Pitfalls in flow cytometric analyses of surfactant-exposed human leukocytes.

BACKGROUND: Surfactant replacement treatment is the standard of care for the prevention and treatment of neonatal respiratory distress syndrome in preterm infants and may also improve oxygenation in acute respiratory distress syndrome in children, adolescents and adults. Beside surface tension- and mechanical shear-reducing functions, natural surfactants have been ascribed immunomodulatory capacities. Current in vitro studies on immunomodulatory effects of pulmonary surfactant preparations on human leukocytes rely on ELISA, Western blot and polymerase chain reaction. Data obtained by flow cytometry are missing, so far, most likely due to confounding phospholipid residues. Intracellular cytokine flow cytometry in surfactant-exposed immune cells would provide information on pro- and anti-inflammatory immunomodulation at the single-cell level and would allow for integrating detailed immunophenotyping, functional assays and assessment of viability. AIM: We implemented a flow cytometry protocol for reliable quantitative assessment of in vitro intracellular cytokine production in surfactant-exposed human lymphocytes (CD4(+)) and monocytes (CD14(+)). METHODS: Two different permeabilization techniques were tested for their ability to provide intracellular cytokine staining in surfactant-exposed CD14(+) monocytes and CD4(+) lymphocytes. Both a commercially available solution containing saponin and ice-cold methanol were used as permeabilization reagents. RESULTS: For both cell types, flow cytometry following saponin-based permeabilization revealed pronounced unspecific fluorescence signals in surfactant-exposed samples overlapping with the fluorescence spectra of the majority of conjugates. Autofluorescence of surfactant phospholipid particles interfered significantly with reliable quantification of fluorochrome-specific signals and conclusive analysis. Implementation of a methanol-based permeabilization protocol resulted in the elimination of confounding non-cell particle signals allowing for an accurate quantification of intracellular cytokine production. CONCLUSION: Reliable detection of intracellular cytokines by flow cytometry may be challenging in surfactant-exposed cell samples due to significant autofluorescence of aggregated phospholipid particles. This issue has been addressed for the first time and may be of high relevance for all types of surfactant research. We demonstrate that a methanol-based permeabilization approach completely removes interfering fluorescent surfactant micelles and allows for correct evaluation of data. The successful removal of confounding surfactant phospholipids opens up a wide variety of multiparameter flow cytometry; a method that has not been applied in the field of surfactant research, yet.

Mesna or cysteine prevents chloroacetaldehyde-induced cell death of human proximal tubule cells.

Chloroacetaldehyde (CAA) is formed in the body from the chemotherapeutically used drug ifosfamide (IFO). CAA leads to cell death in proximal tubule cells mainly through the mechanism of necrosis rather than apoptosis. During chemotherapy, 2-mercaptosulfonic acid (mesna) is used with IFO to protect the urothel from cell damage. Little is known of the effect of mesna on renal proximal tubule cells, the primary site of damage after IFO treatment. Mesna contains a sulfhydryl (SH) group. To clarify whether SH-group-containing molecules can prevent CAA-induced cell death, we studied the effect of mesna and cysteine on necrosis, apoptosis, and protein content in a human proximal tubule-derived cell line (IHKE cells) treated with CAA. Both substances prevented CAA-induced necrotic cell death and protein loss and restored CAA-inhibited caspase-3 activity. CAA also prevented cisplatin-induced apoptosis. This inhibition was reversible in the presence of glutathione (GSH). We conclude that SH-containing molecules can protect proximal tubule cells from cell death because they interact with CAA before CAA can disturb other important cellular SH groups. A sufficient supply of intra- and extracellular SH groups during IFO chemotherapy may therefore have the ability to protect renal tubule cells from cell death.

Chloroacetaldehyde as a sulfhydryl reagent: the role of critical thiol groups in ifosfamide nephropathy.

Chloroacetaldehyde (CAA) is a metabolite of the alkylating agent ifosfamide (IFO) and putatively responsible for renal damage following anti-tumor therapy with IFO. Depletion of sulfhydryl (SH) groups has been reported from cell culture, animal and clinical studies. In this work the effect of CAA on human proximal tubule cells in primary culture (hRPTEC) was investigated. Toxicity of CAA was determined by protein content, cell number, LDH release, trypan blue exclusion assay and caspase-3 activity. Free thiols were measured by the method of Ellman. CAA reduced hRPTEC cell number and protein, induced a loss in free intracellular thiols and an increase in necrosis markers. CAA but not acrolein inhibited the cysteine proteases caspase-3, caspase-8 and cathepsin B. Caspase activation by cisplatin was inhibited by CAA. In cells stained with fluorescent dyes targeting lysosomes, CAA induced an increase in lysosomal size and lysosomal leakage. The effects of CAA on cysteine protease activities and thiols could be reproduced in cell lysate. Acidification, which slowed the reaction of CAA with thiol donors, could also attenuate effects of CAA on necrosis markers, thiol depletion and cysteine protease inhibition in living cells. Thus, CAA directly reacts with cellular protein and non-protein thiols, mediating its toxicity on hRPTEC. This effect can be reduced by acidification. Therefore, urinary acidification could be an option to prevent IFO nephropathy in patients.

Chloroacetaldehyde- and acrolein-induced death of human proximal tubule cells.

Ifosfamide (ifo) is a commonly used drug in chemotherapy. It is metabolized to acrolein (acro) and chloroacetaldehyde (CAA), which are thought to be responsible for renal side effects. We studied the effects of ifo and cyclophosphamide (cyclo) as well as their metabolites, acro and CAA, on cellular protein content, necrosis, apoptosis and cytosolic calcium concentration using a human proximal tubule cell line. The protein content decreased during acro or CAA administration (15 to 300 micromol/l), but not during ifo or cyclo exposure over a time period of up to 72 h. Mild apoptosis was induced only by high acro (150, 300 micromol/l) and low CAA concentrations (15, 75 micromol/l) and only in a narrow time window (24 h). Necrosis was increased after exposure to acro or CAA at all concentrations. CAA was more potent than acro. Ifo and cyclo did not induce necrosis or apoptosis. Glutathione abolished CAA-induced cell death. Cytosolic calcium concentrations increased after acro or CAA administration and showed an oscillating pattern. Cytosolic Ca(2+) chelation did not prevent necrosis. We conclude that neither ifo nor cyclo induce cell damage, but that their metabolites acro and CAA induce cell death. This cell death occurs mainly by necrosis and not by apoptosis.

Melatonin secretion and increased daytime sleepiness in childhood craniopharyngioma patients.

Craniopharyngioma is a rare dysontogenetic benign tumor. Patients frequently suffer from endocrine deficiencies, sleep disturbances, and obesity due to pituitary and hypothalamic lesions. A self-assessment daytime sleepiness questionnaire (German version of the Epworth Sleepiness Scale) was used to evaluate 79 patients with childhood craniopharyngioma. Because hypothalamic lesions may explain daytime sleepiness in craniopharyngioma patients, salivary melatonin and cortisol concentrations were examined in obese and nonobese craniopharyngioma patients (n = 79), patients with hypothalamic pilocytic astrocytoma (n = 19), and control subjects (n = 30). Using a general linear model procedure analyzing the influence of body mass index (BMI) and tumor diagnosis on diurnal salivary melatonin, we found that morning salivary melatonin levels were related to BMI (by F test, P = 0.004) and tumor diagnosis (by F test, P = 0.032). Also for nighttime salivary melatonin levels significant relations with BMI (by F test, P < 0.001) and tumor diagnosis (by F test, P = 0.025) were detectable. Melatonin concentrations in saliva of craniopharyngioma patients collected at night or in the morning showed a negative correlation (night: Spearman's rho = -0.42; P = 0.001; morning: Spearman's rho = -0.31; P = 0.020) with the patient's Epworth Sleepiness Scale score. Severely obese craniopharyngioma patients and severely obese hypothalamic tumor patients had similar patterns of melatonin secretion. Differences in terms of diurnal salivary cortisol concentrations were not detectable when patient groups and controls were compared. We speculate that hypothalamic lesions might be responsible for both obesity and daytime sleepiness. As decreased nocturnal melatonin levels were associated with increased daytime sleepiness, BMI, and hypothalamic tumor diagnosis, further studies on the beneficial effects of melatonin substitution on daytime sleepiness and weight control in these patients are warranted.