Sándor Szucs

Single-cell measurement of superoxide anion and hydrogen peroxide production by human neutrophils with digital imaging fluorescence microscopy

Besides flow cytometry, fluorescence microscopy combined with computerized image analysis offers an alternative tool for assessing phagocyte oxidant generation at the single-cell level. This technique provides an opportunity for the direct visualization of cells and simultaneous measurement of cellular fluorescence intensity. Thus, we developed a simple method for the quantitative evaluation of intracellular superoxide anion and hydrogen peroxide production with image cytometry by using hydroethidine and dihydrorhodamine 123 dyes, respectively. Human neutrophils stimulated with phorbol dibutyrate and labeled by these fluorogenic substrates showed intense, well recognizable red or green fluorescence. The intensity of signals from individual granulocytes of cytospin preparations were quantitatively measured in digitized images. There was a great heterogeneity in response to the stimulus within the granulocyte population as shown by the integrated fluorescence intensity values. In agreement with the results of parallel flow cytometric experiments, this simple image analysis performed on cells of cytospin preparations was able to detect the defects in the oxidative metabolism of neutrophils from patients with cervix carcinoma. We demonstrated that even minor alterations in superoxide anion/hydrogen peroxide generation can be detected by image cytometry as efficiently as by flow cytometry. This result validates imaging microscopy as an alternative to flow cytometry in such experiments. In addition, the image cytometric technique allows the observation of the kinetics of free radical production in individual cell under adherent conditions. Therefore, we carried out image analysis of the oxidative burst of neutrophils adherent to uncoated glass and fibronectin- and type IV collagen-coated surfaces in response to stimulation with phorbol dibutyrate or N-formyl-methionyl-leucyl-phenylalanine. We elaborated a calibration technique for the quantitative measurement of the ethidium bromide generation mediated by superoxide anion within individual adherent granulocytes. The ethidium bromide production varied between 0.48 and 1.17 amol/cell/min.

Age-dependent changes in transmembrane signalling: Identification of G proteins in human lymphocytes and polymorphonuclear leukocytes

In human neutrophils (PMNLs) we found that in the elderly IP3 formation was significantly decreased compared to that of young subjects. For FMLP receptor binding affinity and number no measurable differences occurred upon ageing, studying both the low or the high affinity receptors. The amount of ADP-ribosylated G proteins, catalysed by pertussis toxin (PT) or cholera toxin (CT), was significantly increased in PMNLs of the elderly. In lymphocytes, the PT-catalysed ADP ribosylation of G proteins was also increased with ageing, while the CT-catalysed ribosylation was decreased. The autoradiogram of [32P]ADP-ribosylated proteins by CT in lymphocytes of young individuals showed a major polypeptide of 40,000 M(r). In contrast, in lymphocytes of the elderly, the major polypeptide was 45,000 M(r). In PMNLs, CT labelled quite strongly the 45,000 M(r) band, mainly in the elderly. When PT was used, no age-related pattern changes could be demonstrated, while differences could be observed between the two types of cells. The use of antiserum P680 (G alpha common) showed no age-related pattern changes, while the intensity of the labelled proteins varies with age and cell type. The antiserum U46 (Go alpha) could identify in lymphocytes of young subjects two polypeptides 68,000 and 41,000 M(r). The prominent polypeptide in lymphocytes of the elderly was the 70,000 M(r) and no other polypeptides could be recognized. In PMNLs of young subjects the U46 and serum identified a range of species. In PMNLs of the elderly all these bands were weakly labelled. The present data indicate changes in the pattern and the quantity of G proteins in lymphocytes and PMNLs of elderly subjects.

Stage-related superoxide anion production of granulocytes of gynecologic cancer patients

OBJECTIVE To measure superoxide anion production of polymorphonuclear leukocytes (PMNLs) in 58 gynecologic cancer patients and compare to that of healthy controls. METHODS PMNLs were separated from peripheral blood samples by Ficoll and subsequent Percoll gradient sedimentation. Baseline and phorbol-dibutyrate (100 nmol/l) stimulated superoxide anion production was measured spectrophotometrically as superoxide dismutase inhibitable reduction of ferricytochrome c (50 micromol/l) absorbance. Differences between the mean superoxide anion production of different patient groups and the control group were assessed by Students t-test. RESULTS The mean superoxide anion production of PMNLs of healthy controls was 1.855 nM/min/3 x 10(5) cells (SD=0.211 nM/min/3 x 10(5) cells). Superoxide anion production of gynecologic cancer patients and healthy controls varied in a wide range. PMNLs of patients had lower baseline and stimulated activity than those of healthy volunteers. The frequency of a mean superoxide production at least 2 x SD below the control showed a parallel increase with advancing stage. CONCLUSION Granulocytes of gynecologic cancer patients have reduced capacity and inducibility of superoxide anion production already at an early stage of disease.

Preeclamptic Superoxide-Anion Production: Is There an Increase or a Failure of Reduction?

To the Editor: We read with interest the article by Tsukimori et al1 analyzing the relationship between the superoxide-anion (O2−) production of neutrophils during pregnancy in women with preeclampsia and essential hypertension. The results of the study demonstrated that n -formyl-methionyl-leucyl-phenylalanine stimulated O2− production of neutrophils during pregnancy was increased in women with preeclampsia compared with healthy pregnant women. There was no significant difference in the neutrophil O2− production in normal pregnancy between pregnant and postpartum women. In …

Effects of Various Stimulators and Inhibitors on the Respiratory Burst of PMNL with Aging

It is well known that the incidence of certain diseases such as infections (Gardner, 1980), atherosclerosis (Stout, 1987), diabetes mellitus (DeFronzo, 1981), and tumors (Doll et al., 1970) is increased with aging. It can be assumed that the cause of this increased incidence is multifactorial, but the decrease of the immune response certainly plays an important role (Makinodan and Kay, 1980; Corberand et al., 1981). It was established that the oxidative burst plays an essential role in the host defense against pathogens and other microorganisms (Johnston et al., 1976; Babior and Crowley, 1983); therefore, an alteration of the respiratory burst could contribute to the increased incidence of infectious diseases with aging.