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Localized juvenile periodontitis (ljp) is an early onset form of periodontal disease characterized by unique localization to first molars and incisors and a high prevalence of neutrophil abnormalities, particularly chemotaxis. The intracellular transduction mechanisms that follow receptor-ligand coupling on the neutrophil surface and lead to chemotaxis are not clearly established. Chemotaxis and phagocytosis are modulated by a variety of receptors and involve several activation pathways; the role of intracellular calcium as a presumptive second messenger and mediator of these events is well established. The putative effector mechanisms for the chemotactic receptor of neutrophils also include the possible activation of a phospholipase, protein kinase C, methyltransferase, or adenylate cyclase. In normal neutrophils, a phosphoinositide pathway initiated by phospholipase C, which results in the activation of protein kinase C via diacylglycerol and the generation of IP3, has been implicated. In order to better understand the stages of neutrophil transduction, fluorescent probes were used to monitor neutrophil calcium changes. Chlorotetracycline (CTC) was used as an indirect probe of intracellular membrane-bound pool of calcium stores, and Quin-2 was used to monitor cytosolic free calcium levels of FMLP stimulated normal and LJP neutrophils. The results indicate that the early phase of the calcium response affiliated with the release of intracellularly sequestered calcium appears intact in LJP neutrophils, as the CTC fluorescence changes were similar to control values. The second phase of the calcium response, associated with membrane channel activation and an influx of extracellular calcium, appeared compromised in the neutrophils of the LJP population.(ABSTRACT TRUNCATED AT 250 WORDS)

An in vivo assay was recently developed to monitor the crevicular leucocyte response to chemotactic agents, e.g., casein and N-formyl peptides. This method was used to monitor humans with little or no gingival disease (C group), gingivitis (G group), chronic periodontitis (CP group) and localized juvenile periodontitis (LJP group). Casein (0.2 microliters, 2 mg/ml) was placed into an isolated gingival crevice of each subject with a calibrated wire loop and the time recorded (t = 0). Leucocytes were counted in crevicular washes (10 microliters) 15 minutes later and every 5 minutes thereafter up to t = 50 minutes. This protocol was repeated for the crevice of an adjacent tooth except that the crevicular fluid flow response to the chemotactic challenge was monitored. The C, G and CP subjects showed a similar pattern of response to the chemoattractant with a single "peak" of leucocytes at approximately t = 25 minutes. However, the peak cell count was much greater in the G and CP groups than in the C group. LJPs showed an abnormal pattern with two leucocyte peaks, one at approximately 25 minutes and the other at 45 minutes. Both peaks tended to be higher than the single peak seen in Cs but were significantly lower than that in Gs or CPs, even at similar levels of inflammation. In addition, the peak leucocyte response (to casein) in LJPs did not increase with increasing leucocyte counts in the unchallenged (resting) crevice, whereas a positive relationship was seen in the other groups of subjects. These data suggest that this new assay may provide important diagnostic information on in vivo neutrophil migration in the gingival crevice and on susceptibility to periodontal disease.

Previous studies have implied that chemotaxis defects of neutrophil polymorphonuclear leukocytes (PMNs) can be found in approximately 75% of patients with juvenile periodontitis (JP). In the present study, the Leading Front (LF) method was used to study whether the chemotactic response of PMNs from JP-patients differed from that of adult periodontitis (AP) patients and periodontally healthy control individuals (C). Sixteen JP-patients, 21 AP-patients, and 13 C-individuals were studied. PMNs from each individual, and from a daily reference person were tested against three chemoattractants (N-f-Met-Leu-Phe (FMLP), casein (CA), bacterial chemotactic factor (BCF] and a neutral buffer (Gey's solution (GEY]. Regardless of the test solution a greater difference among individuals could be observed in the JP-group than in the other groups. Apart from this, there were no differences among the groups as regards CA, BCF, and GEY. However, with FMLP, the PMNs of the JP-group had a significantly greater migration distance as compared to the other groups. This finding can probably be ascribed to the fact that the LF method detects other aspects of the PMN response than do the methods used for earlier studies of JP. The finding, in this study, of an enhanced PMN response in JP as regards FMLP may be a reflection of the presence of a non-uniform PMN population whose composition in JP differs from that of the other groups.

Despite some reports to the contrary, it is generally assumed that early-onset forms of periodontal disease (including both juvenile and rapidly progressive periodontitis) are associated with a defect in neutrophil (PMN) chemotactic behaviour. Using the Boyden chamber technique and N-formyl-methionyl-leucylphenylalanine (FMLP) to assess locomotion by the leading front method, we have failed to show any evidence for such depressed PMN locomotion. Indeed, when PMN chemotaxis and chemokinesis are considered in response to a range of chemoattractant doses our results indicate significant enhancement of all but random locomotion. When taken together with other studies, our results suggest that PMNs from patients with early-onset periodontitis may show abnormal locomotory behaviour which can either be enhanced or reduced in nature. The extent to which these results reflect in vitro methodology in uncertain and, furthermore, their in vivo significance is unclear.

The locomotory behaviour of peripheral blood neutrophils (PMNs) from patients with juvenile (JP) and rapidly progressive (RPP) forms of early-onset periodontal disease was studied using the under agarose technique and n-formyl-methionyl-leucyl-phenylalanine (FMLP) as the chemotractant. PMNs from experimental patients showed normal random, chemotactic and chemokinetic locomotory behaviour when compared with control subjects. Further investigation of single-cell movements using time-lapse video analysis also failed to show any significant differences in locomotory behaviour between the PMNs of experimental and control individuals. We conclude that differences in technique may account for much of the variation which exists in the literature with respect to PMN locomotion in periodontal disease. In the final analysis, it is difficult to dispute direct observation of moving cells, and using this approach, we have been unable to confirm the presence of any PMN locomotory defect in our series of patients with early-onset periodontal disease.

Localized juvenile periodontitis (LJP) is characterized by severe, early-onset, molar and incisor bone loss; neutrophil chemotaxis disorders; and a high prevalence of Actinobacillus actinomycetemcomitans infection. LJP is further characterized by significant familial aggregation of the disease. Recent work in our laboratory has demonstrated the selective depletion of a surface glycoprotein of 110,000 Mr (GP110) from LJP neutrophils by using surface labeling with [14C]formaldehyde and autofluorography. The function of GP110 is unknown; however, it does not appear to be a chemotactic factor receptor. Rather, it is bound by a monoclonal antibody (NCD-1) that recognizes a neutrophil differentiation antigen and which itself alters neutrophil chemotactic and secreting functions. To quantify GP110 on LJP and normal neutrophils, fluorescein-labeled NCD-1 was bound to neutrophils and the amount of fluorescence was evaluated by using cytofluorography. Our results indicate that there is a quantifiable reduction (40%) of GP110 on the surface of LJP and GJP neutrophils, compared with controls. Other patients with neutrophil defects express normal quantities of GP110, suggesting disease specificity. Our data suggest that GP110 may be a useful disease marker for LJP and may provide a useful probe for the study of neutrophil chemotactic function and dysfunction.

Previous studies have demonstrated that certain local juvenile periodontitis (LJP) patients possess neutrophils with an intrinsic chemotactic defect to the peptide chemoattractants f-met-leu-phe (FMLP) and C5a. In this investigation, the in vitro response of neutrophils to the chemotactic agent leukotriene B4 (LTB4) was examined. Those LJP patients who possessed defective chemotactic responses to FMLP and endotoxin activated serum (EAS) also had impaired chemotaxis to LTB4. Exogenous LTB4 failed to augment the impaired response to FMLP. Evidence for a global membrane defect in LJP neutrophils resulting in hyporesponsiveness to chemically diverse chemotactic agents is presented.

PMN chemotactic function and oxydative metabolism were investigated in 13 patients with rapidly progressive periodontitis (RPP) and 9 patients with localized juvenile periodontitis (LJP) using a chemotaxis assay (CHA), luminol chemiluminescence (CL) and nitroblue-tetrazolium (NBT) tests. In the LJP patients there was a significantly lower CHA than in the RPP patients (less than 40%). Three out of the 13 RPP patients displayed a cell-dependent increase of CHA and in 3, the reduced CHA measured was serum-associated whilst among the 9 LJP patients, a cell-dependent defect of CHA was present in 5 patients, a serum-associated defect in one patient and a combined cell- and serum-associated defect was found in another patient. The average values of CL in the RPP patients were significantly lower (1309.3 +/- 814.2) than those measured in the LJP group (2208 +/- 826.9). In contrast, the mean NBT test values indicated a lower phagocytic function in the LJP patients than in the RPP ones suggesting also the presence of a serum-dependent factor in RPP and that of a combined cell- and serum-response in LJP. According to these results, the neutrophil dysfunction in RPP, as indicated by both the chemotaxis and the oxydative metabolism nature, was frequently associated with a serum factor.

Nearly 70-75% of patients with localized juvenile periodontitis (JP) have abnormal polymorphonuclear leukocytic (PMN) chemotaxis. The objective of this study was to determine whether the lower chemotactic response in PMNs from JP patients is associated with a defect in intracellular signal transduction, as measured by stimulus-induced changes in free cytosolic calcium (Ca2+) mobilization. We report that peptide chemoattractants such as N-formyl-methionyl-leucyl-phenylalanine (fMLP) and the complement fragment C5a in direct comparative studies induced lower amounts of initial Ca2+ mobilization in PMNs from JP patients than healthy controls, as monitored by intracellular fura-2 fluorescence. The initial resting levels of free cytosolic Ca2+ in PMNs from JP patients and normal individuals were found to be similar. fMLP and C5a both mobilized Ca2+ in PMNs in a dose-dependent manner. Treatment of PMNs from 0.16 to 20 nM fMLP and 0.2 to 20 nM C5a resulted in elevated levels of free cytosolic Ca2+. However, above 20 nM fMLP and 5 nM C5a concentrations the extent of total Ca2+ mobilization did not differ significantly. Although fMLP and C5a caused Ca2+ mobilization in PMN cells from JP and healthy control subjects, fMLP stimulation induced higher levels of free cytosolic Ca2+ mobilization in PMN cells from healthy control subjects (141.29 +/- 25.55 nM/2 x 10(6) PMNs), than PMNs from JP patients (62.33 +/- 23.76 nM/2 x 10(6) PMNs). Similarly C5a induced higher levels of Ca2+ mobilization in PMNs from healthy control individuals (130.43 +/- 18.26 nM Ca2+/2 x 10(6) PMNs)O, when compared to JP patients (49.92 +/- 14.92 nM Ca2+/2 x 10(6) PMNs).(ABSTRACT TRUNCATED AT 250 WORDS)