Table 2.

Ehrlichial contact, but not internalization, and ehrlichial carbohydrate, but not protein, are required for inhibition of O2 release by neutrophils in response to PMA

TreatmentaReduction of ferricytochromec (nmol of O2/106neutrophils)b
Donor 6 (Transwell)c
 PMA13.9  ± 0.3*
 HGE agent (separated from neutrophils by membrane) + PMA  12.6  ± 0.4*
 HGE agent (in contact with neutrophils) + PMA0.4  ± 0.3
Donor 7
 Medium control0.6  ± 0.6
 PMA16.7  ± 3.4*
 HGE agent + PMA4.7  ± 0.4
 Trypsin-treated HGE agent + PMAd 4.5  ± 1.2
 Periodate-treated HGE agent + PMAd 14.4  ± 0.3*
 Pronase treatment of neutrophilsepreincubated with the HGE agent + PMA  18.7  ± 1.8*
 Pronase treatment of neutrophils + PMA16.8  ± 0.1*
Donor 8
 Medium control0.1  ± 0.1
 PMA13.3  ± 0.3*
 HGE agent lysate + PMAf 3.9  ± 0.2
  • a Neutrophils were pretreated with the HGE agent with or without reagent for 30 min prior to PMA stimulation.

  • b Mean ± standard deviation (n = 3). Results are representative of more than three independent experiments. ∗, P < 0.01 compared to neutrophil-alone (no PMA) control (Student's t test).

  • c Neutrophils in one chamber were incubated without or with the HGE agent added to the same or another chamber separated by a Nuclepore polycarbonate porous membrane, using two-compartment Transwell plates. PMA was added to all wells.

  • d Host cell-free HGE agent was treated with 0.25% trypsin for 15 min at room temperature or with 20 mM sodium periodate for 1 h prior to addition to cytochrome creduction assay mixtures and PMA stimulation.

  • e Neutrophils were incubated with host cell-free HGE agent for 30 min at 37°C, incubated with pronase E (2 mg/ml) for 30 min at 37°C to remove uninternalized bacteria, then added to cytochrome c reduction assay mixtures, and stimulated with PMA.

  • f Neutrophils were incubated for 30 min at 37°C with lysed HGE agent prior to PMA stimulation.