In vitro modulation of normal and diseased human neutrophil function by pentoxifylline.

The influence of pentoxifylline on normal and diseased neutrophil function has been studied in vitro. In high concentrations pentoxifylline stimulated human neutrophil chemotaxis toward both bacterial oligopeptides and complement components. Pentoxifylline was also shown in vitro to restore the normal chemotactic capacity of neutrophils from patients with known functional defects, i.e. myelodysplastic syndromes, lazy leucocyte syndrome, juvenile parodontitis, hyper-IgE-syndrome and liver cirrhosis. Pentoxifylline was also shown to strongly inhibit the release of primary and secondary granule release of granulocytes. Moreover, pentoxifylline inhibits both basal and stimulated neutrophil adhesion to both aortic and pulmonary artery calf endothelium. The mechanism whereby pentoxifylline exerts this action is not adequately understood. While our results partially imply interference of pentoxifylline with neutrophil cyclic AMP and/or prostaglandin metabolism, down-regulation of neutrophil functional antigen (e.g. CD11, CD18) expression seems to play a key role in the observed drug effects. Finally, these results indicate that pentoxifylline may be useful in the treatment of granulocyte mediated diseases and symptoms.

In vitro and in vivo evaluation of granulocyte labeling with [99mTc]d,1-HM-PAO.

The functional integrity of white blood cells labeled with [99mTc]d,1-HM-PAO containing variable amounts of the ligand or of the 99mTc activity was evaluated by enzymatic tests and by measuring random migration, chemotaxis, phagocytosis, killing, and adhesion. The ultrastructure of labeled cells was studied by electron microscopy. The tracer dose and the HM-PAO concentration did not significantly affect phagocytosis and killing. The results of the other tests remained normal. A maximum labeling efficiency of 80% was reached by incubating the granulocytes for 20 min with 10-20 mCi of [99mTc]d,1-HM-PAO containing 50 micrograms of the ligand in 1 ml of saline. There was only a slow washout of 20% of activity from the labeled cells in 24 hr. The ultrastructure was not influenced by the labeling technique. Proven infection sites of 17 orthopedic patients were clearly visualized. After a short transient lung uptake, there was a clear spleen and moderate liver uptake with early bladder and late prominent colon visualization. Because of the lower cost, favorable radiation dose and more suitable tracer characteristics, this technique is a promising alternative for 111In labeling of white blood cells.

Evaluation of 111In labelled white blood cells by in vitro functional tests and electron microscopy. Comparison of three labelling methods.

We have studied the influence of granulocyte labelling with commercially available 111In-oxine, tropolone (trop) or home made 111In-Mercapto pyridine (Merc) prepared by the method of Thakur (1985) on the cell structure by electron microscopy and on the cell function by enzymatic tests, random migration, chemotaxis, phagocytosis and bactericidal activity. The granulocytes were labelled with 400 microCi 111In-oxine in saline or 111In-trop or Merc in plasma. The effect of the chelating agents with and without addition of the tracer was studied (n = 4) with varying concentrations: 5-10 micrograms/ml oxine, 10-160 micrograms/ml trop and 1-4 micrograms/ml Merc. Chemotaxis and random migration were not affected by 111In-trop and clearly suppressed by 111In-oxine and Merc; the other tests were normal. The cell structure was disturbed by Merc. The labelling efficiency was excellent with oxine (90%), acceptable with trop (30%-80%) and poor with Merc (10%-25%). Without 111In, chemotaxis and random migration were normal up to a concentration of 80 micrograms/ml trop, 8.5 micrograms/ml oxine and 1 microgram/ml Merc. With addition of 111In, chemotaxis and random migration were unaffected up to 80 micrograms/ml by trop and markedly suppressed by Merc and oxine. It is concluded that labelling with 111In-trop assures intact cells.

Platelets augment granulocyte aggregation and cytotoxicity: uncovering of their effects by improved cell separation techniques using Percoll gradients.

The 'standard' technique of granulocyte preparation for in vitro studies uses dextran removal of erythrocytes and Ficoll-Hypaque gradient centrifugation to increase granulocyte purity. The procedure is lengthy, approximately 150 min in our hands, and provides granulocytes significantly contaminated with platelets (approx. 5 platelets/PMN). We report a technique that replaces dextran with hydroxy-ethylstarch and Ficoll-Hypaque with Percoll. Preparation time is reduced by approximately 40% and platelet contamination by more than 80%. Granulocytes, so prepared, function metabolically (O2-generation, chemiluminescence, HMP-shunt maxima) and, in motility/phagocytosis assays, identically to 'standard' preparations. However, an augmentatory effect of platelets in granulocyte aggregation responses and their mediation of cytotoxicity is uncovered. Ficoll-Hypaque purified cells (platelet-rich) aggregate to a significantly greater degree with FMLP or activated complement lectins and excessively kill 51Cr-labelled target cells when compared to Percoll-preparations (platelet-poor). Re-addition of purified platelets or of platelet release supernatants to the latter reproduces results using the 'standard' preparations.

Effects of quinolones on granulocyte function in vitro.

Granulocytes (PMN) play an important role in antibacterial host defence. The modulation of PMN-function by different antibiotics has been shown previously. Therefore, we investigated the possible immuno-modulatory effects of four new quinolone antibiotics: ciprofloxacin, norfloxacin, pefloxacin, ofloxacin. Pure PMN-preparations were obtained from healthy volunteers and incubated for 30 min at 37 degrees C with the different quinolones at different concentrations (0.1-1-5-10 mg/l). A complete PMN-functional profile was assayed. At all concentrations tested no significant differences were seen for myeloperoxidase content, superoxide production and chemiluminescence. A significant stimulation of phagocytosis and killing occurred at 10 mg/l of norfloxacin and at 10 mg/l of pefloxacin. An increase of chemotaxis towards formyl-methionyl-leucyl-phenylalanine was noted at 10 mg/l of ofloxacin, while stimulation of hexose monophosphate shunt was seen at 1 mg/l of ciprofloxacin and 10 mg/l of ofloxacin. The mechanism whereby both subinhibitory and inhibitory concentrations of quinolones moderately influence PMN-responsiveness is not yet known. Further studies are needed to elucidate the nature of this quinolone-effect and to assess its relevance in vivo.