Neutrophil (PMN) elastase assay for in-vivo chemotaxis (CTX)

PMN elastase activity (EA) is detectable in gingival crevicular fluid (GCF).  Its role as inflammatory marker has widely been recognized.  The source of the enzyme are PMN from periodontal tissues or the gingival crevice.  Objective: of this study was to establish an in-vivo CTX-assay and correlate EA with PMN counts.  Methods: after IRB-approval and informed written consent, 15 healthy individuals participated in this single-blinded, prospective clinical trial.  After a 2 week oral hygiene regimen GCF was assayed with Periopaper^® and volumes assessed with a calibrated Periotron 8000^® (Vetter-Laborbedarf, Ammerbuch/Germany). In the upper jaw, in a randomized split mouth design either CTX-stimulus 2 mg/ml casein at a test tooth (TT) or a placebo (PBS^{(-Mg++/+Ca++)}) at a collateral tooth (CT) were applied into the gingival crevice.  GCF was sampled at BL and every 10 up to 50 min.  Storage conditions were evaluated and GCF immediately frozen (-80 °C) until EA was quantified in a MT-plate reader Fluostar^® (SLT, Crailsheim/Germany).  Assay buffer composition and saturating (10^-3M) substrate solution for EA (MeO-Suc-Ala-Ala-Pro-Val-AMC) yielded activity from all samples.  Ex-vivo naïve PMN from venous blood were correlated with EA.  Results:  20 min after stimulation with 2 mg/ml casein median EA_TT increased from 223 to 289 μU/μl; in contrast, using placebo median EA_CT dropped from 212 to 62 μU/μl, respectivley.  The maximum ΔEA (EA_TT vs. EA_CT) was highly significant (p<0.001;Wilcoxon).  However, for both TT as well as CT the assayed GCF volumes declined over time indicating changes in PMN concentration.  Consequently, EA_tt after its peak and EA_CT diminished to converge at 40 min.  3^rd degree regression analysis allowed to quantify PMN based on EA (r²=0.992;p<0.001).  Conclusions:  Employing casein stimulation we here present a calibrated, non invasive in-vivo CTX assay which permits the accurate calculation of PMN concentrations and reflects their chemotactic migration into GCF.