Sistina Minnucci

Detection of Eosinophils in Whole Blood Samples by Flow Cytometry

A flow cytometric method to detect and study human eosinophils in whole blood was established. Normal subjects and patients with various types of eosinophilia (hypereosinophilic syndromes, allergic diseases, dermatitis, Hodgkins Disease, parasitosis) were studied. Whole blood samples were treated for 10 minutes at room temperature with a commercially available reagent (FACS Lysing Solution, Becton Dickinson) which acts both as a fixative and as a lysing agent. Eosinophils were identified as a granulocytic subpopulation with higher SSC and FSC properties. This cell population was characterized by evident autofluorescence and hypodiploid DNA features after propidium iodide staining. The purity of the eosinophil population sorted after electronic gating was close to 100%. A very significant correlation between eosinophil counting by our whole blood method and other two assays, namely routine automatic counting by the H*3 Bayer System and eosinophil detection by depolarized SSC, was obtained. The phagocytic properties of eosinophils were also studied by means of a commercially available diagnostic kit, thus demonstrating that our method is also suitable for the study of those granulocytic functions which can be evaluated by flow cytometry.

Motility of rhG‐CSF‐induced neutrophils in patients undergoing chemotherapy: evidence for inhibition detected by image analysis

The motility of circulating neutrophils from seven patients affected by intermediate and high‐grade non‐Hodgkins lymphoma was investigated before and after rhG‐CSF administration (5μg/kg/d for 5 d subcutaneously) in the course of chemotherapy. Random motility and bacterial lipopolysaccharide‐induced chemotaxis were studied by the micropore filter technique in a Boyden chamber. These functions were evaluated by a very sensitive technique, based on a computer‐assisted image processing system, capable of giving several parameters about the kinetics of cell migration. Along with a significant increase in neutrophil number, a significant decrease both in random and stimulated motility was found. The kinetics of cell migration showed that the cells maintained the typical gaussian pattern of random motility. On the contrary, neutrophils were found to have lost the typical stimulated migration peak. These findings are consistent with a rhG‐CSF‐induced impairment of the directional movement, rather than of the ability of moving at random. These effects were found in patients who, in the same experimental conditions, had displayed an enhanced phagocytosis and phagocytosis‐associated chemiluminescence along with an enhanced CD32 expression, not due to an aspecific cell manipulation. Two hypotheses may be taken into account: (i) an increased adhesiveness due to a direct or an indirect activity of the cytokine; (ii) an abnormality in the cytoskeleton maturation and/or rearrangement during the accelerated bone marrow transit of myeloid cells. These findings emphasize that rh‐GCSF administration can modulate several functions which play an important role in host defence, and suggest the utility of carrying out further studies to investigate the optimum dosage both to correct neutrophil number and preserve neutrophil functional activities.

FcRIII (CD16) expression on neutrophils from chronic myeloid leukemia. A flow cytometric study

FcRIII (CD16) expression on neutrophils from 17 patients with chronic myeloid leukemia (CML) was studied by flow cytometry using monoclonal antibodies. A variable proportion of CD16-negative neutrophils were found both in CML patients in chronic phase (3 out of 8 patients) and in CML patients in hematological remission (3 out of 9 patients). Neutrophils with reduced FcRIII expression showed more defective chemiluminescence and phagocytosis than neutrophils with normal FcRIII expression. Circulating myeloid cells from three patients in chronic phase, showing a normal percentage of CD16-positive neutrophils, were isolated and fractionated by discontinuous Percoll gradients. This study showed that CD16 appears at the stage of metamyelocyte, that band cells and segmented neutrophils display an identical pattern of membrane FcRIII, and that the fluorescence intensity shown by metamyelocytes is different from that displayed by more mature cells. The association between low FcRIII expression and function abnormality could be suggestive of a defect in CML neutrophil maturation.

Inhibitory Effects of Human Neutrophil Functions by the 45-kD Glycoprotein Derived from the Parasitic Nematode Trichinella spiralis

Aim: We evaluated the effect of the 45-kD protein of Trichinella spiralis (gp45), purified by affinity chromatography, on random migration and chemotaxis, the oxidative metabolism of human neutrophils and on the CD11b upregulation induced by formyl-methionyl-leucyl-phenylalanine (f-MLP). Methods: Donor neutrophils incubated with different amounts of gp45 (0.5, 1, 1.5, 2 μg/ml) or buffer and the random migration and chemotaxis, evaluated by means of a special technique of image analysis, and the chemiluminescence response to f-MLP or phorbol myristate acetate (PMA) were analyzed. The effect on CD11b upregulation was assessed incubating cells with the protein, when activating them with f-MLP. Results: The results showed that gp45 inhibited both random and stimulated migrations, and reduced the response to f-MLP and PMA. Furthermore, gp45 significantly reduced the upregulation of the CD11b induced by f-MLP. Conclusion: The results show that gp45 inhibits PMN in different functions, suggesting an anti-inflammatory action.

Actin polymerization in neutrophils from patients affected by myelodysplastic syndromes—A flow cytometric study

In this study F-actin polymerization in neutrophils from 21 patients affected by myelodysplastic syndromes (MDS) was evaluated by means of a flow cytometric assay. Neutrophils were stimulated with formyl-methionyl-leucyl-phenylanaline (fMLP; 10(-8) M final concentration) for 15, 30, 60 and 120 sec, and F-actin content was determined using fluorescein-isothiocyanate phallacidin as a specific probe. Eight normal subjects were studied as controls. We found that F-actin polymerization was defective in ten patients, with very impaired values after 60 and 120 sec of stimulation with fMLP. The remaining 11 patients showed a prevalent neutrophil population with normal F-actin polymerization and neutrophil sub-populations with either defective or undetectable F-actin polymerization. In the first group, patients with very poor prognosis (refractory anemia with excess blasts, refractory anemia with excess blasts in leukemic transformation, trisomy 8, multiple karyotypic abnormalities) were present, although patients with aberrations of karyotype were present in the second group. It is possible that defects in neutrophil F-actin polymerization may be responsible for neutrophil dysfunction, which has frequently been observed in MDS.

Inhibition of neutrophil oxidative metabolism by trichinellosis patient sera. Parasite origin or host induction

The presence of sera factors able to inhibit both neutrophil chemotaxis and phagocytosis was observed in all patients studied at two months from infection caused by Trichinella britovi and in most of them after one year. Human neutrophils with eosinophils are able to kill T. spiralis newborn larvae in an ADCC system and their major cytotoxic mechanism is oxidative metabolism products. We evaluated the effect of trichinellosis sera on neutrophil oxidative burst to determine if neutrophils are affected by circulating factors during infection. Cells were incubated with sera from trichinellosis patients. Basal or stimulated Superoxide Anion (SA) production and chemilumines‐cence in response to different stimulation (PMA, f‐MLP, opsonized yeasts) of neutrophils incubated with trichinellosis sera were evaluated and compared with those of cells incubated with control sera. The results show that basal SA production was inhibited by 66% of sera and stimulated by 11%. On the contrary f‐MLP stimulated production was significantly increased by 22% sera, and inhibited by none. Chemiluminescence in response to f‐MLP or PMA was inhibited by 46 and 80% of sera, respectively. These results show that trichinellosis sera can modulate not only SA production but also other steps of the oxidative burst, irrespective of the stimulating agent, so suggesting that different neutrophil activation pathways are affected. Increased IL‐2 levels observed in most of the sera did not correlate with the inhibiting capacity of sera. The hypothesis of a parasite origin of the inhibiting factors is discussed in the light of host‐parasite relationship.,

Platelet-Neutrophil Interactions in Uremic Patients: Effects on Neutrophil Superoxide Anion Production and Chemiluminescence

Isolated resting platelets are able to limit neutrophil activation and then can control the tissue-damaging potential of activated neutrophils. In the present study, platelet-neutrophil interactions have been evaluated in 10 uremic patients; the blood samples have been collected before the hemodialysis session. Twelve normal subjects served as controls. Platelets and neutrophils have been isolated and recombined in an autologous ex vivo system. Anion superoxide production and chemiluminescence (which is related to hypochlorous acid production) have been evaluated after stimulation with N-formyl-methionyl-leucyl-phenylalanine. Coincubation of platelets from normal subjects with autologous neutrophils led to a dose-dependent inhibition of both superoxide anion generation induced by N-formyl-methionyl-leucyl-phenylalanine and chemiluminescence. Instead, platelets from uremic patients have not affected superoxide anion production by autologous neutrophils. The chemiluminescence was reduced by coincubation with autologous platelets only at the highest platelet-neutrophil ratio (100:1). In conclusion, the modulation exerted by platelets towards neutrophil activation can be impaired in chronic uremia. Therefore, the tissue-damaging potential of circulating neutrophils, due to toxicity by superoxide anion and hypochlorous acid, may be increased.

Interactions between platelets and neutrophils in essential thrombocythaemia. Effects on neutrophil chemiluminescence and superoxide anion generation

Abstract. Essential thrombocythaemia (ET) is frequently associated with neutrophil and platelet dysfunction, and with increased incidence of vascular complications (thrombosis, haemorrhage). Several interactions between platelets and neutrophils have been reported, and the reciprocal actions between these cells may have an important role both in thromboregulation and in diseases such as those caused by uncontrolled neutrophil activation. In the current paper the authors studied 15 patients affected by ET and 10 normal subjects as controls. Circulating neutrophils and platelets were purified and were re‐combined in constant ratios (50:1, 100:1 and 200:1) and the individual platelet to neutrophil ratio. Super‐oxide anion (O‐2) generation and luminol‐enhanced chemiluminescence (CL) were studied after neutrophil stimulation with fMLP. In normal subjects both O‐2 generation and CL were inhibited by autologous platelets in a dose‐dependent manner. In ET patients, on the contrary, platelet‐dependent inhibition of O‐2 generation did not occur, while a dose‐dependent inhibition of CL was observed. Two groups of ET patients were found: patients with neutrophil O‐2 generation and CL within the normal range, and patients with significantly reduced neutrophil respiratory burst. However, no differences were found between these two groups of patients in terms of platelet effects towards fMLP‐stimulated neutrophils. Therefore, platelets from ET patients were not able to exert the homeostatic control towards neutrophil O‐2 generation shown by platelets from normal subjects, and this phenomenon may have a role in the clinical setting. In fact, O‐2 has been shown to be a very strong direct platelet activator, is able to inactivate nitric oxide (which is a powerful inhibitor of platelet aggregation and adhesion to endothelium), and is directly involved in neutrophil‐mediated tissue damage.

Luminol-enhanced, whole blood chemiluminescence of human neutrophils evaluated by means of an automated, computer-assisted, and high-sensitivity luminescence analyzer

SummaryLuminol-enhanced whole blood chemiluminescence of human neutrophils was studied using opsonized zymosan as a stimulus. Heparinized blood (0.5μl) was used, and the chemiluminescence signals were recorded by a very sensitive, automated, and computer-assisted luminometer (LB 950, Berthold, Wildbad, Germany). The following parameters were provided: integral values over the total measuring time, peak values, the time to reach maximum value, and the time to reach half maximum value. Normal subjects, neutropenic patients, subjects with total or partial myeloperoxidase deficiency, patients with recurrent infections, phagocytic defects, thrombocythemic patients, and those with non-Hodgkins lymphomas undergoing therapy with recombinant human granulocyte colony-stimulating factor were studied. The integral response of chemiluminescence and the time of reach half maximal value were useful indicators of chemiluminescence defects; the assay, was able to detect chemiluminescence responses in neutropenic subjects with neutrophil levels as low as 0.6×109/I; differences between cellular and plasma defects could be identified; the quenching effect of exerted by erythrocytes was negligible.

Neutrophil functions in essential thrombocythemia

Chronic myeloproliferative diseases, such as chronic myeloid leukemia and polycythemia vera, are associated with neutrophil dysfunction. Very little data is available on essential thrombocythemia (ET). In the current study we evaluated 21 patients with ET. All patients were studied at least 16 weeks after any cytostatic therapy and 10 days after any other therapy. Neutrophil functions were investigated as follows: flow cytometric evaluation of whole blood phagocytosis of opsonized FITC-conjugated E. coli; whole blood chemiluminescence after stimulation with opsonized zymosan and evaluation by an automated, computer-assisted luminometer (LB 950, Berthold); and chemiluminescence and superoxide anion generation by purified neutrophils after f-MLP and PMA stimulation. Chemiluminescence and superoxide anion generation after f-MLP stimulation were found to be significantly lower than in normal subjects, whereas values within the normal ranges were registered after PMA stimulation. Phagocytosis-associated chemiluminescence was found to be impaired both by using zymosan opsonized with autologous plasma and zymosan opsonized with normal plasma, despite a normal phagocytic activity. These data show the presence in ET of a complex neutrophil dysfunction that may be related to an impaired signal transduction during both the phagocytic process and f-MLP stimulation.