Effect of LPS from Actinobacillus actinomycetemcomitans JP2 on PMN Function

Actinobacillus actinomycetemcomitans (A.a.) is the key microorganism in LAP patients. The JP2 clone of A.a. has a 530-bp deletion from the promoter region of leukotoxin gene operon; produce high levels of leukotoxin, and is most frequently detected in individuals of African descent with a positive association to the occurrence of severe periodontal destruction in LAP. Objective: To study the effect of whole cell preparations and LPS from A.a.JP2 on neutrophil function. Methods: PMN were isolated from 10 healthy individuals by Ficoll-Hypaque density gradient centrifugation. A.a.JP2 was grown on either solid agar GC medium or trypticase soy broth supplemented with 10% serum. LPS was extracted by hot phenol-water extraction and purified by CsCl gradient. Purified LPS was analyzed by SDS-PAGE and amino acid sequencing. LPS activity was tested by Limulus Amebocyte Lysate (LAL) assay using E. coli LPS as positive control. PMN were cultured with whole bacteria, LPS, or leukotoxin and were studied for superoxide generation and chemotaxis. Mutant A.a.JP2 that does not produce the leukotoxin was used as a control. Results: PMN cultured with JP2 were lysed as early as 15 minutes. Superoxide generation in response to fMLP by PMN preincubated with JP2 LPS was significantly increased compared to those that were not treated with LPS (2.78nmol/10⁷PMN/min vs. 1.78 nmol/10⁷PMN/min, p<0.05). This effect was similar to that of E. coli LPS. A.a.JP2 LPS led to a statistically significant and dose-dependent (20-2000ng/ml) inhibition of PMN chemotaxis (41.5-60.2%). Conclusions: LPS from A.a.JP2 affects PMN function by suppressing chemotaxis and increasing superoxide generation, acting as a modifier of PMN response. Supported by USPHS Grant DE13499 and GCRC Grant MO1 RR00533.