Nitric oxide synthase activity in neutrophils from patients with localized aggressive periodontitis.

BACKGROUND: Localized aggressive periodontitis (LAgP) is associated with neutrophil dysfunction including defective chemotaxis and reduced calcium influx factor activity. Nitric oxide (NO) and its enzyme, nitric oxide synthase (NOS), have been suggested to be involved in chemotaxis. Some reports, however, were unable to detect either NO or NOS in human neutrophils. In this study, we focused on NOS activity in LAgP neutrophils and examined the involvement of NOS in chemotaxis of normal neutrophils and NOS activity in neutrophils from normal subjects and patients with LAgP. METHODS: Neutrophils from 10 normal subjects and 10 LAgP patients were isolated from peripheral venous blood. Membrane associated-NOS (MA-NOS) and soluble NOS (S-NOS) were extracted from cells with or without FMLP stimulation. NOS activity was measured using the radiolabeled L-arginine to L-citrulline conversion assay. RESULTS: N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly inhibited FMLP-induced chemotaxis (P<0.01) and dibutyryl cGMP, an activator of cGMP-dependent protein kinase, significantly attenuated the inhibition by L-NAME (P<0.01). Unstimulated and FMLP-stimulated MA-NOS activity in LAgP neutrophils was statistically significantly higher than that in normal neutrophils (P<0.05). S-NOS activity in LAgP neutrophils was higher than that in normal neutrophils. CONCLUSIONS: This study suggests that NOS is present in human neutrophils and may be involved in FMLP-induced chemotaxis in normal neutrophils. NOS activity is increased in LAgP and is negatively correlated to chemotaxis response.

Defective calcium influx factor activity in neutrophils from patients with localized juvenile periodontitis.

BACKGROUND: Localized juvenile periodontitis (LJP) is an early-onset periodontal disease associated with neutrophil dysfunction, including defective chemotaxis, reduced protein kinase C (PKC) activity, and reduced calcium entry. These observations are important because reduced availability of cytosolic-free calcium concentration in the cell will have detrimental consequences for the numerous cytosolic calcium concentration-dependent pathways. In particular, there is a direct relationship between Ca2+ flux and the cell activation enzyme PKC. In this report, we focused on the mechanism of calcium entry, investigating a newly described molecule, calcium influx factor (CIF). CIF is thought to be a second messenger for the opening of membrane calcium channels when intracellular calcium stores are depleted. We examined CIF activity in neutrophils from normal subjects and LJP patients. METHODS: Neutrophils from 11 LJP patients, 3 adult periodontitis (AP) patients, and 12 normal subjects were isolated from peripheral venous blood. CIF was extracted with thapsigargin, a Ca2+-ATPase inhibitor, from isolated neutrophils and CIF activity measured using a 45CaCl2 uptake assay. RESULTS: The CIF activity in neutrophils from LJP patients ranged from 98.9 to 281.5 units/mg protein (mean = 180.2 +/- 56.3) and from 291.9 to 755.5 units/mg protein (mean = 528.8 +/- 153.8) in non-periodontal disease controls. CIF activity in AP patients was also measured and found to be similar to controls. The CIF activity in LJP patients was statistically significantly reduced compared to that in normal subjects (P <0.001). CONCLUSIONS: This study suggests that CIF activity may be an important determinant in neutrophil abnormalities in LJP.

Cytoskeletal actin reorganization in neutrophils from patients with localized juvenile periodontitis.

Localized juvenile periodontitis (LJP) is an early-onset periodontal disease associated with a polymorphonuclear neutrophil (PMN) defective migratory response. Kinetics of actin polymerization-depolymerization determine the shape changes occurring in the plasma membrane-associated cytoskeleton and provide the driving force for directed cell migration (chemotaxis). Therefore, we investigated the relation between an abnormality in LJP PMN chemotaxis and an altered reorganization of the actin filament network. PMNs isolated from peripheral blood of LJP patients (n=14) and matching control subjects (n=12) were evaluated for random and directed migration in a Boyden chamber assay, and the kinetics of actin polymerization were studied by flow cytometry. Three groups of LJP patients could be distinguished on the basis of their PMN-chemotactic response compared to their matched control: depressed (n=6), normal (n=4), and elevated (n=4). The abnormal (depressed or elevated) chemotaxis was generally not related to abnormal random migratory response, except for two patients. Since the kinetics of formyl-methionyl-leucyl-phenylalanine-induced F-actin response were highly variable from one subject to another, means were calculated at each timepoint with the values obtained from each group of subjects and compared by a general factorial design analysis. No statistically significant differences were detected between the control group and the LJP patient group. Furthermore, the data did not show a correlation between the kinetics of actin polymerization-depolymerization and the abnormal chemotactic response observed in LJP PMNs. Hence, the chemotaxis defect in LJP PMN appears to be mediated by signaling events that carry their effect independently of an intact cytoskeleton.

Calcium-dependent protein kinase C activity of neutrophils in localized juvenile periodontitis.

Protein kinase C is a key molecule in neutrophil signal transduction after receptor stimulation by soluble bioactive molecules. It has been reported that neutrophils from most patients with localized juvenile periodontitis (LJP) do not have a normal response after stimulation with a chemotactic ligand such as N-formylmethionylleucylphenylalanine (FMLP). To further clarify the mechanism of this altered response and to confirm and expand earlier observations, the calcium-dependent protein kinase C activity of neutrophils from patients with LJP was evaluated. Peripheral blood neutrophils from 12 patients and 12 healthy subjects, age, sex, and race matched, were sonicated and subsequently subfractionated by ultracentrifugation into a soluble fraction (cytosol rich) and a particulate fraction (membrane rich). The calcium-dependent protein kinase C activity was evaluated in each fraction by phosphorylation of histone with radiolabeled ATP in the presence or in the absence of phorbol 12-myristate 13-acetate stimulation. Results revealed that the total calcium-dependent protein kinase C activity of neutrophils from patients with LJP and depressed chemotactic migration to FMLP (201.0 +/- 63.6 pmol/min/10(7) cells) was lower than that of neutrophils from healthy subjects (287.6 +/- 55.7 pmol/min/10(7) cells) (P < 0.01). The calcium-dependent protein kinase C activity in neutrophils from patients with LJP exhibited a positive correlation with chemotactic migration to FMLP (P < 0.05). The low activity of calcium-dependent protein kinase C in neutrophils from the patients reflected the low activity in the soluble fraction from the neutrophils. After stimulation with phorbol 12-myristate 13-acetate, the calcium-dependent protein kinase C activity was found to be lower from patients with LJP than from healthy subjects. These results suggest that lower calcium-dependent protein kinase C in neutrophils is a predisposing factor for LJP.

Superior leukocyte separation with a discontinuous one-step Ficoll-Hypaque gradient for the isolation of human neutrophils.

A discontinuous gradient system composed of two commercially available Ficoll-Hypaque mixtures (Mono-Poly resolving medium (MPRM) and Histopaque 1.077) is described for the purification of mononuclear and polymorphonuclear leukocytes from human blood. Like the original one-step Hypaque-Ficoll procedure (Ferrante and Thong, 1978), a single centrifugation at 500 X g for 30 min resulted in the formation of two distinct leukocyte fractions. In contrast to MPRM alone, the discontinuous system (MPRM-HP) was capable of resolving leukocyte fractions from blood volumes as small as 1 ml with excellent purity and yield. MPRM-HP was also compatible with a wider range of anticoagulants and permitted fractionation of specimens resistant to MPRM.