Neutrophil-mediated cellular cytotoxicity triggered by immobilized aggregated IgG: Anin vitro model of cell injury during immune complex diseases

Normal human neutrophils were found to destroy ox red blood-cell targets when incubated on micropore filters coated with aggregated IgG, as determined by the⁵¹Cr release method. An intact neutrophil oxidative metabolism was essential for the cytotoxic event, since cells from patients with chronic granulomatous disease failed to exert any cytolysis. The target-cell destruction was prevented by catalase, azide, and cyanide and was enhanced by superoxide dismutase, suggesting involvement of the myeloperoxidase-hydrogen peroxide system. Neutrophil-mediated cytotoxicity was markedly amplified by the chemotactic peptideN-formyl-methionyl-leucylphenylalanine, as a result of an increased activity of the myeloperoxidase-hydrogen peroxide cytolytic system itself. This system of cytotoxicity provides a direct evidence for the neutrophil capacity of destroying bystander target cells under conditions simulating thein vivo immunologically mediated tissue injury and offers an excellent model to study events occurring during immune complex diseases.Supported by Grant 83.00902.96/115.11547 from the Italian CNR-PFCCN.     

Repeated exposure to heterologous hepatitis C viruses associates with enhanced neutralizing antibody breadth and potency

A prophylactic hepatitis C virus (HCV) vaccine that elicits neutralizing antibodies could be key to HCV eradication. However, the genetic and antigenic properties of HCV envelope (E1E2) proteins capable of inducing anti-HCV broadly neutralizing antibodies (bNAbs) in humans have not been defined. Here, we investigated the development of bNAbs in longitudinal plasma of HCV-infected persons with persistent infection or spontaneous clearance of multiple reinfections. By measuring plasma antibody neutralization of a heterologous virus panel, we found that the breadth and potency of the antibody response increased upon exposure to multiple genetically distinct infections and with longer duration of viremia. Greater genetic divergence between infecting strains was not associated with enhanced neutralizing breadth. Rather, repeated exposure to antigenically related, antibody-sensitive E1E2s was associated with potent bNAb induction. These data reveal that a prime-boost vaccine strategy with genetically distinct, antibody-sensitive viruses is a promising approach to inducing potent bNAbs in humans.     

Role of the oxidative metabolic burst in the antibody-dependent cellular cytotoxicity mediated by neutrophil polymorphonuclears

The purpose of the present study was to investigate the mechanisms by which neutrophil polymorphonuclears (PMN) mediate antibody-dependent cellular cytotoxicity (ADCC). Under experimental conditions which allow target cell phagocytosis, PMN were found to efficiently kill IgG-sensitized ox erythrocytes, as determined by the 51Cr release assay. Inhibition of the target cell ingestion by colchicine did not affect the PMN cytotoxic activity, suggesting that target cell phagocytosis does not represent an essential step in the PMN-mediated ADCC against erythrocytes. PMN from patients with Chronic Granulomatous Disease, who have defective oxidative metabolic burst, displayed an impaired ADCC activity, which was unaffected by changes in the phagocytic capacities induced by colchicine. The results indicate that, under the experimental conditions employed, both the intracellular and the extracellular target cell destruction by PMN involve oxygen-dependent mechanisms.     

Arterial and venous Thrombelastograph variables differ during cardiac surgery

The Thrombelastograph (TEG; Haemoscope Corp., Skokie, IL) coagulation analyzer is an effective point-of-care monitor for routine clinical practice and clinical research. Prior investigators have used either arterial or venous samples of blood for TEG measurements. We conducted this prospective cohort study to determine potential differences in TEG variables between arterial and venous blood samples. Arterial and venous samples were drawn from 40 cardiac surgical patients, yielding 134 pairs for comparison. Twenty-nine comparisons (control) were between arterial and arterial samples and were not significantly different. For the arterial and venous comparisons (n = 105), mean (+/-sd) arterial and venous values were the following: reaction time, 10 +/- 2 mm vs 13 +/- 4 mm, P = 0.004; maximum amplitude, 59 +/- 9 mm vs 49 +/- 12 mm, P < 0.001; alpha angle, 61 +/- 10 degrees vs 51 +/- 14 degrees, P < 0.001; K, 5 +/- 2 mm vs 8 +/- 4 mm, P = 0.007; and lysis, 2.5 +/- 1.7 vs 2.5 +/- 2.0 (not significant), arterial versus venous, respectively. Arterial blood samples demonstrated TEG values reflecting stronger (larger maximum amplitude) and faster (shorter reaction time and K value, wider alpha angle) clot formation. The results suggest that users of TEG coagulation analyzers should be consistent with the site of blood sampling given the potential differences obtained. IMPLICATIONS: Thrombelastograph (TEG) values obtained from venous blood samples differ from values obtained from arterial blood samples. When the TEG coagulation analyzer is used for clinical purposes, it is important to be consistent in the blood collection site.     

Naturally-occurring cellular cytotoxicity mediated by neutrophil polymorphonuclears: requirements for the target cell lysis

The purpose of the present study was to investigate the general conditions under which neutrophil polymorphonuclears (PMN) mediate antibody-independent cytolysis in the presence of normal human serum (NHS). Normal PMN were found to kill rabbit red blood cells (RRBC) only when cultured with 1% NHS. NHS was per se incapable of lysing RRBC. PMN from a patient with Chronic Granulomatous Disease did not destroy RRBC targets even in the presence of 1% NHS. In addition, cytotoxicity by normal PMN was significantly reduced by scavengers of oxygen metabolites. The results suggest that the target cell lysis by PMN in the presence of NHS requires a synergistic interaction between at least two mediators: serum factor(s) and oxygen metabolite(s).     

Role of hypochlorous acid and chloramines in the extracellular cytolysis by neutrophil polymorphonuclear leukocytes

The lysis of human red blood cells (HRBC) by neutrophil polymorphonuclear leukocytes (PMN), triggered with opsonized zymosan (OPZ) particles, was inhibited by azide, catalase, Cl- -free medium and amino acids indicating the involvement of myeloperoxidase (MPO), hydrogen peroxide (H2O2), Cl- ions and hypochlorous acid (HOCl) respectively. Thus, the cytolytic process depends on the following reaction: (Formula: see text). Because the oxidizing agent HOCl is also the precursor of the chloramines, a group of oxidants formed by the reaction between HOCl and PMN-derived ammonia (NH4+) or amines (R-NH2), the observed HRBC lysis can be theoretically due to HOCl and/or chloramines. Nevertheless, we found that PMN-mediated cytotoxicity occurs as an unidirectional process, being HRBC targets lysed and PMN unaffected. This finding indicates that the cytotoxin must be relatively more efficient against HRBC as compared with PMN. In fact, reagent HOCl (used at concentrations comparable to those generated by PMN) but not chloramines displayed such a type of property. Taken together, the data suggest that HRBC are killed by PMN-derived HOCl without the requirement for chloramines: this implies that NH4+ and R-NH2, released by PMN, act as down-modulators of the cytotoxic process, serving as HOCl trapping agents.     

Cellular cytotoxicity mediated by granule-depleted neutrophil cytoplasts

Summary Neutrophil-derived nucleus- and granule-free cytoplasts, consisting of cytosol enclosed by an intact plasma membrane, were able to destroy ⁵¹Cr-labelled ox red blood cells (ORBC) in the presence of phorbol myristate acetate (PMA). The slope of the target cell lysis vs the log of the cytoplast number was similar to that observed with neutrophils as effector cells. Nevertheless, a number of cytoplasts 60–80 times higher than that of neutrophils was required to obtain a common level of cytotoxicity. The ability of cytoplasts and neutrophils to lyse ORBC was completely abolished by catalase and unaffected by superoxide dismutase and mannitol, suggesting the involvement of hydrogen peroxide in the target cell damage. Addition of myeloperoxidase (MPO) to cytoplasts increased lysis. The MPO inhibitor azide significantly reduced the cytolysis by neutrophils, but not the cytolysis by cytoplasts, except when experiments were carried out in the presence of MPO. The results indicate that neutrophil cytosol and plasma membrane represent the basic requirement for the PMA-dependent cytolytic process, whereas MPO behaves as a device to amplify lysis.