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(Journal of Leukocyte Biology. 2000;68:662-668.)
© 2000 by Society for Leukocyte Biology

Monoclonal Lym-1 antibody-targeted lysis of B lymphoma cells by neutrophils. Evidence for two mechanisms of Fc{gamma}RII-dependent cytolysis

Luciano Ottonello*, Alan L. Epstein{dagger}, Marina Mancini*, Massimo Amelotti*, Patrizia Dapino* and Franco Dallegri*

* Department of Internal Medicine, University of Genova Medical School, Genova, Italy; and
{dagger} Department of Pathology, University of Southern California, Los Angeles, California

Correspondence: Prof. Franco Dallegri, Dipartimento di Medicina Interna e Specialità Mediche, Viale Benedetto XV n.6, I-16132 Genova, Italy. E-mail: MACROBUTTON HtmlResAnchor otto{at}csita.unige.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human neutrophils incubated with the anti-HLA-DR mAb Lym-1, plus PMA, induced significant cytolysis of B lymphoma cells compared with Lym-1 and PMA alone. The effect of PMA was independent of the ability of the compound to stimulate neutrophil-respiratory burst. In fact, first, neutrophils from a patient with chronic granulomatous disease were cytolytically effective in spite of their inability to produce oxidants. Second, various kinase inhibitors exerted different effects on the PMA-stimulated cytolytic system and neutrophil-oxidative burst. Previous studies have shown the involvement of the Fc{gamma}RII, CD11b-CD18 integrins, and CD66b glycoproteins in the Lym-1 mAb-dependent cytolysis by GM-CSF-stimulated neutrophils. The present PMA-stimulated system was inhibited by the anti-Fc{gamma}RII mAb IV.3, the anti-CD18 mAb MEM 48, and the anti-CD11b mAb 2LPM19c but not by the anti-CD66b mAb 80H3 and N-acetyl-D-glucosamine. Furthermore, the PMA- and GM-CSF-stimulated cytolysis was insensitive and sensitive to inhibition by pertussis toxin, respectively. Thus, the use of PMA and GM-CSF as neutrophil stimulants uncovers the existence of distinct mechanisms of Lym-1 mAb-mediated cytolysis.

Key Words: human neutrophils • ADCC • Lym-1 • Fc receptors • GM-CSF • phorbol myristate acetate


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lym-1 is a murine mAb well-suited for targeted immunotherapy of human malignant lymphomas [1 , 2 ]. Indeed, it is indeed specific for a human class II molecule variant [1 ] expressed on the surface of B-lymphoma cells and incapable of undergoing shedding or modulation after binding [1 ]. Initial clinical trials with Lym-1 used intravenously in patients with refractory B cell lymphomas showed only modest reductions in lymph node size in some cases [3 ], stimulating investigators to use 131I-labeled products with superior results [4 ]. Although various factors can contribute to these partial responses with naked antibody [2 , 5 ], the inadequacy of host-immune effector systems is likely to play a major role. Among these systems, neutrophils appear to be appropriate targets to improve monoclonal antibody (mAb)-dependent host-antitumor activities [2 , 6 7 8 ]. Indeed, these phagocytes have displayed cytokine-stimulated antineoplastic function in various mouse models [9 , 10 ]. Moreover, they have been found to mediate mAb-dependent tumor cell lysis via a process sensitive to stimulation by various biological response modifiers [7 , 8 , 11 12 13 ]. Nevertheless, the mechanisms whereby distinct mediators exert the same stimulatory action are largely obscure. We have recently shown that granuloctye-macrophage colony-stimulating factor (GM-CSF)-stimulated neutrophils mediate Lym-1 mAb-dependent lysis via Fc receptor for immunoglobulin G (IgG) type II (Fc{gamma}RII), with the concomitant and absolute requirement for CD11b-CD18 integrins and carcinoembryonic antigen (CEA)-like CD66b glycoproteins [14 ]. In the present study, the replacement of GM-CSF with phorbol 12-myristate 13-acetate (PMA) uncovers the existence of alternative mechanisms of cytolysis, which is CD66b- and pertussis toxin (PT)-independent.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Culture medium and reagents
The following culture medium was used: RPMI 1640 (Irvine Scientific, Santa Ana, CA), supplemented with 10% heat-inactivated (56°C, 45 min) fetal calf serum (FCS; HyClone Europe, Cramlington, England), and 2 mM glutamine (Irvine Scientific; RPMI-FCS). Hanks’ balanced saline solution (HBSS) was obtained from Irvine Scientific. Ficoll-Hypaque was purchased from Seromed, Berlin, Germany. Sodium chromate 51Cr was obtained from the Radiochemical Center, Amersham, England. Triton X-100, ethidium bromide, PMA, ferricytochrome c type VI horse heart, superoxide dismutase (SOD) type I from bovine blood, genistein (GST), wortmannin (WMN), PT, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), N-acetyl-D-glucosamine (NADG), bovine serum albumin (BSA), and mouse IgG2a were purchased from Sigma Chemical Co., St. Louis, MO. Heparin was obtained from Roche, Milano, Italy. Human recombinant GM-CSF was from Genzyme, Cambridge, MA.

mAbs
The previously described anti-B cell lymphoma mAb Lym-1 (IgG2a) [1 ] was used for the cytolytic assay. Lym-1 has been shown to bind to a discontinuous epitope on the HLA-DR-10 ß chain, overexpressed on human B cell lymphomas and leukemia [15 ]. Moreover, the following mAbs were used: anti-CD32 IV.3 (IgG2b, Fab fragments, Medarex, Annandale, NJ), anti-CD16 3G8 (IgG1, native mAb and F(ab')2 fragments, Medarex), anti-CD18 MEM 48 (IgG1) and anti-CD11a MEM 25 (IgG; kindly provided by V. Horejsi, Institute of Molecular Genetics, Academy of Science, Prague, Czech Republic), anti-CD11b VIM12 (IgG1 kindly provided by W. Knapp, Vienna, Austria), anti-CD11b 2LPM19c (IgG1, Dako A/S, Denmark), and anti-CD66b 80H3 (IgG1, Serotec, Torino, Italy). Fluorescein isothiocyanate (FITC)-conjugated anti-CD11b (44, IgG) was from BioSource, Camarillo, CA. The appropriate control, isotype-matched, FITC mAbs were from Immunotech, Marseille, France. FITC rabbit anti-mouse IgG F(ab')2 fragment was from BioSource.

Neutrophil preparation
Heparinized venous blood (heparin 10 U/mL) was obtained from healthy volunteers (20–45 years old) after informed consent. No donor had an infectious disease or was under medication at the time of and for two weeks before sampling. Blood was also obtained from a previously described [16 ] patient with chronic granulomatous disease (CGD). Neutrophils were prepared by dextran sedimentation, followed by centrifugation (400 g, 30 min) on Ficoll-Hypaque density gradient, as previously described [8 ]. Contaminating erythrocytes were removed by hypotonic lysis [8 ]. Neutrophils resuspended in RPMI-FCS were >97% pure, as determined by morphologic analysis of Giemsa-stained cytopreps [8 ] and greater than 98% viable. Experiments were also carried out using neutrophils pretreated with 10 ng/mL PMA. These cells were prepared as previously described [17 ]. Briefly, neutrophils were incubated (15 min, 4°C) with 10 ng/mL PMA, subsequently centrifuged at 4°C, and then washed twice in 50 mL vol ice-cold RPMI [17 ].

Target cells
Burkitt’s lymphoma Raji cells [8 ] were used as targets in the cytolytic assays. The Raji cell line was grown in RPMI-FCS and subcultured every 3 days. The capacity of these cells to bind Lym-1 antibody was measured by indirect immunofluorescence with flow cytometry using a rabbit anti-mouse IgG F(ab')2 polyclonal antibody conjugated with FITC (Dako A/S) [8 ]. For the cytolytic assays, 4 x 106 Raji cells were labeled with 100–200 µCi sodium chromate 51Cr by incubating for 1 h at 37°C (final vol, 0.5 mL; medium, RPMI 1640+5% FCS). After washing, the labeled cells were resuspended in RPMI-FCS.

Cytolytic assays
Cytolytic activity of neutrophils was measured as described elsewhere in detail [8 , 18 ]. Briefly, target cells (2x104) were mixed with neutrophils at an effector:target ratio of 20:1, with and without 10 µg/mL Lym-1 mAb [1 ] and/or 10 ng/mL PMA appropriately diluted in RPMI-FCS. Experiments were carried out also using 1 ng/mL GM-CSF instead of PMA. The effector:target ratio of 20:1 was chosen on the basis of preliminary experiments, also taking into account previous observations [8 ]. Experiments were carried out in the absence or presence of the various mAbs and reagents used to probe the cytolytic process. The replacement of Lym-1 with 10 µg/mL mouse IgG2a, in the presence of 10 ng/mL PMA, resulted in no lysis. The assays were carried out in triplicate and in a final vol of 150 µL, using round-bottom microplates (Falcon, Becton-Dickinson Italia, Milano, Italy). After 14 h of incubation in a humidified atmosphere of 95% air and 5% CO2, the 51Cr-release was determined by the formula 100 x (E-S)/(T-S), where E is the cpm released in the presence of effector cells, T is the cpm released after target cells with 5% Triton X-100, and S is the cpm spontaneously released by target cells incubated with medium alone (<18%).

Superoxide release assay
The release of superoxide anions (O2-) was studied in duplicate by using a modification of the method of Babior et al. [19 ], as previously described [20 ]. Briefly, neutrophils (5x105 cells) in a final vol of 0.5 mL HBSS were incubated (37°C, 20 min) with 10 ng/mL PMA. Incubations were carried out in the presence of 80 µM ferricytochrome c. Parallel tubes, with the same components, included 150 U/mL SOD also. After incubation, the tubes were centrifuged (400 g, 10 min) at 4°C, and the optical density of the supernatants was measured. The O2- production was determined from the A550 of the samples without SOD, minus the A550 of matched samples with SOD, using an extinction coefficient of 2.1 x 104 M-1cm-1.

Immunofluorescence analysis
Purified neutrophils (1x106 cells in HBSS) were preincubated (15 min, 4°C) with 10 ng/PMA and subsequently incubated for 30 min at 4°C in the presence or absence of 4 µg/mL anti-CD66b mAb 80H3 [14 ]. After washing twice with cold HBSS, the cells were incubated (30 min, 4°C) with 20 µg/mL goat anti-mouse F(ab')2. The cells were washed with HBSS containing 0.1% sodium azide and then incubated (30 min, 37°C) with FITC-conjugated, anti-CD11b mAb 44. The cells were examined using a fluorescence Nikon Optiphot-2 microscope and images were collected by a Hamamatsu Color-chilled 3 CCD camera. The ability of neutrophils to bind mAb 80H3 (anti-CD66b) and the capacity of Raji cells to bind Lym-1 mAb were measured by indirect immunofluorescence with flow cytometry (EPICS XL flow cytometer, Coulter, Hialeah, FL), using a rabbit anti-mouse IgG F(ab')2polyclonal antibody conjugated with FITC.

Statistical analysis
Results were expressed as mean ± 1 SE and/or a median with the 95% confidence interval. Statistical differences were analyzed by the Mann-Whitney test. Significance was accepted when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Raji cell lysis by PMA-stimulated neutrophils
As shown in Figure 1 , incubation of human neutrophils with 51Cr-labeled Raji cells at an effector:target cell ratio of 20:1 did not result in 51Cr release after 14 h of incubation. Therefore, neutrophils appear to be incapable of mediating spontaneous cytotoxicity toward Raji target cells. Moreover, neither PMA (10 ng/mL) nor Lym-1 mAb (10 µg/mL) had consistent lytic activity against Raji cells (percent cytolysis, 1.1±0.7, 1.4±0.8, and 0.6±0.6 by PMA, Lym-1, and PMA+Lym-1, respectively; mean±1 SE, n=3). Conversely, the addition of 10 ng/mL PMA or 10 µg/mL Lym-1 to neutrophils caused low but significant target-cell lysis (Fig. 1) . Moreover, the simultaneous addition of PMA and Lym-1 to neutrophil target-cell cocultures resulted in consistent amplification of the lysis (Fig. 1) . In each case, the lysis induced by PMA + Lym-1 was higher than that calculated by adding the values of lysis observed with PMA and Lym-1 used as single stimulating agent. Thus, PMA and Lym-1 cooperate synergistically to stimulate neutrophil-mediated cytolysis. Experiments were then planned to understand if PMA has an impact on tumor-cell sensitivity to neutrophil-mediated cytotoxicity. Therefore, parallel assays were carried out by studying Lym-1 mAb-dependent Raji cell lysis by neutrophils in presence of PMA and by neutrophils pretreated with, and then tested in the absence of, soluble PMA. PMA-pretreated neutrophils and native neutrophils exposed to soluble minimum Eagle’s PMA caused comparable levels of lysis (32.1±3.1 and 36.6±3.5, respectively; mean±1 SE, n=4). Consequently, it appears that PMA increases neither the susceptibility of target cells to the lysis nor the amount of Lym-1 that sensitizes target cells and is available for neutrophil binding. Consistent with this last finding, the exposure of Raji cells to PMA for 6 h did not affect the surface density of bound Lym-1, as judged cytofluorimetrically (unpublished results). In conclusion, PMA appears to act primarily on neutrophils in our setting. Because PMA is the most potent activator of the neutrophil-oxidative burst, the possibility that its cooperation with Lym-1 results in the oxidative lysis of Raji cells by neutrophils seems to be a reasonable hypothesis. Nevertheless, neutrophils from a patient with CGD, i.e., genetically incapable of activating the oxidative burst, were effective in causing PMA/Lym-1-stimulated Raji cell lysis (Fig. 2 ). This is consistent with the concept of target-cell lysis as an oxygen-independent event.



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  		Figure 1. Neutrophil-mediated cytolysis in the absence or presence of 10 µg/mL Lym-1 and/or 10 ng/mL PMA. 51Cr-labeled Raji target cells were at 2 x 104. The neutrophil:Raji cell ratio was 20:1. The incubation time was 14 h. Results are expressed as mean ± 1 SE (Nil, Lym-1, and Lym-1+PMA: n=15; PMA: n=9). PMA vs. Nil, p < 0.05; Lym-1 vs. Nil, p < 0.01; Lym-1 + PMN vs. PMA, p < 0.05; Lym-1 + PMA vs. Lym-1, p < 0.01.

 


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  		Figure 2. Cytolysis mediated by neutrophils from a patient with CGD and by neutrophils from two normal controls (N1 and N2). 51Cr-labeled Raji cells were at 2 x 104. The neutrophil:target cell ratio was 20:1. The incubation time was 14 h. Lym-1 = 10 µg/mL; PMA = 10 ng/mL. Results are mean ± 1 SE from one experiment performed in triplicate.

 
Intervention of neutrophil CD32 and CD11b-CD18.
To identify neutrophil-surface molecules involved in the process, mAbs specific for Fc{gamma}RII (CD32) and III (CD16), as well as mAbs specific for CD11b-CD18 integrins, were tested in a PMA/Lym-1-stimulated cytolytic system. As shown in Figure 3 , mAb IV.3 toward CD32 molecules inhibited significantly and efficiently neutrophil-mediated cytolysis. On the contrary, the anti-CD16 mAb 3G8 was ineffective (Fig. 3) . These data are consistent with the intervention of Fc{gamma}RII (CD32) without the involvement of Fc{gamma}RIII. Moreover, the lysis was inhibited by mAb MEM 48 and mAb 2LPM19c, which, respectively, recognize CD18 and CD11b (Fig. 3) . Conversely, mAb MEM 25 (4 µg/mL) against CD11a [lymphocyte function-associated antigen-1 (LFA-1)] had no effect (% cytolysis, 34.9±4.5 and 32.1±1.4 in the presence and absence of MEM 25; mean±1 SE, n=6). This suggests an absolute requirement for CD11b-CD18 integrins in the process. In agreement with an active role of these adhesion molecules in the present cytolytic system, the lysis was enhanced significantly by the anti-CD11b mAb VIM-12 (Fig. 4 ), a mAb known to activate these integrins [21 ]. Using GM-CSF-stimulated neutrophils, Lym-1 mAb-mediated lysis was found to be dependent strictly on the intervention of CEA-like CD66b glycoproteins, which cooperate with CD11b-CD18 via saccharide-inhibitable, i.e., lectin-like, interactions [14 ]. In the present PMA system, the lysis was found to be unaffected by the addition of anti-CD66b mAb 80H3 (lysis, 33.17±3.2 and 35.10±0.7 in the absence or presence of 4 µg/mL mAb 80H3; mean±1 SD, n=4). Moreover, the use of 8 instead of 4 µg/mL mAb 80H3 did not produce an inhibitory effect (% inhibition, 4.5±4.0, mean±1 SE, n=3). The effect of the anti-CD66b mAb was tested simultaneously also in the GM-CSF and PMA-stimulated system. As shown in Figure 5A , the PMA system was confirmed to be insensitive to mAb 80H3, whereas the GM-CSF system was inhibited efficiently. Consistent with this conclusion, the GM-CSF but not the PMA system was inhibited by the saccharide NADG (Fig. 5B) . Conversely, neutrophil exposure to PMA results in the CD66b surface-expression upregulation and allows the CD66b/CD11b interaction (unpublished results). This suggests that PMA-treated neutrophils express high amounts of functionally active CD66b molecules, which, however, do not intervene in PMA-stimulated cytolysis.



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  		Figure 3. Effect of the anti-CD32 (Fc{gamma}RII) mAb IV.3, anti-CD16 (Fc{gamma}RIII) mAb 3G8, anti-CD18 mAb MEM 48, and anti-CD11b mAb 2LPM19c on PMA-stimulated, neutrophil-mediated, Lym-1, mAb-dependent lysis. 51Cr-labeled Raji cells were at 2 x 104. The neutrophil:Raji cell ratio was 20:1. The incubation time was 14 h. (A) Lysis in the absence vs. presence of IV.3: p = 0.0286. (B) Lysis in the absence vs. presence of 3G8: p = 0.200. (C) Lysis in the absence vs. presence of MEM 48: p = 0.0006. (D) Lysis in the absence vs. presence of 2LPM19c: p = 0.0286.

 


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  		Figure 4. Effect of the anti-CD11b mAb VIM12 on the PMA-stimulated, neutrophil-mediated, Lym-1, mAb-dependent lysis. 51Cr-labeled Raji cells were at 2 x 104. The neutrophil:target cell ratio was 20:1. The incubation time was 14 h. Lysis in the absence vs. presence of VIM12: p = 0.0043.

 


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  		Figure 5. Effect of the anti-CD66b mAb 80H3 (A) and NADG (B) on the PMA- (solid bars) and GM-CSF- (open bars) stimulated Lym-1, mAb-dependent lysis by neutrophils from five (A) and four (B) donors. 51Cr-labeled Raji cells were at 2 x 104. The neutrophil:target cell ratio was 20:1. The incubation time was 14 h. Results are shown as mean ± 1 SE. (A) PMA: lysis in the presence vs. absence of 80H3: p = 0.1508. GM-CSF: lysis in the presence vs. absence of 80H3: p = 0.0079. (B) PMA: lysis in the presence vs. absence of NADG: p > 0.9999. GM-CSF: lysis in the presence vs. absence of NADG: p = 0.0286.

 
Effects of inhibitors of postreceptor signaling
To gain insight into mechanisms of postreceptor neutrophil activation of the cytolytic potential, various agents were used. As shown in Figure 6 , treatment with PT did not affect neutrophil-lytic efficiency after PMA/Lym-1 stimulation. On the contrary, it was effective with neutrophils exposed to GM-CSF/Lym-1 (Fig. 6) . This suggests that activation of the neutrophil-lytic machinery by PMA/Lym-1 and GM-CSF/Lym-1 involves distinct signaling pathways. In addition, other compounds were tested: GST, an inhibitor of tyrosine kinases [22 ]; H-7, which has been shown to inhibit various kinases [23 ]; and WMN, which inhibits phosphatidyl-inositol-3-kinase and phospholipase D [24 ]. As shown in Figure 7 , these inhibitors reduced neutrophil-lytic efficiency in a dose-dependent manner in the PMA- and GM-CSF-stimulated systems. Moreover, doses of agents herein shown to inhibit PMA/Lym-1-stimulated cytolysis (Fig. 7) were tested on the PMA-triggered neutrophil-oxidative response. As shown in Figure 8 , exposure of neutrophils to PMA resulted in O2- production. This production was completely inhibited by H-7 and unaffected by GST and WMN (Fig. 8) at concentrations equally effective in inhibiting cytolysis (Fig. 7) . Therefore, these data are consistent with the ability of PMA to synergize with Lym-1 to induce neutrophil-mediated and oxygen-independent target-cell attack.



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  		Figure 6. Effect of PT on the PMA- and GM-CSF-stimulated, neutrophil-mediated, Lym-1, mAb-dependent lysis. 51Cr-labeled Raji cells were at 2 x 104. The neutrophil:target cell ratio was 20:1. The incubation time was 14 h. Neutrophils (2x106/mL) were incubated with PT or control medium for 2 h at 37°C and washed twice before use. (A) PMA: lysis by PT-treated neutrophils vs. control neutrophils: p = 0.222. (B) GM-CSF: lysis by PT-treated neutrophils vs. control neutrophils: p = 0.0022.

 


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  		Figure 7. Effect of GST (A and D), H7 (B and E), and WMN (C and F) on PMA-stimulated (left panels) and GM-CSF-stimulated (right panels), neutrophil-mediated, Lym-1, mAb-dependent lysis. 51Cr-labeled Raji cells were at 2 x 104. The neutrophil:target cell ratio was 20:1. The incubation time was 14 h. Concentrations in abscissa are expressed as µM. Results are shown as mean ± 1 SE of four (A, C, E, and F), five (B), or six (D) determinations for each point.

 


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  		Figure 8. Superoxide anion generation by neutrophils stimulated with PMA in the absence or presence of GST, H7, or WMN. PMA = 10 ng/mL; GST = 25 µM; H7 = 100 µM; WMN = 0.1 µM. Results are expressed as mean ± 1 SE, n = 3. PMA + H7 vs. PMA: p < 0.001.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study shows that mAb Lym-1 and PMA interact synergistically to trigger the neutrophil-cytolytic machinery against B lymphoma cells. Moreover, the investigation of events underlying this cytolytic system suggests that 1) PMA/Lym-1-stimulated target-cell lysis occurs as a result of a neutrophil-delivered, oxygen-independent attack; 2) the lytic process involves Fc{gamma}RII and CD11b-CD18 integrins, without the intervention of CD66b glycoproteins; and 3) the activation of neutrophil-cytolytic potential proceeds through PT-insensitive but kinase-dependent pathways.

It is well-known that PMA is the most potent stimulant as far as the neutrophil-respiratory burst is concerned [25 , 26 ]. Nevertheless, in the present setting, different findings suggest that the stimulatory activity of PMA is independent completely on its ability to induce oxidant production in neutrophils. First, the effects of several inhibitors of the cytolytic system differed from those exerted on PMA-induced oxidative burst. Second, neutrophils from a patient with CGD-mediated cytolysis in spite of their incapacity to generate oxidants. Therefore, the PMA-dependent effect on neutrophil Lym-1 mAb-mediated cytolysis resembles that exerted on IgG-induced cell phagocytosis [27 , 28 ]. Conversely, the independence of the lysis from oxygen metabolites is in agreement with our previous observation carried out using Raji cells sensitized with rabbit antiserum as targets [18 ]. In this regard, defensins are the major candidates for the role of cytolytic mediators [29 , 30 ].

Previous studies dealing with the mechanisms of Lym-1, mAb-dependent cytolysis by GM-CSF-stimulated neutrophils [14 ] have shown that the process involves Fc{gamma}RII without the intervention of other Fc{gamma}Rs and strictly requires CD11b-CD18 integrins and CEA-like CD66b glycoproteins to physically interact in a lectin-like manner on the neutrophil surface. Similar to these findings, PMA-stimulated neutrophils exerted Lym-1, mAb-mediated lysis via Fc{gamma}RII and CD11b-CD18 integrins. Nevertheless, we were unable to prove the intervention of CD66b molecules. This is suggested by the incapacity of the anti-CD66b mAb 80H3 and NAGD to block PMA-stimulated cytolysis. Nevertheless, the ability of PMA-triggered neutrophils to exert cytolysis independently of CD66b is not related to the PMA-induced glycoprotein’s shedding or inactivation. Indeed, in agreement with previous observations [31 ], activated neutrophils underwent upregulation of CD66b on their surface. In addition, upregulated CD66b maintain the ability to undergo physical association with CD11b-CD18 integrins. Therefore, PMA stimulation appears to bypass the CD66b requirement observed for GM-CSF-exposed neutrophils. Finally, it is known that neutrophil activation via Fc{gamma}RII proceeds through two pathways, only one of which is regulated by PT-sensitive G proteins [32 , 33 ]. Because of the observed different susceptibility to PT, the GM-CSF/Lym-1 and the PMA/Lym-1 cytolytic systems appear to use the toxin-sensitive and -insensitive Fc{gamma}RII signaling pathway, respectively. Indeed, the inhibitory activity of the toxin cannot be attributed to interference with GM-CSF cell stimulation or CD11b-CD18/CD66b-delivered signals [34 35 36 37 ].

Cooperation among plasma membrane receptors, such as Fc{gamma}R and CD11b-CD18 molecules, in activating phagocyte-effector functions is a well-recognized phenomenon, particularly with neutrophil phagocytosis and related cell responses [38 , 39 ]. In fact, such a type of cooperation has been investigated extensively, primarily in terms of the ability of CD11b-CD18 integrins to serve as a signaling partner for the glycosyl-phosphatidyl-inositol-anchored Fc{gamma}RIII [39 , 40 ]. The present study suggests a collaboration of CD11b-CD18 with Fc{gamma}RII as well, in a manner similar to that observed in certain models of Fc{gamma}RII-mediated phagocytosis or cell activation [41 , 42 ]. In this regard, the known association of these receptors with various intracellular kinases, including tyrosine kinases [43 , 44 ], is consistent with the observed susceptibility of the cytolytic process to suppression by various inhibitors. Nevertheless, as the specificity of these inhibitory compounds can be questioned, the present findings do not allow a detailed identification of the signal-transducing pathway responsible for activation of the neutrophil-cytolytic potential. However, the ability of PMA to stimulate neutrophil-cytolytic activity is consistent with the intervention of protein kinase C, although the involvement of a phorbol ester receptor lacking kinase activity cannot be excluded [45 ].

In conclusion, the present data, coupled with the findings obtained using GM-CSF instead of PMA as stimulant [14 ], suggest the existence of at least two mechanisms underlying neutrophil, mAb-dependent cytolysis. In fact, although the PMA and GM-CSF systems share the requirement for Fc{gamma}RII and CD11b-CD18 integrins, only the GM-CSF system involves CD66b glycoproteins and PT-sensitive signaling pathways. These findings, enlightening the molecular mechanisms that govern neutrophil-cytotoxic activities, may provide rational bases for improving mAb-dependent antilymphoma therapies by pharmacological manipulation of effector cell efficiency. In this context, the present data obtained in the PMA system may inspire new attempts for developing novel immunotoxins such as protein kinase activators conjugated with antitarget mAb, including Lym-1 mAb.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from Cancer Therapeutics, Inc., Los Angeles, California, to A.L.E. and by a grant from M.U.R.S.T. to F.D.

Received December 16, 1999; revised April 14, 2000; accepted June 9, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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