Leukocyte activation is known to involve cell membrane potential changes. Phenobarbital, an anesthetic and anticonvulsant that can inhibit neuronal membrane depolarization, may also affect leukocyte activation. Measuring membrane potential, actin polymerization, chemotaxis, superoxide production, lymphocyte proliferation, intracellular calcium concentration, and cytokine production, we found that phenobarbital at a concentration of 15-30 micrograms/ml, which is considered a therapeutic serum level for controlling seizures, did not affect polymorphonuclear neutrophil (PMN) activation. At levels higher than 100 micrograms/ml, phenobarbital significantly suppressed formylmethionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis. Concentrations greater than 300 micrograms/ml also inhibited phorbol myristate acetate-stimulated membrane potential change. In contrast, 30 micrograms/ml phenobarbital significantly inhibited lymphocyte proliferation stimulated by phytohemagglutinin (PHA) and pokeweed mitogen. This concentration of phenobarbital also suppressed the increase of intracellular free calcium induced by PHA. However, only a higher concentration of phenobarbital (300 micrograms/ml) was able to inhibit PHA-induced interleukin-2 (IL-2) production and suppress the proliferation of PHA-induced IL-2 receptor-bearing lymphocytes. These results suggest that concentrations of phenobarbital associated with anticonvulsive levels do not affect PMN activation but suppress lymphocyte activation, possibly by affecting intracellular signal transduction.

We have investigated the effects of a novel neutrophil-activating factor released by Trichomonas vaginalis (TV-NAF) on neutrophil chemotaxis. TV-NAF was present in the supernatant from 107 T. vaginalis (STV) cultured in 1 ml of serum-free Hanks' balanced salt solution (HBSS) at 37°C for 30 min. With a multichamber chemotactic assay, we found that there were 112 + 15 migrated neutrophils (mean ± standard deviation, n = 7) for STV and 11 ± 4 for HBSS per high-power field (x400). STV was also able to induce neutrophil actin assembly (increased 1.5-fold), enhance expression of complement receptor type 3 (increased 5-fold), and promote intracellular calcium mobilization (increased 2.5-fold). There was no chemotactic activity in the preparation of STV from killed trichomonads. The fact that heating up to 100°C or deproteinization by treatment with proteinase K at 65°C for 1 h did not abolish its chemotactic activity suggests that the TV-NAF involved was not a protein. The chemotactic activity of TV-NAF was associated with the fraction containing small molecules of less than 3,000 Da. Therefore, the possibility that eicosanoid production by trichomonads is responsible for neutrophil activation was investigated. Leukotriene B4 (LTB4; 500 pg/ml) but not thromboxane B2 (<20 pg/mi) or prostaglandin E2 (<8 pg/ml) was found in the STV by radioimmunoassay. Production ofLTB4 by trichomonads was time dependent and increased twofold when arachidonic acid (100 ,uM) was added but was not decreased when eicosanoid inhibitors were present. Evidence for the presence of LTB4 in STV was further provided by the fact that rabbit anti-LTB4 antiserum could abolish the chemotactic activity of STV. These studies suggest that the spontaneous release of TV-NAF, which is most likely LTB4, may activate neutrophils, presumably through a different arachidonate metabolic pathway than that in mammalian cells. * Corresponding author. cellular calcium mobilization. The results from these studies provide more information about the inflammatory response induced by T. vaginalis. MATERIALS AND METHODSOrganism. Seven local isolates, axenically cultivated, were maintained in a modified medium identical to the TYI-S-33 medium of Diamond et al. (7), except that 0.5% Panmede (Paines & Byrne Limited, Greenford, England) was added. The number of organisms per culture was determined with a Coulter counter (model D industrial; Coulter Electronics, Inc., Hialeah, Fla.) with a 70-,um aperture tube.Preparation of STV. Each isolate of T. vaginalis grown at 37°C in 15-ml tubes containing TYI-S-33 medium was harvested during the logarithmic growth phase after 36 h of cultivation. They were centrifugally washed three times in HBSS (GIBCO, Grand Island, N.Y.), and then viable cells were counted in a hemacytometer with trypan blue-saline. HBSS without phenol red was used as the wash buffer and diluent throughout this study. The medium-free flagellates (107) were incubated in 1 ml of HBSS at 37°C for 30 min. Motile flagellates were then removed by gentle centrifugation (500 ...

Introduction Synovial cells are potential sources of inflammatory mediators in bacterial-induced arthritis but their involvement in the inflammatory response to Candida albicansinduced septic arthritis is largely unknown.

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