Johan Glette

Factors important for the measurement of chemiluminescence production by polymorphonuclear leukocytes

Chemiluminescence (CL) production by phagocytosing polymorphonuclear leukocytes (PMNLs) was measured by an automatic photoluminometer with built-in mixing and temperature controls. Agitation of the vials with PMNLs and opsonized zymosan particles influenced both the lag time and the CL production. Maximal production was obtained by continuous mixing of the samples, the reaction peak occurring within 6 min. Increasing the temperature from 20 to 40 degrees C also increased the CL production, and in further experiments 37 degrees C was used. Aggregation of the PMNLs was avoided by washing the cells in PBS containing gelatin 1 g/l. Glucose, Ca2+ and Mg2+ in the final reaction mixture were necessary for maximal CL responses. The measurements of CL per s up to 4 min, the peak CL value, or the integral below the CL curve up to 6 min were all linearly proportional to the number of PMNLs in the reaction mixture. Since the lag time and the time before reaching peak CL may vary, the integral below the curve up to 6 min was chosen as the mode of CL measurement. On repeated measurements the coefficient of variation was 6.3%. The mean CL integral value for PMNLs from 14 healthy individuals was 205 +/- 19 mVs, indicating a good reproducibility of the standardized assay.

PORPHYRIN‐INDUCED PHOTODAMAGE TO ISOLATED HUMAN NEUTROPHILS

Abstract— Human neutrophils were irradiated with light at 340–380 nm in the presence of low concentrations of protoporphyrin or uroporphyrin. At increasing light doses or increasing concentrations of protoporphyrin, the neutrophils rapidly lost the ability of locomotion. Also, neutrophil chemiluminescence and hexose‐monophosphate shunt activity rapidly declined. An early event was leakage of endogenous K+ followed by lactate dehydrogenase and at a later stage leakage of particle‐bound acid phosphatase. A number of cellular enzymes were inactivated, the susceptibility to inactivation decreased in the order: succinate dehydrogenase > lactate dehydrogenase > glutamate dehydrogenase > acid phosphatase. Uroporphyrin had no effect on neutrophil functions, leakage of K+, or cellular enzymes. The results suggest that photodamage to the plasma membrane and the mitochondria are earlier events than photodamage to lysosomes.

Doxycycline induced photodamage to human neutrophils and tryptophan.

Abstract— Neutrophil functions were studied following irradiation (340–380 nm) of the cells in the presence of 22 mUM doxycycline. At increasing light fluence the locomotion, chemiluminescence and glucose oxidation (by the hexose monophosphate shunt) of the neutrophils steadily decreased. The photodamage increased with increasing preincubation temperature and time and was enhanced in D2O, reduced in azide and abolished in anaerobiosis. Superoxide dismutase, catalase or mannitol did not influence the photodamage. Photooxidation of tryptophan in the presence of doxycycline was increased9–10‐fold in D2O and nearly abolished in the presence of 0.25 mM NaN3, indicating that singlet oxygen is the most important reactive oxygen species in the doxycycline‐induced photodamage. The results may explain some of the features of tetracycline‐induced photosensitivity and why other authors have obtained diverging results when studying the influence of tetracyclines on neutrophil functions.

Phototoxicity of tetracyclines as related to singlet oxygen production and uptake by polymorphonuclear leukocytes.

The photo-induced singlet oxygen production of six tetracyclines was measured as tryptophan degradation. Demethylchlortetracycline was the most efficient singlet oxygen producer followed by doxycycline. The least efficient producer was minocycline. Doxycycline, however, was the most potent inducer of photodamage to polymorphonuclear leukocytes (PMNLs) followed by demethylchlortetracycline. Accordingly, the singlet oxygen production during irradiation did not correlate with the induction of photodamage to the PMNLs. However, the uptake of doxycycline by the cells was 3 times higher than that of demethylchlortetracycline, and the tetracycline-induced photodamage to the PMNLs correlated with the product of singlet oxygen production during irradiation and the drug uptake by the cells.

Influence of tetracyclines and light on the release of lysozyme from human granulocytes and monocytes

Doxycycline inhibited the release of lysozyme from human granulocytes and monocytes exposed to non-opsonized zymosan particles. This effect was more marked for granulocytes than for monocytes. Oxytetracycline, however, did not influence the release. The difference between the drugs can be explained by differences in their lipid solubilities. The divalent cation chelator, EDTA, also reduced the release of lysozyme from leukocytes exposed to non-opsonized zymosan. Accordingly, the selective release of lysozyme from human leukocytes is divalent cation dependent. The inhibition of release by doxycycline is most likely also due to binding of these ions. When the cells were exposed to UVA light in the presence of doxycycline, the inhibition of lysozyme release was potentiated. Using irradiated cells, maximal inhibition was obtained at 20 micrograms doxycycline/ml. However, it is not clear whether these results have clinical relevance.

Influence of fever temperatures and of some cytoactive drugs on in vitro lysozyme release from monocytes and granulocytes

The selective in vitro release of lysozyme from human monocytes and granulocytes was not greatly influenced by temperatures above 37 degrees C and up to 40 degrees C. The release was markedly inhibited by preincubation with phenylbutazone, oxyphenylbutazone, colchicine and vincristine. A water-soluble hydrocortisone complex also inhibited lysozyme release, but at high concentrations, lysis of the cells occurred. Although methotrexate had a weak inhibiting effect, no appreciable influence on release was observed with cyclophosphamide or cytarabine. Thus, release of lysozyme from blood leukocytes is likely to be dependent on cellular functions involving the stability of both microtubules and membranes.

Effect of Doxycycline and PUVA Light on Human Polymorphonuclear Leukocyte Function

Polymorphonuclear leukocytes (PMNLs) are a cornerstone of host defense against infection, and the interaction of PMNLs with antimicrobial agents has been extensively studied [10, 14, 15]. Reports on the effect of tetracyclines on PMNL functions are contradictory. Chlortetracycline and doxycycline seem to markedly inhibit the phagocytic activity of the cells [5, 18] while no such effect has been demonstrated with tetracycline hydrochloride [9]. More recent reports [16, 17] have demonstrated only a slight decrease in phagocytosis of 32P-labeled Escherichia coli by human and rat PMNLs. Doxycycline administrated to healthy individuals has also been shown to slightly inhibit the migration to skin chambers [4].