Gingival Crevicular Neutrophils Research Articles

Targeting of Porphyromonas gingivalis with a bispecific antibody directed to FcαRI (CD89) improves in vitro clearance by gingival crevicular neutrophils

Phagocytosis and killing of pathogens by polymorphonuclear neutrophils (PMN) from gingival crevicular fluid (GCF) is diminished in chronic periodontitis patients. As an approach to improve targeting of PMN toward a periodontopathogen, Porphyromonas gingivalis, the efficacy of a bispecific antibody (BsAb) directed against both recombinant 130 kDa hemagglutinin domain (r130k-HMGD) of P. gingivalis, and PMN Fc receptor (FcR) was evaluated. GCF PMN exhibited higher IgA FcR (FcαRI) levels, and lower IgG FcR (FcγRIIa and FcγRIIIb) levels than PB PMN. Functional studies revealed that GCF PMN exhibited a higher capacity to phagocytose and kill P. gingivalis opsonized with a BsAb targeting P. gingivalis r130k-HMGD to FcαRI as compared to an anti-r130k-HMGD antibody. However, phagocytosis and killing activity of PB PMN that were incubated with the two antibodies proved comparable. These data support targeting of pathogens toward FcαRI as an option to improve antibacterial immunity in chronic periodontitis patients.

Effective in vitro clearance of Porphyromonas gingivalis by Fc alpha receptor I (CD89) on gingival crevicular neutrophils.

Porphyromonas gingivalis has been implicated as a causative pathogen in periodontitis. Immunotherapeutic approaches have recently been suggested to aid in the clearance of P. gingivalis from disease sites. Because antibody-Fc receptor (FcR) interactions play a role in the effector functions of polymorphonuclear neutrophils (PMN), we evaluated which FcR on PMN from gingival crevicular fluid (GCF) serves as an optimal target molecule for FcR-directed immunotherapy. GCF PMN and peripheral blood (PB) PMN from adult periodontitis patients were analyzed for their immunoglobulin G (IgG) and IgA FcR (Fc gamma R and Fc alpha R, respectively) expression and function by studying IgG- and IgA-mediated elimination of P. gingivalis. GCF PMN exhibited higher Fc alpha RI and Fc gamma RI levels and lower Fc gamma RIIa and Fc gamma RIIIb levels than PB PMN. Functional studies revealed that GCF PMN exhibited less of a capacity to phagocytose and kill IgG1-opsonized P. gingivalis than PB PMN. IgA1-mediated phagocytosis and killing capacity was, however, comparable between GCF PMN and PB PMN. In summary, these in vitro results document that Fc alpha RI represents a candidate target for FcR-directed immunotherapy for the clearance of P. gingivalis.

Purification of human neutrophil collagenase, establishment of its monoclonal antibodies and application to gingival crevicular neutrophils

In order to investigate the role of neutrophil collagenase in the periodontal disease, human neutrophil collagenase was purified and two monoclonal antibodies against this enzyme were obtained. This enzyme was purified by four step-affinity chromatography: heparin-aminocellurofine, gelatin Sepharose 4B, collagen-Sepharose and collagenase inhibitor column chromatographies. To produce the monoclonal antibody against the enzyme, the Balb/c mouse was immunized and its spleen cells were fused with the mouse myeloma cells. Two monoclonal antibodies to the enzyme, 2F3 (IgG1) and 3F12 (IgG1), which recognized a conformational structure of the enzyme apart from its catalytic site were obtained. Both antibodies were monospecific to leukocyte collagenase and did not cross-react with the other metalloproteinases such as leukocyte gelatinase, skin fibroblast collagenase, gelatinase and stromelysin. Using these monoclonal antibodies, collagenase was stained granularly in gingival crevicular neutrophils.

In vitro release of elastase from human blood and gingival crevicular neutrophils

In vitro release of elastase from human blood and gingival crevicular neutrophils

In vitro release of elastase from human blood and gingival crevicular neutrophils

Peripheral PMNs were collected from blood, and crevicular PMNs separated by filtration from gingival washings in 13 patients, aged 22–75 y, who had varying degrees of gingivitis and periodontitis. After pre-incubation with cytochalasin B, the same number of crevicular and peripheral cells were incubated either in PBS (with Ca 2+ and Mg 2+) (spontaneous release) or in the same buffer containing increasing concentrations of FMLP (stimulated release); elastase activity was measured in the supernatant by a fluorometric technique. There was a higher spontaneous release of enzyme from crevicular than from peripheral neutrophils. The average elastase activity in the supernatant of 1 × 10 4 crevicular cells was more than five times higher than that obtained from the same number of peripheral cells. However, stimulated crevicular PMNs liberated smaller amounts of enzyme than did stimulated peripheral PMNs. These results suggest that crevicular PMNs are already releasing elastase, and are consistent with the possibility that lysosomal enzymes contribute to tissue damage during gingivitis and periodontitis.

The effect of dichloromethylene bisphosphonate on human gingival crevicular neutrophil myeloperoxidase activity

Neutrophil myeloperoxidase is an important component of the oxygen-dependent microbicidal system and is enzymatically activated in crevicular cells. The purpose of this study was to investigate the effect of dichloromethylene bisphosphonate, a drug used to modify bone resorption, on extracted neutrophil myeloperoxidase activity directly, and indirectly, by means of a luminol-dependent chemiluminescence assay. Enzyme activity was increased, up to a drug concentration of 1 × 10 2 μg/ml: 1 × 10 5 neutrophils. Inhibition occurred only at high concentrations, supporting the evidence for low toxicity. This cellular model warrants further investigation with respect both to possible modulation of inflammatory mechanisms relevant to the pathogenesis of periodontitis and other diseases, and to a better understanding of the pharmacological activity of bisphosphonates.

Chemotaxis of gingival neutrophils: scanning electron microscopy.

Gingival neutrophils, tested in the chemotaxis-under-agarose assay, were examined with the scanning electron microscope. Non-motile cells were round with narrow surface ridges. In response to the chemoattractant, zymosan-activated serum, the cells elongated and became polarized with an anterior lamellipodium and a posterior uropod. Cells flattened as they emigrated beneath the agarose. These studies show that gingival crevicular neutrophils from healthy subjects are capable of responding vigorously to external stimuli and hence may participate in the pathophysiology of periodontal disease.

Reduced oxidative function in gingival crevicular neutrophils in periodontal disease.

Measurable amounts of viable and functional polymorphonuclear neutrophils (PMNs) are recovered from pooled washings of the gingival crevice of healthy individuals. In the present study, we have assessed the ability of the PMNs removed from single healthy or diseased pocket sites to mount an oxidative burst when challenged with phorbol myristate acetate (PMA) and compared these activities with each other and with those obtained with autologous peripheral-blood PMNs. The oxidative burst after PMA stimulation was evaluated by using methods developed for the flow cytometer. The results showed that the PMNs collected from untreated disease sites were minimally responsive to PMA when compared with peripheral-blood PMNs collected at the same time from the same individual. Thus, whereas the peripheral-blood PMNs exhibited significantly lower resting oxidative product formation and a 500% increase when stimulated with PMA, all gingival-crevicular PMNs exhibited significantly higher resting formation of oxidized products but only a 150% increase after PMA stimulation. PMNs obtained from a consistently healthy site had significantly higher resting production of oxidized products and were able to mount the greatest absolute increase in oxidized products after PMA stimulation when compared with PMNs collected from diseases sites. Mechanical debridement of these diseased sites, which both reduced the bacterial numbers and restored clinical health, resulted in the recovery of gingival-crevicular PMNs that exhibited an oxidative burst more typical of that observed in PMNs obtained from healthy gingival sites and from the peripheral blood. This suggested that the PMNs collected from the diseased sites either had been exhausted by the large numbers of bacteria present in these sites or had been specifically inhibited by these bacteria.

Myeloperoxidase activity in human gingival crevicular neutrophils

Myeloperoxidase (MPO) activity in neutrophil leucocytes from gingival crevicular exudate and peripheral venous blood was assayed both spectrophotometrically and cytochemically in 50 healthy subjects with and without clinically evident gingival inflammation. All blood neutrophil MPO levels were within the normal range; gingival neutrophil MPO levels were normal in all subjects with inflamed tissue but enzyme activity was not detected in approximately 50 per cent of those with no inflammation. There was positive correlation ( p < 0.0003) between the gingival index and MPO activity. The gingival crevice is an accessible example of a functional site in host defence and it may be that subpopulations of neutrophils are selectively sequestered into such areas in response to chemotactic stimuli.

Functional integrity of gingival crevicular neutrophil polymorphonuclear leucocytes as demonstrated by nitroblue tetrazolium reduction.

Gingival crevicular neutrophils were collected from 20 healthy subjects and their ability to reduce nitroblue tetrazolium was investigated by means of a semi‐quantitative histochemical test. 78.04 % (± s.e.m. 0.8 %) of the cells were vital. 42.9 % (± s.e.m. 3.3 %) of the crevice neutrophils were NBT positive as compared to 5.6% (± s.e.m. 0.89%) of unstimulated peripheral venous blood neutrophils from the same patients (p &lt; 0.001). No difference between male and female was found. The results indicate a stimulation of crevicular neutrophils by bacteria. Furthermore, a slight correlation between blood and crevicular neutrophil activity in each individual was found, suggesting that even in the normal state some antigen or other bacterial product in the circulation is capable of stimulating the neutrophils.One patient with hereditary cyclical neutropenia, shown to have a greatly reduced number of crevicular cells, had normal activity of both blood and crevicular neutrophils as measured by this test. These findings, compatible with known immunological results, are compared with the neutrophil function test results in this patient and two other neutropenic subjects.This study supports the view that the gingival crevicular neutrophil is a functional cell capable of contributing towards host defense.