Group II phospholipase A2 in human gingiva with periodontal disease

Fourteen kilodalton phospholipase A2 molecules (PLA2) are classified into two groups, I- and II-PLA2, and only the latter has been considered to play a pathogenetic role in various forms of tissue inflammation. Previously we demonstrated high PLA2 activity in gingival crevicular fluid (GCF) of patients with periodontal disease, without determining the group of the enzyme involved. In this study, the activity, groups and levels of enzyme in gingiva taken from 13 sites of periodontal disease were determined using both biochemical and radioimmunological methods. A linear correlation between the activity and the level of II-PLA2 was observed. No I-PLA2 was found in any of the samples tested. These data suggest that the PLA2 activity found in the GCF of patients with periodontal disease does not belong to the I-PLA2 but to the II-PLA2 group.


Introduction
Intracellular phospholipase A (PEa2; EC 3.1.1.4) catalyses the hydrolysis of the ester bound at the sn-2 position of glycerophospholipids and is a key enzyme in the production of potent inflammatory mediators, including prostaglandins, leukotrienes and platelet activating factor. Recently, it has been demonstrated that mammalian PEA can be classified into at least three groups on the basis of their primary structure. Two of these groups are designated Iand II-PLA2, and are found in pancreatic juice and a variety of tissues other than pancreas, respectively. Both groups have a molecular mass of 14 kDa. The third group is present in the cytosolic fraction of various tissues and has a molecular weight of 85 kDa. Among these enzyme groups, only II-PLA has been considered to play an important role in the process of inflammation, for example, in rheumatoid arthritis or inflammatory bowel disease. In our previous studies, it was demonstrated that rat gingival cells had II-PLA activity and that this activity was induced by the inflammatory cytokines interleukin-l]3 (IL-113) and tumour necrosis factor-cz (TNF-00, but inhibited by the anti-inflammatory cytokine transforming-growth factor-]3 (TGF-[). 6,7 In addition, we also demonstrated PLA activity in human gingival crevicular fluid (GCF) from patients with periodontal disease and indicated that this activity appeared to reflect periodontal disease conditions (active or resting), suggesting that its potential usefulness as a marker for the assessment of disease activity. However, the maximum volume of GCF was too small to determine whether the activity if contained belonged to the II-PLA2 group, even with the sensitive method we employed. Since GCF contains infiltrates from adjacent gingival tissue that enter the gingival crevice, the enzyme activity in GCF reflects that in the gingival tissue.
To clarify the type of PEa present in GCF from patients with periodontal disease, the activity and level of PEA were measured in the gingival tissue using both biochemical and radioimmunological assay systems, respectively.

Materials and Methods
Gingival tissue: Gingival tissues from seven patients (mean age 42.0 + 2.2 years) who had at least five teeth with a pocket depth of more than 6 mm were used. The depth of the periodontal pocket (gingival crevice), which usually indicates the degree of periodontal tissue destruction, and its measurement is considered to be the most reliable tool for the diagnosis and assessment of periodontal disease. A tooth with healthy periodontal tissue has a pocket depth of less than 2 mm, whereas a tooth with a periodontal pocket depth of more than 4 mm is considered to be affected by periodontal disease. None of the patients were receiving or had previously received any medication or periodontal treatment before the study. Gingival tissue preparation: Inflammatory gingival tissues were taken from 13 sites in the seven patients at the time of periodontal surgical therapy. The excised tissues were rinsed thoroughly with ice-cold isotonic solution and weighed. Then they were minced and homogenized in 100 mM Tris-HCl (pH 7.4) using a glass homogenizer at 2 000 rpm for 30 s, followed by centrifugation at 20 000 x g. An aliquot of each supernatant was used as the enzyme source for either biochemical determination of PLA2 activity or radioimmunological PEA assay.
Biochemical assay for PLAe activity: Phospholipase A activity was determined according to the method described by Tojo et al. using 0.8 mM 1-palmitoyl-2 oleoyl-sn-glycero-3-phosphoglycerol as a substrate in the presence of 5 mM cholate. Fatty acids released by PLA were labelled with 9-anthryldiazomethane and then separated by high-performance liquid chromatography column, followed by quantitation using marganic acid as an internal standard. Calcium-dependent PEt was estimated as the difference between the activity assayed in the presence of 5 mM CaCL and that in the presence of 10 mM ethylenediaminetetraacetate.
Radioimmunoassayfor I-and II-PLA2: The level of Iand II-PEt in the supernatant was measured using a radioimmunoassay kit (Shionogi Pharmaceutical Ltd, Osaka, Japan) and a monoclonal antibody against human pancreatic group Ior splenic II-PLA as described elsewhere. 1 Results and Discussion Figure 1 shows the sensitivity of the RIA used in this study and representative II-PLA levels as a function of wet weight of gingival tissue. As can be seen, a minimum gingival tissue wet weight of about 1 mg was enough for detection of II-PLA 2. The level of II-PLA increased with gingival wet weight over a range of I to 25 mg in a dose-dependent manner. It was not possible to determine whether the activity belonged to Ior II-PEt in GCF, because usually the volume of GCF is less than 3 ILtl, which is insufficient for RIA. However, the results suggested that the PLA2 activity detected in GCF was classifiable by examining biopsy samples of gingival tissue. It would be acceptable and meaningful not only for periodontologists but also for patients for about 1 mg wet weight of gingiva to be taken as a biopsy sample to obtain precise and predictable information on the diseased sites. Table 1 shows the levels of PEA determined by the RIA in each supernatant of gingival tissue from 13 different sites. The mean level of II-PLA was 83.7 pg/ mg wet weight, with wide ranges in the enzyme level from 5 to 216 pg/mg wet weight. This wide range of values was not surprising, because our previous data showed that PLA activity was well correlated only with periodontal disease activity, and not with the degree of destruction caused by the previous disease activity. It has also been shown that II-PLA2 activities found in inflamed sites other than those due to periodontitis, such as rheumatoid arthritis, Crohn's disease and septic shock, 11 are well correlated with disease activity, and thus II-PLA2 is believed to play an important role in the initiation and propagation of the inflammatory process in these diseases. Recently, we showed that GCF sampled from sites of active periodontitis had higher PEA activity than those from quiescent sites, although we could not identify whether the activity belonged to group or II. These activities detected in GCF were thought to be those induced in the inflamed gingival tissues followed by secretion into the gingival crevice. In contrast, none of the samples tested had any detectable levels of I- PLA using this assay system. These results suggest that the PLA2 activity found in the GCF of patients with periodontal disease is not I-PLA but II-PLA2.
It has been reported that serum contains many factors that modulate the activity of PEA2, for example, an activating protein such as PLA2-activating protein 12 and an inhibitory protein such as C3dg. 13 Up to now it has not been clarified whether the elevated PEA2 activity in GCF and gingival tissue is due to an increase in enzyme concentration or to direct activation of the enzyme. Figure 2 shows the relationship between the level of II-PLAz determined by RIA and the activity determined by biochemical assay in the 13 samples. As can be seen, a highly significant correlation (r 0.907, p < 0.001) between the level and activity of II-PLA2 was observed. These findings indicate that the PLA2 activity detected in GCF was attributable primarily to an increase in the protein concentration of II-PLA2, probably via de novo protein synthesis in the gingiva, and not to activation of the enzyme. This close correlation between the level and activity of II-PLA agrees well with previous reports on septic shock, 11 malaria, 14 or Crohn's disease and ulcerative colitis. 15 We showed that inflammatory cytokines such as IL-1l] and TNF-{x induced, whereas an anti-inflammatory cytokine, TFG-{], inhibited the synthesis and secretion of II-PLA2 in cultured rat gingival fibroblastic cells. 6,7 It is well known that these cytokines are present in GCF and levels of IL-I in gingival tissue correlate well with disease activity of periodontitis. 16 Although it was not possible to identify the source of II-PLA2 in gingival tissue from this study, it is considered that gingival cells in the inflamed sites are one of the most likely candidates. In summary, the present results suggest that PLA activity detected in GCF does not belong to I-PLA2, but to II-PLA2. It is also suggested that measurement of either the level of II-PLA in biopsy samples of inflamed gingiva or the enzyme activity in GCF may be useful for detecting sites of active disease in periodontitis.