The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kindzelskii, A. L.
Right arrow Articles by Petty, H. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kindzelskii, A. L.
Right arrow Articles by Petty, H. R.
The Journal of Immunology, 1999, 162: 3188-3192.
Copyright © 1999 by The American Association of Immunologists

Early Membrane Rupture Events During Neutrophil-Mediated Antibody-Dependent Tumor Cell Cytolysis1

Andrei L. Kindzelskii and Howard R. Petty2

Department of Biological Sciences, Wayne State University, Detroit, MI 48202


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although cell-mediated cytolysis is a fundamental immune effector response, its mechanism remains poorly understood at the cellular level. In this report, we image for the first time transient ruptures, as inferred by cytoplasmic marker release, in tumor cell membranes during Ab-dependent cellular cytolysis. The cytosol of IgG-opsonized YAC tumor cells was labeled with tetramethylrhodamine diacetate followed by the formation of tumor cell-neutrophil conjugates. We hypothesized that tumor cell cytolysis proceeds via a series of discrete membrane rupture/resealing events that contribute to marker release. To test this hypothesis, we occluded the fluorescence image of the labeled tumor cells by passing an opaque disk into a field-conjugated plane between the light source and the sample. Multiple small bursts of fluorescent label release from tumor cells could be detected using a photomultiplier tube. Similarly, multiple fluorescent plumes were observed at various sites around the perimeter of a target. These findings support a multihit model of target cytolysis and suggest that cytolytic release is not focused at specific sites. Cytolytic bursts were generally observed at 20-s intervals, which match the previously described reduced nicotinamide-adenine dinucleotide phosphate and superoxide release oscillation periods for neutrophils; we speculate that metabolic oscillations of the effector cell drive the membrane damage of the target.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cytolysis, the lytic disruption of target cells, is a fundamental immunological effector function. Cell-mediated cytolytic responses are conducted by, for example, cytotoxic T cells, NK cells, macrophages, and neutrophils. One mechanism of cytolysis, which we shall focus on in the present study, is Ab-dependent cellular cytolysis (ADCC).3 Numerous in vitro studies have shown that ADCC requires opsonization of a target with Ig and the engagement of effector cell surface receptors for the Fc domains of Ig molecules 1, 2, 3, 4 . Indeed, cell surface Fc receptors of inflammatory cells play a key role in providing host protection 2 . The level of in vitro cytotoxicity can be affected by numerous factors, such as cytokines 5, 6 . Although much research has focused on the role of Ab-independent recognition of tumor cells by cytotoxic T cells, recent studies support an in vivo role for Ab-dependent tumor cell destruction 7, 8, 9, 10 . For example, in the murine system, both passive immunization with mAb directed against glycoprotein 75 and active immunization with glycoprotein 75 offer substantial protection in melanoma metastasis models 11, 12 .

Although the central role of cytolytic phenomena in host defense has been supported by thousands of scientific studies describing their biological features, the elementary physical mechanisms of these phenomena are unknown. Most cytolysis studies rely upon the release of a cytoplasmic marker, such as 51Cr, to detect cytolysis. The kinetics of marker release in these experiments are, of course, a population average and consequently provide no detailed information regarding events occurring at the level of individual cells. For instance, it is widely presumed that membrane ruptures or tears are an integral feature of cytolysis, yet no direct evidence for membrane rupture/cytoplasmic leakage exists in nucleated target cells. Because tumor cells are fairly large, one would anticipate that only a small fraction of their cytoplasmic contents would be released during a cytolytic event. To detect these comparatively weak signals, we occluded tumor cell illumination by passing an opaque disk into a field-conjugated plane between the light source and the sample. We now quantitatively and qualitatively show the kinetics of ADCC on a single-cell level, thereby demonstrating the formation of pericellular cytolytic plumes during target destruction and their oscillatory nature.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Neutrophil preparation

Peripheral blood was obtained from healthy individuals by venipuncture using heparinized tubes. Neutrophils were isolated from venous blood using Ficoll-Hypaque solutions (Sigma, St. Louis, MO) and density gradient centrifugation 13 . Cells were washed twice by centrifugation and then resuspended in HBSS (Life Technologies, Grand Island, NY). Trypan blue staining indicated that 95–99% of the cells were viable.

Latex bead samples

Fluorescent latex beads (Fluoresbrite YG, diameter = 1.14 µm) were obtained from Polysciences (Warrington, PA). To minimize diffusional displacement of the beads during imaging studies, beads were mixed with 2% fluid-phase gelatin at ~40°C (type B; Sigma). The mixture was placed on a microscope slide, a coverslip was added, and the gelatin was allowed to harden at room temperature before observations.

YAC tumor cells

YAC cells were grown in suspension culture using RPMI 1640 medium (Life Technologies) containing 10% FCS and 10 µg/ml penicillin-streptomycin in a humidified CO2 incubator at 37°C 14 . YAC cells were thoroughly washed by centrifugation and then resuspended in HBSS. The cells were >95% viable as judged by trypan blue staining.

YAC cell labeling

YAC cells were labeled with tetramethylrhodamine diacetate (TMR) (Molecular Probes, Eugene, OR). Briefly, stock solutions of TMR were prepared by mixing 1.5 mg of TMR with 200 µl of DMSO and then diluting with 50 ml of HBSS. Next, YAC cells were labeled with a 1/10 dilution of the stock solution for 1 h at 37°C. Cells were thoroughly washed by centrifugation 14 .

Opsonization

Labeled YAC cells were opsonized with a rabbit anti-mouse thymocyte IgG fraction (adsorbed) (Accurate Chemicals, Westbury, NY) as described previously 14 . Next, the cells were washed three times with HBSS. The opsonized and labeled YAC cells were mixed with neutrophils at an E:T ratio of 1:2 and then incubated at 37°C for 30 min.

Optical microscopy

Cells were examined using an axiovert inverted fluorescence microscope (Carl Zeiss, New York, NY) with mercury illumination interfaced to a Perceptics Biovision workstation (Knoxville, TN) 15, 16 . The fluorescence images were collected by an intensified charge-coupled device camera (ICCD) (model XC-77; Hamamatsu Photonics, Bridgewater, NJ). A narrow bandpass discriminating filter set was used with excitation at 540/20 nm and emission at 590/30 nm (Omega Optical, Brattleboro, VT). A long-pass dichroic mirror was used at 560 nm. To occlude the bright cytoplasm of labeled tumor cells, a special adapter (Zeiss) for the fluorescence light source was employed. An opaque disk on a glass substrate held on an aluminum slider was placed into a field-conjugated plane of the adapter. Differential interference contrast photomicrographs were collected using Zeiss polarizers and a charge-coupled device camera (Model 72; Dage-MTI, Michigan City, IN). The images were stored on hard disk and photographed using a freeze-frame video recorder (Polaroid, Boston, MA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In the present study, we tested the hypothesis that membrane rupture and consequent cytoplasmic leakage are early and transient events in tumor cell cytolysis. Inasmuch as preliminary studies suggested that the fluorescence of cytolytic labels released from cells at early timepoints was a small fraction of the total cellular fluorescence, it was necessary to increase the relative contrast of the released label. To do this, we occluded the bright fluorescence of the target cells. Occultation is an optical strategy employed by astronomers to visualize the corona of the sun, planetary oblateness, etc. (e.g., 17 . Thus, an opaque disk on a transparent substrate was placed in a field-conjugated plane near the mercury epifluorescence lamp. With the bright cell occluded, the ICCD camera operates at maximal gain, thus allowing detection of the weak fluorescence leakage in real time. To confirm the rigor of the approach, we imaged fluorescent latex beads in the presence and absence of an occultation disk. Fig. 1GoA shows a fluorescence micrograph of latex beads using conventional illumination. In Fig. 1GoB, this same field of beads is shown again after the occultation disk is in place. As this figure illustrates, illumination was effectively blocked for a circular region in the image plane corresponding to the disk in the field-conjugated plane of the adapter without a loss in overall image quality. During quantitative intensity measurements, nonspecific background fluorescence (from other cells or prior membrane ruptures) was reduced by reducing the diameter of the field diaphragm (illustrated in Fig. 1GoC). Thus, we minimize background fluorescence while maximizing the contrast of cytoplasmic marker release using pericellular illumination.



View larger version (72K):
[in this window]
[in a new window]
  		FIGURE 1. Illustration of the imaging technique. To exclude the bright fluorescence of labeled tumor cells while retaining the ability to detect pericellular fluorescence, a "doughnut-like" pattern of concentric circles was created on the sample. A, A field of fluorescent beads is shown. B, The same field of beads is shown again after inserting an opaque disk into a field-conjugated plane between the sample and the lamp. C, The extent of perimeter detection was limited by the field diaphragm. Magnification is x560.

 
Single-cell marker-release studies

IgG-coated and TMR-labeled YAC cells were incubated with human neutrophils at a 1:2 E:T ratio for 30 min at 37°C and then transferred to a microscope slide and held at 37°C using a heated microscope stage. Target to effector conjugates were selected and then aligned with the occultation disk by adjusting the stage and/or the slider holding the disk. The intensity of released pericellular fluorescence was monitored continuously using a photomultiplier tube and was recorded by a computer. Fig. 2Go shows several representative quantitative marker-release experiments. In Fig. 2Go, trace a, a long timebase was used to show an entire time course of early cytolysis, including background levels before and after marker release. As this illustrates, a series of intensity spikes was observed. Fig. 2Go, b–g, shows the results of similar single-cell marker-release experiments depicting the heterogeneity of the responses. In general, between three and eight marker-release spikes were observed. The background level of pericellular fluorescence, which increases during marker release, decreases following the last spike. As illustrated in the kinetic studies of Fig. 2Go, marker release was often observed at 20-s intervals.



View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 2. Quantitative fluorometry of pericellular fluorescence of tumor cells in neutrophil-tumor cell conjugates. Neutrophil-tumor cell conjugates were observed at 37°C. The fluorescence of the tumor cell was optically occluded, and the pericellular fluorescence intensity was recorded using a photomultiplier tube apparatus, amplifier, and computer. In these experiments, the fluorescence intensity (ordinate) is plotted against time (abscissa). Trace a shows a representative kinetic profile of pericellular fluorescence intensity over a 7.5-min period. Note the onset and termination of oscillatory marker-release events. Traces b–g show six additional quantitative marker-release studies for different effector cell to tumor cell conjugates. These traces illustrate the heterogeneity of the responses observed. In general, at least three to eight pulses of marker release can be resolved. Similar results were obtained on 15 different days with a total of 113 kinetic traces.

 
To ascertain the spatial characteristics of marker release, we acquired images of marker release using an ICCD camera. Images were captured as the fluorescent marker diffused into the pericellular illumination region. Fig. 3Go shows several representative qualitative micrographs of marker release during neutrophil-mediated ADCC. Plumes of label released from the cells were observed. A variety plume shapes were observed. This may be due, for example, to membrane ruptures near the edge of the occultation disk (excitation cone of light) vs membrane ruptures near the apical or basal surfaces, which would be out of the focal plane, dimmer, and more diffuse. Interestingly, no preferred orientation of the plume was found relative to the position of the effector cell; a similar observation was made previously for ADCC of E 18 . Thus, the marker was released as pericellular plumes of fluorescence that occur randomly around the perimeter of a target.



View larger version (46K):
[in this window]
[in a new window]
  		FIGURE 3. A gallery of photomicrographs illustrating marker release. Labeled target cells were occluded during ADCC as described above. A total of 18 examples of fluorescent marker-release experiments are shown to illustrate the heterogeneity of the responses observed. Marker release is associated with a plume of fluorescence intensity, presumably associated with a point of cytolytic membrane rupture. The site of marker release did not correlate with the position of the effector cell. Similar results were obtained on 8 different days; a total of 80 different cells were photographed. Magnification is x500.

 
We subsequently confirmed the relationship between the quantitative intensity measurements and the qualitative imaging results. Fig. 4Go shows a time-correlated intensity and imaging experiment of a neutrophil-tumor cell conjugate. In this experiment, ~50% of the light was directed to a photomultiplier tube (thus accounting for the slight reduction in the signal/noise ratio in Fig. 4GoA), whereas the remainder was sent to an ICCD camera. Fig. 4GoA shows a quantitative kinetic measurement of fluorescence intensity vs time. The timepoints labeled 1, 2, and 3 correspond to the images shown in Fig. 4Go, B, C, and D, respectively. Thus, the bursts of fluorescence intensity illustrated in Fig. 4GoA correspond to separate plumes of marker release. For example, the burst of intensity labeled as 1 in Fig. 4GoA corresponds to the appearance of a bright plume labeled as 1 in Fig. 4GoB. Similar relationships hold for bursts 2 and 3. These data also illustrate the longitudinal features of individual marker-release events. For example, the bright burst labeled as 1 in Fig. 4GoB dissipates within several seconds as shown in Fig. 4GoC and cannot be observed in Fig. 4GoD. Because the rate of rupture at these early times is faster than diffusion of the marker from the illumination area, the background level of fluorescence increases during the observations. In some cases, this can begin to obscure the individual events (e.g., Fig. 2Go).



View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 4. Time-correlated fluorometric and imaging experiments. A, Kinetic single-cell fluorescence tracing showing pulses of marker release from labeled tumor cells during neutrophil-mediated ADCC. The fluorescence intensity (photocurrent) is listed at the ordinate, whereas time (in seconds) is given at the abscissa. Multiple temporally distinct marker-release events are shown. Optical micrographs of fluorescence were acquired at three points in time (labeled 1, 2, and 3) in A. B–D correspond to the timepoints labeled 1, 2, and 3 in A. Note that the bursts of marker release illustrated quantitatively in A correspond to distinct marker-release plumes at different positions in B, C, and D, respectively. Similar results were obtained on 3 separate days. For B–D, magnification is x585.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The quantitative analysis of immunological cytolysis can be traced to the work of Brunner et al. 19 , who demonstrated that Ag-specific T lymphocytes promote the cytolytic release of 51Cr from target cells. Early studies also showed that the cytolytic process can be divided into distinct phases. For example, the release of cytoplasmic 86Rb and small molecules (e.g., ATP and nicotinamide) precedes that of 51Cr, which is bound to proteins 20, 21 . The label used in the present study, TMR, is a small molecule. Because our data represent the earliest detectable TMR release, it seems likely that the cytolytic plumes observed (Figs. 3Go and 4Go) are early marker-release events. Moreover, this early cytolytic damage occurs at roughly the same time as the first committed cellular steps in cytolytic programming 20 and may contribute to subsequent membrane damage (e.g., colloid-osmotic effects). Cytolysis, the release of cytoplasmic contents, consequently begins with multiple small bursts of cytoplasmic content release around the perimeter of a target.

Although cytolysis represents a major form of immunological effector function, its details have remained obscure in nucleated cells due to an inability to detect individual cytolytic events. Previous microscopic studies have observed individual cytolytic events during Ab-dependent lysis of lipid vesicles and sheep E 18, 22 . Lysis in both of these systems demonstrated a catastrophic breakdown of the target membrane and a complete loss of cytoplasmic contents. A previous report has indirectly studied cytolysis by following the cytoplasmic calcium concentration of a tumor cell target; this concentration presumably increases due to calcium influx and CTL contact 23 . However, cytolytic events were not photographed, and release kinetics were not quantitated. In contrast, our approach is to occlude the fluorescence of bright tumor cells, thereby allowing the weak fluorescence leaking from the cell to be detected. The strategy demonstrated pericellular cytolytic plumes of fluorescent marker release. Our kinetic studies of tumor cell cytolysis indicate that relatively small marker-release events take place without catastrophic membrane rupture at the timepoints examined. This finding is consistent with the fact that conventional imaging techniques cannot detect cytolytic plumes around tumor cells. Thus, in contrast to other cytolytic systems, tumor cell cytolysis is not an all-or-none biological response.

Another key finding in the present study is that multiple marker-release events take place, as illustrated by the multiple fluorescence intensity peaks associated with each target cell (Fig. 2Go). Again, this observation contrasts sharply with the single marker-release events of liposomes and E 18, 22 . Nonetheless, it is consistent with prior indirect studies of complement-mediated lysis of nucleated target cells that supported a multihit hypothesis 24 . Because cytolysis is defined as cytoplasmic marker release, we interpret the multiple marker-release events as corresponding to multiple cytolytic events. Each cytolytic event comprises membrane rupture and resealing events, as suggested by their finite duration. Hence, each cytolytic event is composed of a membrane rupture event that allows the fluorescent label to leak from a cell and accumulate as a pericellular plume of fluorescence followed by a resealing event and dissolution of the label into the environment.

Our quantitative kinetic experiments (Fig. 2Go) show that cytolytic burst peak intensities occur at ~20-s intervals. This time interval corresponds to that observed for oscillations in other neutrophil functions and properties such as actin assembly, shape and velocity changes, receptor oscillations, and reduced nicotinamide-adenine dinucleotide phosphate oscillations (reviewed in 25 . Moreover, the oscillatory release of reactive oxygen metabolites and pericellular proteolysis take place at 20-s intervals 16 . Several functional properties of neutrophils appear to be driven by their oscillatory metabolism 15, 16 . Furthermore, reactive oxygen metabolite release has been linked to tumor cell cytotoxicity 26, 27, 28 . Thus, both oscillatory enzymatic and oxidative capacities of neutrophils could contribute to the timing of the oscillatory cytolytic bursts observed in the present study. In either case, it seems likely that oscillatory metabolite concentrations within the effector cell drive the oscillatory cytolytic damage of the target cell.

Studies of the time-dependent formation and dissipation of cytolytic marker-release plumes show their occurrence at multiple sites around the perimeter of tumor cells (Fig. 4Go, B–D). This suggests that there is no unique membrane site associated with cytolysis. Hence, cytolytic marker release is not necessarily found at sites of effector-to-target cell contact. We have reported similar findings for neutrophil-mediated E lysis 18 . The contact zone between target and effector cells may, in fact, be stabilized by links and cross-links among cytoskeletal components, membrane Ags, IgG, and Fc receptor complexes. Furthermore, macrophages form exclusionary zones around sites of contact with IgG-opsonized targets 29 . Thus, it may be difficult to damage a contact zone, and such zones may impede the diffusion of cytosolic labels into the extracellular environment.

In addition to providing the first direct evidence regarding the subcellular mechanism of tumor cell cytolysis and dramatic evidence in support of the multihit hypothesis, our studies also provide a new tool for dissecting the temporal elements of tumor cell destruction. It should become possible to image later events during cytolysis by taking advantage of green fluorescent proteins, microscope flow chambers 13 , and ICCDs possessing greater dynamic ranges. Moreover, when emerging tools in biology such as imaging spectrophotometry and extracellular fluorescence labels that report their chemical environment (e.g., 16 are used, it should become possible to temporally correlate well-defined chemical reactions with membrane rupture. Thus, it may be possible to mechanistically associate the molecules participating in host defense with specific cytolytic plumes. It should also be possible to examine additional cell types that may display different cytolytic release mechanisms.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI27409 (to H.R.P.). Back

2 Address correspondence and reprint requests to Dr. Howard R. Petty, Department of Biological Sciences, Wayne State University, Detroit, MI 48202. E-mail address: Back

3 Abbreviations used in this paper: ADCC, Ab-dependent cellular cytolysis; TMR, tetramethylrhodamine diacetate; ICCD, intensified charge-coupled device. Back

Received for publication October 8, 1998. Accepted for publication December 9, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bright, R. K., M. H. Shearer, R. C. Kennedy. 1994. Immunization of BALB/c mice with recombinant simian virus 40 large tumor antigen induces antibody-dependent cell-mediated cytotoxicity against simian virus 40-transformed cells: an antibody-based mechanism for tumor immunity. J. Immunol. 153:2064.[Abstract]
  2. Clynes, R., Y. Takechi, Y. Moroi, A. Houghton, J. V. Ravetch. 1998. Fc receptors are required in passive and active immunity to melanoma. Proc. Natl. Acad. Sci. USA 95:652.[Abstract/Free Full Text]
  3. Kushner, B. H., N.-K. V. Cheung. 1992. Absolute requirement of CD11/CD18 adhesion molecules, FcRII, and the phosphatidylinositol-linked FcRIII for monoclonal antibody-mediated neutrophil antihuman tumor cytotoxicity. Blood 79:1484.[Abstract/Free Full Text]
  4. Ralph, P., I. Nakoinz, B. Diamond, D. Yelton. 1980. All classes of murine IgG antibody mediate macrophage phagocytosis and lysis of erythrocytes. J. Immunol. 125:1885.[Abstract]
  5. Kerst, J. M., M. de Haas, C. E. van der Schoot, I. C. M. Slaper-Cortenbach, M. Kleijer, A. E. G. von dem Borne, R. H. J. van Oers. 1993. Recombinant granulocyte colony-stimulating factor administration to healthy volunteers: induction of immunophenotypically and functionally altered neutrophils via an effect on myeloid progenitor cells. Blood 82:3265.[Abstract/Free Full Text]
  6. Vaickus, L., W. Biddle, D. Cemerlic, K. A. Foon. 1990. Interferon {gamma} augments lym-1-dependent, granulocyte-mediated tumor cell lysis. Blood 75:2408.[Abstract/Free Full Text]
  7. Schreiber, G. J., K. E. Hellstrom, I. Hellstrom. 1992. An unmodified anticarcinoma antibody, BR96, localizes to and inhibits the outgrowth of human tumors in nude mice. Cancer Res. 52:3262.[Abstract/Free Full Text]
  8. Iliopoulos, D., C. Ernst, A. Steplewski, J. A. Jambrosic, U. Rodeck, M. Herlyn, W. H. Clarck, H. Koprowski, D. Herlyn. 1989. Inhibition of metastases of a human melanoma xenograft by monoclonal antibody to the GD2/GD3 gangliosides. J. Natl. Cancer Inst. 81:440.[Abstract/Free Full Text]
  9. Kasprzyk, P. G., S. U. Song, P. P. Di Fiore, C. R. King. 1992. Therapy of an animal model of human gastric cancer using a combination of anti-erbB-2 monoclonal antibodies. Cancer Res. 52:2771.[Abstract/Free Full Text]
  10. Heijnen, I. A. F. M., L. J. M. Rijks, A. Schiel, B. Stockmeyer, H. A. van Ojik, M. Dechant, T. Valerius, T. Keler, A. L. Tutt, M. J. Glennie, et al 1997. Generation of HER-2/neu-specific cytotoxic neutrophils in vivo: efficient arming of neutrophils by combined administration of granulocyte colony-stimulating factor and Fc{gamma} receptor I bispecific antibodies. J. Immunol. 159:5629.[Abstract]
  11. Naftzger, C., Y. Takechi, H. Kohda, I. Hara, S. Vijayasaradhi, A. N. Houghton. 1996. Immune response to a differentiation antigen induced by altered antigen: a study of tumor rejection and autoimmunity. Proc. Natl. Acad. Sci. USA 93:14809.[Abstract/Free Full Text]
  12. Hara, I., Y. Takechi, A. N. Houghton. 1995. Implicating a role for immune recognition of self in tumor rejection: passive immunization against the brown locus protein. J. Exp. Med. 182:1609.[Abstract/Free Full Text]
  13. Albrecht, E., H. R. Petty. 1998. Cellular memory: neutrophil orientation reverses during temporally decreasing chemoattractant concentrations. Proc. Natl. Acad. Sci. USA 95:5039.[Abstract/Free Full Text]
  14. Zhou, M. J., H. R. Petty. 1989. Novel phagocytic behavior of human neutrophils: micro-scisson of YAC tumor cells during antibody-dependent cellular cytotoxicity. Eur. J. Cell Biol. 50:201.[Medline]
  15. Kindzelskii, A. L., M. M. Eszes, III R. F. Todd, H. R. Petty. 1997. Proximity oscillations of complement receptor type 4 and urokinase receptors on migrating neutrophils are linked with signal transduction/metabolic oscillations. Biophys. J. 73:1777.[Abstract/Free Full Text]
  16. Kindzelskii, A. L., M. J. Zhou, R. P. Haugland, L. A. Boxer, H. R. Petty. 1998. Oscillatory pericellular proteolysis and oxidant deposition during neutrophil migration. Biophys. J. 74:90.[Abstract/Free Full Text]
  17. Lellouch, E., W. B. Hubbard, B. Sicardy, F. Vilas, P. Bouchet. 1986. Occultation determination of Neptune’s oblateness and stratospheric methane mixing ratio. Nature 324:227.
  18. Francis, J. W., L. A. Boxer, H. R. Petty.. 1988. Optical microscopy of antibody-dependent phagocytosis and lysis of erythrocytes by living normal and chronic granulomatous disease neutrophils: a role of superoxide anions in extra- and intra-cellular lysis. J. Cell. Physiol. 135:1.[Medline]
  19. Brunner, K. T., J. Mauel, J.-C. Cerottini, B. Chapuis. 1968. Quantitative assay of the lytic action of immune lymphoid cells on 51Cr-labelled allogeneic target cells in vitro: inhibition by isoantibody and by drugs. Immunology 14:181.[Medline]
  20. Martz, E.. 1976. Early steps in specific tumor cell lysis by sensitized mouse T lymphocytes: electrolyte permeability increase in the target cell membrane concomitant with programming for lysis. J. Immunol. 117:1023.[Abstract/Free Full Text]
  21. Henney, C. S.. 1973. Studies on the mechanism of lymphocyte-mediated cytolysis: the use of various target markers to study cytolytic events. J. Immunol. 110:73.[Abstract/Free Full Text]
  22. Lewis, T. J., H. M. McConnell. 1978. Model lipid bilayer membranes as targets for antibody-dependent, cellular- and complement-mediated immune attack. Ann. N. Y. Acad. Sci. 308:124.[Medline]
  23. Poenie, M., R. Y. Tsien, A.-M. Schmitt-Verhulst. 1987. Sequential activation and lethal hit measured by [Ca2+]i in individual cytolytic T cells and targets. EMBO J. 6:2223.[Medline]
  24. Koski, C. L., L. E. Ramm, C. H. Hammer, M. M. Mayer, M. L. Shin. 1983. Cytolysis of nucleated cells by complement: cell death displays multi-hit characteristics. Proc. Natl. Acad. Sci. USA 80:3816.[Abstract/Free Full Text]
  25. Petty, H. R.. 1998. Oscillatory signals in migrating neutrophils: effects of time-varying chemical and electrical fields. J. Walleczek, ed. Self-Organized Biological Dynamics and Nonlinear Control by External Stimuli Cambridge University Press, Cambridge, U.K.
  26. Cao, D., L. A. Boxer, H. R. Petty. 1993. Deposition of reactive oxygen metabolites onto and within living tumor cells during neutrophil-mediated antibody-dependent cellular cytotoxicity. J. Cell. Physiol. 156:428.[Medline]
  27. Borregaard, N., K. Kragballe. 1980. Role of oxygen in antibody-dependent cytotoxicity mediated by monocytes and neutrophils. J. Clin. Invest. 66:676.
  28. Clark, R. A., S. J. Klebanoff. 1975. Neutrophil-mediated tumor cell cytotoxicity: role of the peroxidase system. J. Exp. Med. 141:1442.[Abstract/Free Full Text]
  29. Wright, S. D., S. C. Silverstein. 1984. Phagocytosing macrophages exclude proteins from zones of contact with opsonized targets. Nature 309:359.[Medline]



This article has been cited by other articles:


Home page
 Infect. Immun.Home page
R. R. Ganta, C. Cheng, E. C. Miller, B. L. McGuire, L. Peddireddi, K. R. Sirigireddy, and S. K. Chapes
Differential Clearance and Immune Responses to Tick Cell-Derived versus Macrophage Culture-Derived Ehrlichia chaffeensis in Mice
Infect. Immun., January 1, 2007; 75(1): 135 - 145.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
A. L. Kindzelskii and H. R. Petty
From the Cover: Apparent role of traveling metabolic waves in oxidant release by living neutrophils
PNAS, July 9, 2002; 99(14): 9207 - 9212.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
E. Di Carlo, G. Forni, P. Lollini, M. P. Colombo, A. Modesti, and P. Musiani
The intriguing role of polymorphonuclear neutrophils in antitumor reactions
Blood, January 15, 2001; 97(2): 339 - 345.
[Full Text] [PDF]

Home page
 J. Immunol.Home page
Y. Adachi, A. L. Kindzelskii, N. Ohno, T. Yadomae, and H. R. Petty
Amplitude and Frequency Modulation of Metabolic Signals in Leukocytes: Synergistic Role of IFN-{gamma} in IL-6- and IL-2-Mediated Cell Activation
J. Immunol., October 15, 1999; 163(8): 4367 - 4374.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kindzelskii, A. L.
Right arrow Articles by Petty, H. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kindzelskii, A. L.
Right arrow Articles by Petty, H. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS