Differential gene expression in neutrophils from chronic generalised periodontitis

Studies demonstrate that peripheral neutrophils from chronic generalised periodontitis (CGP) patients exhibit a hyper-reactive phenotype. However, no studies have analysed large-scale gene expression differences between neutrophils from CGP and control individuals. Objective: To identify genes differentially expressed in peripheral blood neutrophils from CGP patients relative to periodontally healthy controls. Methods: Heparinized blood was obtained from 9 non-smoking CGP patients, who exhibited neutrophil hyper-responsivity to FcγR stimulation by enhanced chemiluminescence (Wright et al. J Dent Res 2004, 83: abstr 1088), and age/sex/smoking-matched controls. Blood was collected before and 3-months after conventional non-surgical therapy. RNA was prepared from isolated neutrophils (Percoll gradient/erythrocyte lysis) by phenol/chloroform extraction. Patient and control RNA pools from the 4 pairs of cells exhibiting the highest hyper-responsive differential were used to screen Affymetrix HG_U133A microarrays. Confirmatory RT-PCR was performed for 9 selected genes using pooled RNA (including post-treatment samples) and individual patient and control RNA samples (n=9). Results: Microarray data identified differential expression (>2-fold) of 46 distinct genes (43 increased, 3 decreased in CGP neutrophils), representing a range of ontological classes. Subsequent RT-PCR analysis of the original pooled samples corroborated these data as did analysis of transcript levels in the 9 patient and control samples. However, these latter results demonstrated heterogeneity of expression in CGP and healthy individuals. Overall the patient/control differences detected by RT-PCR in the 4 sample pairs used for microarray analysis did not differ from the five additional pairs of samples (χ² = 0.043; p>0.975). Furthermore, RT-PCR analysis of post-treatment pooled samples indicated a decrease in transcript expression, which approached that of control values. Conclusions: Peripheral neutrophils from CGP patients exhibit a distinct molecular phenotype, which appears to be altered by therapy. Mechanistically, this suggests involvement of a peripheral stimulus, rather than a constitutional up-regulation of the genes investigated. Supported by MRC UK-G0000797.