Mapping the entry of reactive oxygen metabolites into target erythrocytes during neutrophil‐mediated antibody‐dependent cellular cytotoxicity

Journal Cellular Physiology

1992

Self Cite

Fluorescence intensified/enhanced microscopy has been used to study the metabolic activation of living human neutrophils in time-lapse sequences. The autofluorescence associated with NAD(P)H's emission band was studied within individual quiescent and stimulated cells. Excitation of NAD(P)H-associated autofluorescence was provided by a high-intensity Hg-vapor lamp. The background-subtracted autofluorescence signals were computer enhanced. In some cases the ratio image of NAD(P)H-associated autofluorescence to tetramethyl-rhodamine methyl ester (TRME) fluorescence, which was found to be uniformly distributed within neutrophils, was calculated to normalize autofluorescence intensities for cell thickness. Activation of the NADPH oxidase by phorbol myristate acetate, F-, N-formyl-methionyl-leucyl-phenylalanine (FMLP), or tumor necrosis factor (TNF) dramatically reduced autofluorescence levels. Membrane solubilization with sodium dodecyl sulfate eliminated autofluorescence. Thus, control experiments indicated that most or all of the detectable NAD(P)H-associated autofluorescence was due to NAD(P)H, consistent with previous non-microscopic studies. To understand the metabolic events surrounding the internalization and oxidative destruction of targets, we have imaged the NAD(P)H-associated autofluorescence of neutrophils and the Soret band of antibody coated target erythrocytes during cell-mediated cytotoxicity. Absorption contrast microscopy of the erythrocyte's Soret band is an especially sensitive indicator of the entry of reactive oxygen metabolites into this target's cytosol. Thus, it is possible to spectroscopically dissect and image the substrate (NADPH) and product (O2-) reactions of the NADPH oxidase in living unlabeled neutrophils. During real-time experiments at 37 degrees C, the level of NAD(P)H-associated autofluorescence surrounding phagosomes greatly increases before the disappearance of the target's Soret band. NAD(P)H-associated autofluorescence in the vicinity of phagocytosed erythrocytes is greatly diminished after target oxidation. This suggests that NAD(P)H is translocated to the vicinity of phagosomes prior to the oxidation of targets. The apparent cytosolic redistribution of NAD(P)H was confirmed by ratio imaging microscopy to control for cell thickness. We suggest that NADPH including its sources and/or carriers accumulate near phagosomes prior to target oxidation and that local NADPH molecules are consumed during target oxidation.

Section: Discussionmentioning

confidence: 93%

Imaging neutrophil activation: Analysis of the translocation and utilization of NAD(P)H‐associated autofluorescence during antibody‐dependent target oxidation

Bing

Journal Cellular Physiology

1992

Self Cite

“…2B). As time passes the erythrocytes disappear at this wavelength indicating cytosolic oxidation (data not shown; see Petty et al, 1992 for further kinetic Soret imaging studies). Since leakage of hemoglobin was not observed, the disappearance a t 430 nm is due to hemoglobin oxidation (Francis et al, 1988a).…”

Section: Cell Shapementioning

confidence: 89%

Superoxide‐mediated lysis of erythrocytes: The role of colloid‐osmotic forces

Zhou

Journal Cellular Physiology

1993

Self Cite

Although superoxide anions are a well-known mediator of cytotoxicity, their mechanism of target cell lysis is not clearly understood. In the present study we have used an exogenous source of superoxide to study erythrocyte cytolysis. RBC lysis was studied in buffers containing the cations Li+, Na+, K+, Rb+, and Cs+; superoxide anions were produced and available in these buffers. During this model superoxide-dependent cytolytic process, erythrocytes underwent a shape change from biconcave disk to sphere as shown by scanning electron microscopy. Soret band transmitted light microscopy has confirmed this shape change and shown that it precedes cytosolic oxidation. This evidence is consistent with a colloid-osmotic type lytic mechanism. Erythrocyte lysis was studied by 51Cr-release and light scattering methods. Superoxide-mediated target cytolysis was characterized by: 1) a sigmoidal dose-response curve and 2) a lag time in cytolysis after superoxide addition in kinetic light scattering experiments. The efficacy of cytolysis followed the rank order Cs+ > Rb+ > Na+, Li+ > sucrose = raffinose, which provides additional support for a colloid-osmotic lytic mechanism. Furthermore, the rank order potency correlates with the cations' hydration numbers. We suggest that oxidative events trigger the formation of colloid-osmotic pores approximately 1 nm in diameter.

“…Unfortunately, their mechanism(s) of signal transduction are not understood completely. However, these various transduction mechanisms may all terminate in a calcium-and granule-dependent step required for the deposition of superoxide anions in targets, but not necessarily for the respiratory burst per se [Maher et al, 1993;Petty et al, 1992;Liang and Petty, 19921. In the present study our primary interest is in Fc receptor-triggered superoxide production. Neutrophils possess two classes of plasma membrane Fc receptors which are known as FcyRII (CD32) and FcyRIIIB (CD16).…”

mentioning

confidence: 99%

Influence of polysaccharides on neutrophil function: Specific antagonists suggest a model for cooperative saccharide‐associated inhibition of immune complex‐triggered superoxide production

Zhang

J of Cellular Biochemistry

1994

Self Cite

We have previously shown that certain monosaccharides (N-acetyl-D-glucosamine and mannose) could cooperatively inhibit the ability of neutrophils to release superoxide anions in response to immune complexes. To test the possible origins of the cooperative inhibition of superoxide release, we have examined the effect of a panel of polysaccharides on superoxide release in the presence or absence of immune complexes. Although exposure to particulate beta-glucan and hyaluronan triggered superoxide release from neutrophils, other polysaccharides including chitin and mannan were without effect. Both chitin and mannan, but not other polysaccharides, inhibited the immune complex-mediated stimulation of superoxide release in a dose-dependent fashion. In sharp contrast to the cooperative inhibition mediated by monosaccharides, chitin and mannan exhibited Hill coefficients of 1. This inhibition of superoxide production was not due to simple blockage of Fc receptors since fluorescent immune complexes bound equally well to neutrophils in the presence or absence of mannan or chitin as shown by epifluorescence microscopy and quantitative fluorometry. Furthermore, this inhibition of superoxide release was not observed when neutrophils were stimulated with phorbol myristate acetate and ionophore A23187 or hyaluronan. Therefore, the specific inhibition of superoxide production by mannan and chitin could not be explained by either receptor blockage or by some nonspecific effect on cells. We suggest that these molecules interfere with a step in transmembrane signaling, presumably involving the integrin CR3. The observed Hill coefficients suggest the possibility that one polysaccharide may simultaneously bind to two monosaccharide binding sites yielding a Hill coefficient of 1, whereas individual monosaccharides separately bind yielding a Hill coefficient of 2.