Neutrophil Chemotaxis Dysfunction in Human Periodontitis

Polymorphonuclear leukocyte (PMNL) chemotaxis studies of 32 patients with localized juvenile periodontitis (periodontosis or LJP), 10 adult patients with a history of LJP (post-LJP), 8 patients with generalized juvenile periodontitis (GJP), and 23 adults with moderate to severe periodontitis were performed: (i) to determine the prevalence of a PMNL chemotaxis defect in a large group of LJP patients; (ii) to study PMNL chemotaxis in patients with other forms of severe periodontal disease; and (iii) to determine if the PMNL chemotaxis defect seen in LJP patients is a cell-associated defect or is mediated by humoral factors. The effect of periodontal treatment on PMNL chemotaxis was studied in nine LJP patients. The chemotactic response was measured with the Boyden chamber procedure, and patient's peripheral PMNL were compared with those of control subjects, using endotoxin-activated serum, bacterial factor, N-formylmethionyl-leucylphenylalanine, and leukocyte-derived chemotactic factor as the standard chemoattractants. Based upon statistical analysis of chemotaxis assays, most carried out on at least two and often three or more separate occasions, 26 of 32 LJP patients, 7 of 10 post-LJP patients, and 5 of 8 GJP patients exhibited cellular defects of chemotaxis, whereas only 2 of 23 of the patients with adult periodontitis exhibited depressed chemotaxis. Elevated PMNL chemotaxis was occasionally found in subjects with juvenile periodontitis (2 of 32 with LJP and two of eight with GJP); however, it was found in a significant number (10 of 23) of patients with adult periodontitis. In eight of nine LJP patients, depressed PMNL chemotaxis was observed before and after periodontal therapy. The results indicate that the PMNL chemotaxis defect observed in juvenile periodontitis is due to a cell-associated defect of long duration. These studies suggest that the PMNL plays a major protective role against periodontal infection and that the cellular chemotactic defects and may predispose subjects to LJP.     

Lectin-Mediated Induction of Human Neutrophil Chemotaxis, Chemokinesis, and Cap Formation

Six lectins, including concanavalin A, phytohemagglutinin P, castor bean I, wheat germ agglutinin, peanut agglutinin, and pokeweed mitogen, were studied for their ability to stimulate human neutrophil locomotion and cap formation. Five of these lectins with known monosaccharide specificities, including concanavalin A, phytohemagglutinin, P, castor bean I, wheat germ agglutinin, and peanut agglutinin, were found to stimulate human neutrophil migration in a modified Boyden assay. Pokeweed mitogen showed negligible activity in the locomotion assay as compared with other lectins. Tests were performed to determine if the observed neutrophil migration in response to lectins was directional, and it was found that concanavalin A, phytohemagglutinin P, and peanut agglutinin were both chemokinetic and chemotactic, whereas castor bean I was only chemokinetic. Wheat germ agglutinin could not be declared chemotactic or chemokinetic due to its tendency to agglutinate neutrophils. Studies with fluoresceinated lectins demonstrated that lectins which stimulate neutrophil migration also bind to neutrophil surfaces. Preincubation with specific monosaccharide ligands blocked both stimulated locomotion and fluorescence, suggesting that an available lectin-binding site was required both for lectin binding and the stimulation of migration. Additional experiments indicated that fluoresceinated concanavalin A, phytohemagglutinin P, castor bean I, wheat germ agglutinin, and peanut agglutinin all induce cap formation on the neutrophil.     

Effect of Azelastine Hydrochloride on Macrophage Chemotaxis and Phagocytosis in Vitro

Azelastine, a newly synthesized anti-allergic agent, was tested for its effects on guinea pig macrophage chemotaxis and phagocytosis. As specific macrophage chemo-attractants, we used macrophage chemotactic factors a and c; separated and highly purified from inflamed skin sites. Macrophage chemotaxis induced by skin extract or chemotactic factors was significantly suppressed by a low concentration of the agent (1 microgram/ml); the effect was dose-dependent. The inhibition of chemotaxis was reversible, because chemotactic activity was restored when the agents was removed by washing cells before chemotactic assay. Inactivation of chemotactic factors was not detected by mixing azelastine and factors a and c. Azelastine may directly interact with macrophages to decrease their chemotactic responsiveness. beta-Glucuronidase activity in the medium and macrophages after phagocytosis of polystyrene latex particles was not affected by this agent at concentrations ranging from 1 to 10 micrograms/ml. The phagocytosis of latex particles or sheep red blood cells opsonized with IgG antibodies (EA) and anchoring of macrophages to substrate were not inhibited and azelastine did not damage the macrophages as determined by lactate dehydrogenase (LDH) release assay.     

Effect of Lidocaine on Neutrophil Chemotaxis in Newborn Infants

Anesthetic drugs can influence the immune system, particularly granulocyte function. The goal of the present study was to evaluate if lidocaine used for epidural anesthesia during cesarean section can influence neonatal neutrophil chemotaxis. We measured chemotaxis and plasma cord lidocaine and cortisol levels in (A) 15 infants born by cesarean section with epidural anesthesia, (B) 15 infants born by vaginal delivery, and (C) 20 infants born by cesarean section with general anesthesia. Chemotaxis levels were significantly lower in group A infants (35.5 ± 16.1 m) compared to groups B (54.6 ± 10.5 m) and C (71.4 ± 23 m). The highest cortisol levels were observed in vaginally delivered infants. A significant inverse relationship was observed between chemotaxis and lidocaine levels (r = –0.6, P = 0.016) in infants born by cesarean section after epidural anesthesia, while no significant correlation was observed between chemotaxis and cortisol level. In conclusion, lidocaine, transferred through the placenta to the fetus during epidural anesthesia, may have an inhibitory effect on chemotaxis.     

Cocaine Inhibits Human Neutrophil Phagocytosis and Phagolysosomal Acidification In Vitro

Abstract

Cocaine, used intravenously, increases the risk of infections, but its effects on neutrophil phagocytosis have not been examined in vitro. Human neutrophils were suspended in cocaine hydrochloride 0, 1, 10, 50, 100 or 200 μg/ml in Hank's balanced salt solution to which was added a phagocytic meal of killed Saccharomyces cerevisiae stained with the pH indicator dye bromcresol purple. Yeast per phagocytosing neutrophil and the percent neutrophils phagocytosing yeast were reduced in neutrophils treated with cocaine 100 and 200 μg/ml (P < 0.05). When examined for percent of yeast phagocytosed after 10 minutes, neutrophils treated with cocaine 1-200 μg/ml demonstrated a decrease (P < 0.05). However, at 60 minutes only neutrophils treated with cocaine 50 and 100 μg/ml still showed a decrease in percent of yeast phagocytosed. Phagolysosomal acidification was impaired in neutrophils treated with 50, 100 and 200 μg/ml cocaine. Thus, cocaine inhibits neutrophil phagocytosis and phagolysosomal acidification in vitro, offering a reason for cocaine users/abusers to have impaired host defense and to be potentially at higher risk for infections.     

Leukocyte Migration Agarose Test for the Assessment of Human Neutrophil Chemotaxis

To apply the leukocyte migration agrose test (LMAT) to the in vitro assessment of human neutrophil chemotaxis, effects of different culture conditions on neutrophil migration under agarose were, studied. Presence of either serum or human serum albumin (HSA) in the culture medium was necessary for detectable neutrophil migration. HSA was preferred since heat-stabile chemotactic agents were found to be generated from fresh serum in the presence of agarose additional CO₂ in the assay milieu could be replaced by decreasing the NaHCO₃ concentration of the culture medium. Both the directed and the spontaneous migration rates of neutrophil leukocytes increased when the concentration of agarose was decreased. Area and distance of migration and cumulative cell count of migrated neutrophil leukocytes were suitable for quantitative the neutrophil migration rate.     

The Effect of Mare's Milk Consumption on Functional Elements of Phagocytosis of Human Neutrophil Granulocytes From Healthy Volunteers

We investigated functional parameters of phagocytosis in 18 healthy volunteers drinking 250 mL of mare's milk, deep-frozen (FMM) or lyophilized (LMM), or cow's milk (CM) daily for three weeks. Blood was taken before, weekly during, and one week after intervention. Chemotaxis of isolated polymorphonuclear leukocytes (PMN) was investigated by a micropore filter assay, migration was determined fluorimetrically. The activity of phagocytosis and respiratory burst of PMN in whole blood was analysed with Phago- and Bursttest ® by flow cytometry. Contrary to phagocytosis activity, chemotactic index (CI) and burst activity diminished significantly in the group FMM. One week after intervention, CI rose tendentiously, burst activity significantly. In conclusion, immunostimulating effects ascribed to mare's milk consumption could not be observed in healthy volunteers, at least concerning phagocytosis. Our results suggest that drinking FMM modulates inflammation processes by decreasing chemotaxis and respiratory burst, which might be favourable for giving relief to diseases with recurrent inflammation.     

Humoral Inhibition of Neutrophil Chemotaxis in Crohn''s Disease

In patients with Crohn's disease (CD) we investigated the C3 conversion of zymosan-activated serum (ZAS) and looked for the occurrence of chemotactic factor inactivation (CFI). We also studied the cell-directed inhibitory effect (CDI) of the CD patients' plasma and, in the same group, complement activation and complement-mediated deactivation. The mean value of ZAS C3 conversion in CD was no different from that of healthy controls, but in steroid-treated patients it was lower than in untreated CD. CFI occurred in 1 of the 23 CD sera tested, and CDI was observed in 6 out of the 22 patients tested. EDTA C3 conversion was present in 12 patients, and complement-mediated deactivation was associated with high values of EDTA C3 conversion. Our findings indicate that complement dysfunction and inhibitory factors of neutrophil chemotaxis are present in CD. These findings could explain the defective neutrophil migration into skin windows. Whether they are relevant to the pathogenesis of tissue injury or of infectious complications and are specific for CD, however, remains to be established.     

Inhibition of Neutrophil Chemotaxis by Elastase-Generated IgG Fragments

IgG1 is cleaved in vitro by granulocyte elastase into Fc and Fab fragments. The elastase-specific Fc fragment has been previously detected in vivo. Biological activity of the fragments has been described in modulating neutrophil oxidative metabolism and enzyme release. To investigate further effects granulocyte chemotaxis (CT) was tested. The CT was assayed in Boyden chambers and the chemotactic index (CI) was calculated which represents the mean distance travelled by the activated cells. Stimulation of leucocyte CT by casein, activated serum and FMLP gives maximal values of delta CI = 46.7, 26.4 and 7.2 microns, respectively. Native IgG1 and the elastase-produced IgG fragments do not stimulate leucocyte CT. FMLP-stimulated CT is specifically inhibited by the elastase-produced Fc fragments. Addition of 7 nmol Fc to stimulus concentrations of 16 to 125 nM FMLP results in total inhibition of chemotaxis demonstrated by CI values which are lower than those for unstimulated cells. The inhibition of CT is concentration dependent in the range of 2 to 7 nmol Fc/10(6) PMN. Number and affinity of FMLP receptors are not influenced by Fc fragments, so Fc binds neither to FMLP nor the FMLP receptor. CT stimulated by casein shows a large portion of chemokinesis. Only at suboptimal casein concentrations do Fc and IgG have an inhibitory effect on CT (0.63 mg casein/ml, 10 nmol peptide/10(6) PMN). C5a-stimulated CT is not influenced by IgG or IgG fragments which indicates that the samples are not cytotoxic. So the FMLP and casein-stimulated CT is specifically inhibited by the elastase-produced Fc fragments in a low concentration range.     

Effect of Cyclosporin A on Human Neutrophil and Monocyte Function

The effect of various concentrations of cyclosporin A (CyA), ranging from below peak blood levels to 20 times higher than blood levels of human peripheral blood polymorphonuclear and mononuclear leukocytes, was examined. CyA was found to bind to neutrophils with Kd values in the range of 20-50 nM. CyA at clinically obtainable blood level concentrations had no effect on neutrophil and monocyte chemotaxis, neutrophil oxidative burst, monocyte phagocytosis, or neutrophil bactericidal activity. The data on the release of lactoferrin, a secondary granule substance, from activated neutrophils showed that the calcium ionophore A 23187-induced lactoferrin release was inhibited by treatment of cells with 4 microM CyA, whereas release of lactoferrin from zymosan- or phorbol myristate acetate-activated neutrophils was not affected by the same concentration of CyA. This effect could either be due to differences in the degree of cell membrane perturbation by the various activators or to calcium dependence of neutrophil activation. A third possibility may be that CyA acts at some subsequent steps in the release process of neutrophils. It is concluded that CyA does not interfere with important functions of human phagocytes, the cells that play a major role in the defence against invading microorganisms.     

Impact of Resolvin E1 on Murine Neutrophil Phagocytosis in Type 2 Diabetes

Diabetic complications involve inflammation-mediated microvascular and macrovascular damage, disruption of lipid metabolism, glycosylation of proteins, and abnormalities of neutrophil-mediated events. Resolution of inflamed tissues to health and homeostasis is an active process mediated by endogenous lipid agonists, including lipoxins and resolvins. This proresolution system appears to be compromised in type 2 diabetes (T2D). The goal of this study was to investigate unresolved inflammation in T2D. Wild-type (WT) and genetically engineered mice, including T2D mice (db/db), transgenic mice overexpressing the human resolvin E1 (RvE1) receptor (ERV1), and a newly bred strain of db/ERV1 mice, were used to determine the impact of RvE1 on the phagocytosis of Porphyromonas gingivalis in T2D. Neutrophils were isolated and incubated with fluorescein isothiocyanate-labeled P. gingivalis, and phagocytosis was measured in a fluorochrome-based assay by flow cytometry. Mitogen-activated protein kinase (MAPK) (p42 and p44) and Akt (Thr308 and Ser473) phosphorylation was analyzed by Western blotting. The mouse dorsal air pouch model was used to evaluate the in vivo impact of RvE1. Results revealed that RvE1 increased the neutrophil phagocytosis of P. gingivalis in WT animals but had no impact in db/db animals. In ERV1-transgenic and ERV1-transgenic diabetic mice, phagocytosis was significantly increased. RvE1 decreased Akt and MAPK phosphorylation in the transgenic animals. In vivo dorsal air pouch studies revealed that RvE1 decreases neutrophil influx into the pouch and increases neutrophil phagocytosis of P. gingivalis in the transgenic animals; cutaneous fat deposition was reduced, as was macrophage infiltration. The results suggest that RvE1 rescues impaired neutrophil phagocytosis in obese T2D mice overexpressing ERV1.     

The Effect of Anti-Tuftsin Antibody on the Phagocytosis of Bacteria by Human Neutrophils

Tuftsin (Thr-Lys-Pro-Arg) is a naturally occurring tetrapeptide which stimulates most known functions of the polymorphonuclear and mononuclear phagocytic cell lines. Although tuftsin is a well characterized bioactive peptide, the exact physiological role tuftsin plays remains unclear. Specific mouse anti-tuftsin antiserum generated in our laboratory, is now available for phagocytosis inhibition studies. Monolayers of human neutrophils were prepared on glass coverslips from a few drops of finger prick blood obtained from a single healthy donor. The monolayers were treated with and without mouse anti-tuftsin antiserum at dilutions of 1:1000 or 1:2000. Exogenous tuftsin (1 μg/ml) was also added with and without antibody. Treated and untreated neutrophils were subsequently incubated with unopsonized Staphylococcus aureus. The proportion of cells accomplishing phagocytosis (phagocytic index) and the number of bacteria engulfed per cell (avidity index) were recorded. The results showed that exogenous tuftsin increased phagocytosis while the addition of mouse anti-tuftsin antiserum at a 1:1000 dilution inhibited phagocytosis both with and without exogenous tuftsin. This effect was diminished by the antiserum at the 1:2000 dilution. This study reaffirms that tuftsin plays an important physiological role in phagocytosis.     

The Effect of Anti-Tuftsin Antibody on the Phagocytosis of Bacteria by Human Neutrophils

Tuftsin (Thr-Lys-Pro-Arg) is a naturally occurring tetrapeptide which stimulates most known functions of the polymorphonuclear and mononuclear phagocytic cell lines. Although tuftsin is a well characterized bioactive peptide, the exact physiological role tuftsin plays remains unclear. Specific mouse anti-tuftsin antiserum generated in our laboratory, is now available for phagocytosis inhibition studies. Monolayers of human neutrophils were prepared on glass coverslips from a few drops of finger prick blood obtained from a single healthy donor. The monolayers were treated with and without mouse anti-tuftsin antiserum at dilutions of 1:1000 or 1:2000. Exogenous tuftsin (1 μg/ml) was also added with and without antibody. Treated and untreated neutrophils were subsequently incubated with unopsonized Staphylococcus aureus The proportion of cells accomplishing phagocytosis (phagocytic index) and the number of bacteria engulfed per cell (avidity index) were recorded. The results showed that exogenous tuftsin increased phagocytosis while the addition of mouse anti-tuftsin antiserum at a 1:1000 dilution inhibited phagocytosis both with and without exogenous tuftsin. This effect was diminished by the antiserum at the 1:2000 dilution. This study reaffirms that tuftsin plays an important physiological role in phagocytosis.     

高剂量X射线对人离体外周血中有核细胞DNA的损伤及其修复

目的:应用单细胞凝胶电泳技术检测X射线对离体外周血中有核细胞的DNA的损伤及损伤修复.方法:采用血常规正常的离体人外周血,用能量为6MV的X射线给予0Gy、2Gy、4Gy、6Gy、8Gy、10Gy的剂量照射,分别在0.5h、1h、2h用单细胞凝胶电泳技术检测全血中有核细胞的彗星率、慧尾长度和尾部DNA含量.结果:0Gy...     

Inhibition of Neutrophil Chemotaxis by a Monoclonal Antibody (TM316)

A monoclonal antibody, TM316, IgM kappa, was raised against the human monocytoid leukaemia cell line THP-1, and was shown to inhibit polymorphonuclear leucocyte (PMN) chemotaxis. The molecular weight (MW) of the protein on the PMN membrane with which TM316 bound was about 78,000. TM316 inhibited the chemotactic response of human PMN induced by at least three kinds of chemotactic factors (activated serum, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), and a lymphocyte-derived chemotactic factor (LDCF)) to the same extent. The extent of inhibition of chemotaxis by TM316 was strongly correlated with the quantity of cellular surface antigen recognized by TM316, when a cell sorter was used for analysis. TM316 did not alter the number of Fc or complement receptors of PMN, nor did it affect luminol-enhanced chemiluminescence (CL), lysosomal enzyme release, adherence, or superoxide anion generation by PMN. TM316 seemed to recognize a common surface antigen which was necessary only for the process of chemotaxis.     

不同剂量X射线对人外周血有核细胞DNA及精子DNA损伤的比较

目的:应用单细胞凝胶电泳技术检测不同剂量X射线对人离体外周血有核细胞DNA及精子DNA的损伤,评估外周血有核细胞及精子在高剂量X射线照射后DNA的损伤程度.方法:采用血常规正常的人外周血和采集精液常规正常的人精液,用能量为6MV的X射线给予0Gy,2Gy,4Gy,6Gy,8Gy,10Gy的剂量照射.照射后1h内进行单细...     

Effect of Melanin and Carotenoids of Exophiala (Wangiella) dermatitidis on Phagocytosis, Oxidative Burst, and Killing by Human Neutrophils

The black yeast Exophiala (Wangiella) dermatitidis is an increasingly recognized pathogen and a leading cause of severe pheohyphomycosis. Melanin is thought to contribute to the virulence of E. dermatitidis. Whereas the synthesis and the redox properties of melanin have been studied intensively, the influence of melanin and carotenoids on the phagocytosis, the oxidative burst, and the killing of E. dermatitidis by human neutrophils has not been studied. To study their effects on these phenomena, we applied a combination of flow cytometry and a colony-count-dependent method. Using E. dermatitidis wild-type strain 8565 and several melanin-deficient mutants that have been described previously, we demonstrate that melanin prevents this pathogen from being killed in the phagolysosome of the neutrophils. Melanin did not influence the phagocytosis or the oxidative burst of the neutrophils involved. The carotenoids torulene and torularhodine were not found to contribute to the prevention of killing. The ability of E. dermatitidis to block the effects of the neutrophil oxidative burst may critically impair the potential of the host to sufficiently eliminate this fungal pathogen and thus may play an important role in the pathogenesis of phaeohyphomycosis.     

Streptolysin O Inhibition of Neutrophil Chemotaxis and Mobility: Nonimmune Phenomenon with Species Specificity

The effects of streptolysin O (SO) (1 to 4 hemolytic units) on the mobility of neutrophilic leukocytes from humans, baboons, sheep, and rabbits were compared. After SO treatment, chemotaxis and random mobility of human neutrophils were markedly suppressed, baboon and sheep neutrophils were partially suppressed, and rabbit neutrophils were unaffected and demonstrated normal chemotaxis and mobility. The amounts of SO used in the mobility studies caused no leukocyte lysis or trypan blue uptake by human, baboon, or sheep cells, and minimal lysis or trypan blue uptake by rabbit cells. The possible involvement of immune mediators in the observed inhibition of human neutrophils was considered and excluded by the following studies. White blood cells from humans with humoral or cellular immune deficiencies responded in a manner similar to normal human cells; supernatant solutions from SO-treated human white blood cells did not contain a chemotactic suppressor; preincubation of SO with cholesterol (an inhibitor of SO hemolytic activity) caused loss of the chemotactic suppressive effect of the toxin on human leukocytes; and leukocytes from rabbits preimmunized with SO remained refractory to chemotactic suppression.