminol-mediated chemiluminescence in response to phagocytic and nonphagocytic stimuli. The chemiluminescence response of whole blood to all stimuli increased dramatically and nonlinearly as the infection progressed, and there was a concomitant increase (80%) and decrease (70%) in the total numbers of leukocytes and erythrocytes, respectively. The proportion of polymorphonuclear leukocytes in the total leukocyte population increased threefold. On a per cell basis and at a constant hematocrit, the chemiluminescence response of peripheral leukocytes from infected animals to phorbol myristate acetate or opsonized zymosan was only slightly greater than that of cells from uninfected animals. Polymorphonuclear leukocytes isolated from the blood of infected animals also showed no large increase per cell in chemiluminescence responsiveness. Thus, although leukocyte numbers increase during a murine malarial infection, there appears to be no major change in the capacity of individual peripheral blood leukocytes to produce activated species of oxygen. However, the physiological reduction in the total concentration of hemoglobin at high parasitemia, due to hemolysis and hemoglobin digestion by the parasites, increases the possibility of oxygen radical-mediated damage to tissues and intraerythrocytic parasites as a result of decreased antioxidant protection.