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CD11b mRNA expression in neutrophils isolated from peripheral blood and gingival crevicular fluid
K Watanabe1, B Blew, M Scherer
1Department of Periodontics, School of Public Health, University of Illinois at Chicago, USA.
Abstract:
Adhesion molecule CD11b/CD18 expressed by neutrophils (PMNs) participates in cell migration and phagocytosis of C3bi derivatized bacteria. It is this phagocytic function that eliminates some of the known periodontal pathogens in periodontal pockets. In patients with advanced periodontitis, homotypic aggregation of crevicular fluid PMNs (CF-PMNs) may occur due to overexpression of CD11b/CD18 and this may lead to ineffective elimination of periodontal pathogens. We have previously shown that CF-PMNs isolated from the periodontal pockets overexpress CD11b compared to PB-PMNs. This study tested the hypotheses that (1) overexpression of surface CD11b correlates with expression of CD11b mRNA in CF-PMNs isolated from advanced periodontitis subjects, and (2) the intrinsic capacity of CD11b mRNA upregulation by PB-PMNs from periodontitis patients differs from that of control subjects. CF-PMNs and peripheral blood PMNs (PB-PMNs) were isolated from 13 subjects with healthy gingiva (control group) and 13 subjects with advanced periodontitis (patient group). The surface expression of CD11b was determined by flow cytometry and CD11b mRNA was determined by extraction of mRNA and reverse transcription to cDNA followed by DNA amplification using primers to detect a segment of the cDNA which encodes CD11b. The results of this study confirm that the surface expression of CD11b on CF-PMNs is significantly higher in periodontitis subjects vs control subjects (p = 0.03), whereas surface CD11b expression on PB-PMNs does not differ significantly between groups (p = 0.06). The level of surface CD11b expression on CF-PMNs did not correlate with the amount of mRNA present in CF-PMNs in either group (p = 0.056, 0.07 for control and periodontitis patients, respectively). Most (9 of 13) individuals in the patient group expressed CD11b mRNA whereas very few control subjects (2 of 11) had CD11b mRNA in their CF-PMNs. This difference between groups was statistically significant (p = 0.004). The capacity to upregulate CD11b mRNA upon stimulation with fMLP and/or GM-CSF was highly variable and there was no statistical difference between the 2 groups.
Insights
Neutrophils in periodontitis patients show increased CD11b expression, but surface levels don't correlate with mRNA. However, CD11b mRNA is more prevalent in neutrophils from periodontal pockets of patients with advanced periodontitis.
Area of Science:
- Immunology
- Oral Biology
- Molecular Biology
Background:
- Neutrophils, specifically polymorphonuclear leukocytes (PMNs), utilize adhesion molecule CD11b/CD18 for critical functions like migration and phagocytosis of bacteria.
- In advanced periodontitis, increased CD11b/CD18 expression on PMNs in periodontal pockets may lead to their aggregation, potentially impairing the elimination of periodontal pathogens.
- Previous research indicated that PMNs from crevicular fluid (CF-PMNs) in periodontitis patients overexpress CD11b compared to those in peripheral blood (PB-PMNs).
Purpose of the Study:
- To investigate the correlation between surface CD11b expression and CD11b mRNA levels in CF-PMNs from individuals with advanced periodontitis.
- To determine if peripheral blood PMNs (PB-PMNs) from periodontitis patients have a different intrinsic capacity for CD11b mRNA upregulation compared to controls.
- To elucidate the role of CD11b expression in the context of periodontitis and neutrophil function.
Main Methods:
- Isolation of CF-PMNs and PB-PMNs from subjects with healthy gingiva (control group) and those with advanced periodontitis (patient group).
- Quantification of surface CD11b expression using flow cytometry.
- Measurement of CD11b mRNA levels via RNA extraction, reverse transcription to cDNA, and DNA amplification using specific primers.
Main Results:
- Surface CD11b expression was significantly higher on CF-PMNs from periodontitis subjects compared to controls (p = 0.03).
- No significant difference in surface CD11b expression was observed on PB-PMNs between the two groups (p = 0.06).
- Surface CD11b levels on CF-PMNs did not correlate with their corresponding mRNA levels in either group (p = 0.056, 0.07).
- CD11b mRNA was detected in CF-PMNs from a majority of periodontitis patients (9/13) but only a small fraction of control subjects (2/11), a statistically significant difference (p = 0.004).
- The capacity of PB-PMNs to upregulate CD11b mRNA upon stimulation showed high variability, with no significant difference between periodontitis patients and controls.
Conclusions:
- While CF-PMNs in periodontitis exhibit increased surface CD11b, this does not directly correlate with their mRNA levels, suggesting post-transcriptional regulation.
- The presence of CD11b mRNA in CF-PMNs is significantly more common in periodontitis patients, indicating an altered transcriptional state in neutrophils within the periodontal pocket.
- The intrinsic ability of PB-PMNs to upregulate CD11b mRNA does not differ between periodontitis patients and controls, implying that local factors within the periodontal pocket influence CD11b expression.