Polymorphonuclear Granulocytes Induce Antibody-Dependent Apoptosis in Human Breast Cancer Cells1

Recent studies in HER-2/neu-targeted immunotherapy demonstrated that polymorphonuclear neutrophils (PMN) mediated Ab-dependent cellular cytotoxicity against HER-2/neu-positive breast cancer cell lines. However, the mechanism of cell death remained unclear. We used several assays to analyze the induction of apoptosis in the breast cancer cell line SK-BR-3 via PMN-dependent Ab-dependent cellular cytotoxicity. In the presence of the HER-2/neu Ab 520C9 and PMN from healthy donors, apoptosis occurred as detected by annexin V binding and disappearance of euploid SK-BR-3 nuclei, which can be differentiated from PMN nuclei by their increased DNA contents. Apoptosis induction was observed with E:T cell ratios as low as 10:1. Laser scanning fluorescence microscopy of TUNEL tumor cells or staining for cleaved cytokeratin-18 further confirmed apoptosis of the SK-BR-3 breast cancer cells. Killing via 520C9 was dependent on the interaction with FcR on PMN, because 1) F(ab′)2 fragments of 520C9 mediated no cytotoxicity, 2) target cell death was influenced by a biallelic polymorphism of FcγRIIa on the effector cells, and 3) a bispecific Ab against HER-2/neu and the IgA receptor (FcαRI) expressed on effector cells significantly induced apoptosis. Thus, PMN induce Ab-dependent apoptosis against human breast cancer cells targeted with HER-2/neu-directed mAbs or FcR directed bispecific Abs.

O verexpression of the protooncogene HER-2/neu is observed in approximately one-third of breast cancers and a varying proportion of ovarian, gastric, and other epithelial tumors. HER-2/neu expression is an independent risk factor for early relapse and death in breast cancer (1). The combination of the mAb trastuzumab (Herceptin) directed against HER-2/neu and chemotherapy improved the clinical outcome for patients with HER-2/neu-overexpressing metastatic breast cancers (2). HER-2/ neu forms heterodimers with other members of the epidermal growth factor receptor family, e.g., epidermal growth factor receptor, HER-3 or HER-4 (3). HER-2/neu is a tyrosine kinase signaling molecule and its overexpression results in constitutive and coreceptor-independent kinase activation. We have previously demonstrated that isolated polymorphonuclear neutrophils (PMN) 3 are potent effector cells against a wide range of malignancies in vitro (4 -6). In contrast to T cells, the cytotoxicity of PMN is dependent on the presence of Abs binding to the target cells. Usually, PMNmediated Ab-dependent cellular cytotoxicity (ADCC) is measured using 3-h 51 Cr release assays (7,8) with high E:T cell ratios up to 1000:1 (9). To recruit cell-mediated effector mechanisms, Abs must interact with Ig FcRs, which are classified as Fc␥-, Fc␣-, or Fc⑀-receptors depending on their specificity for IgG, IgA, or IgE, respectively (10). Fc␥R are grouped according to their affinity into low affinity receptors-named Fc␥RII (CD32) and Fc␥RIII (CD16)-and the high affinity Fc␥RI (CD64) (11). In vivo FcRbearing cells like NK cells, monocytes, or PMN are able to infiltrate tumors and to cross-link mAb bound to the surface of tumor cells (12). The myeloid form of Fc␥RII, e.g., the Fc␥RIIa expressed on PMN, contains an immunoreceptor tyrosine-based activation motif in its cytoplasmic domain, whereas the Fc␥RIIb isoforms, e.g., expressed on B cells display immunoreceptor tyrosinebased inhibitory motifs. A genetic polymorphism in the extracellular ligand binding domain of Fc␥RIIa determines reactivity with human IgG2, murine IgG1, and rat IgG2b isotypes (11). The two alleles differ in a single amino acid at position 131. The arginine-containing allotype (R131) displays an increased affinity for murine IgG1, when compared to the allotype with histidine in this position (H131). Binding of ligands to Fc␣RI (CD89), Fc␥RI, and Fc␥RIIIa initiates intracellular signaling by the FcR common ␥-chain, containing immunoreceptor tyrosine-based activation motif. We have recently demonstrated that Fc␣RI triggers PMN-mediated cytotoxicity even better than the established activating molecules Fc␥RI and Fc␥RIIa (13). The pivotal role of FcR for the tumoricidal activities of mAbs has recently been demonstrated in knock-out mice, in which the signaling machinery of FcR was genetically disrupted (14). Recent studies in lymphoma patients showed an association between the Fc␥RIIIA isotype and the clinical and molecular responses to CD20-directed therapy with mAbs (15).
The capacity to mediate ADCC has been demonstrated in vitro for monocytes/macrophages, NK cells, as well as eosinophilic and neutrophilic granulocytes. PMN are increasingly recognized as an important effector cell population for rejection of malignant tumors in vivo (12,16,17). Furthermore, PMN were the predominant effector cell population for the killing of breast cancer cells in the presence of HER-2/neu Abs in vitro (4). However, the mechanism of target cell death remains elusive. Recent therapeutic advances in Ab-mediated HER-2/neu directed cancer therapy have renewed the interest in its mechanism (2).
Apoptotic cell death is characterized by a breakdown of the membrane lipid asymmetry, by the loss of matrix adhesion and mitochondrial membrane potential, by "boiling" of cytoplasm, condensation of chromatin, and finally by internucleosomal cleavage of the nuclear DNA (18). Exposure of phosphatidylserine on the outer leaflet of the cytoplasmic membrane already occurs at an early stage of apoptosis and is detected by the binding of annexin V. In late stages, DNA fragmentation and loss of chromatin result in sub-G 1 DNA content of the dying cells. The latter can be detected by propidium iodide (PI) staining in the presence of detergent. In contrast, the conventional short-term 51 Cr release assay measures release of cytosolic chromate ions, which can only be observed when the cytoplasmic membrane of the target cells are disrupted in either primary or secondary necrosis. The 51 Cr release assay requires high E:T cell ratios, unlikely to occur in vivo. In the present study, we investigated ADCC at low E:T cell ratios. Under these more physiological conditions, we observed an Ab-dependent apoptosis of target cells (ADAC) depending on the presence of both PMN and tumor-specific Abs.

Cells and culture conditions
The human breast cancer cell lines SK-BR-3 and MDA-MB 453 were obtained from the American Type Culture Collection (Manassas, VA). Cells were kept in RF10 ϩ medium consisting of RPMI 1640 (Life Technologies, Paisley, U.K.) supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 U/ml streptomycin, and 3 mM L-glutamine (all from Life Technologies). Experiments reported in this study were in accordance with the Declaration of Helsinki. After informed consent, 10 -20 ml of citrate anticoagulated peripheral blood was drawn from healthy volunteers for effector cell preparation.

Isolation of PMNs and mononuclear cells
Isolated neutrophils were obtained by a method slightly modified from that described in Ref. 19. Briefly, citrate anticoagulated blood was layered over a discontinuous Percoll gradient (Seromed, Berlin, Germany), consisting of 70 and 62% Percoll. After centrifugation, neutrophils were collected between the two Percoll layers, and mononuclear cells from the plasma/ Percoll interface. Remaining erythrocytes were removed by hypotonic lysis. Purity of neutrophils was determined by cytospin preparations and exceeded 95%, with few contaminating eosinophils and Ͻ1% mononuclear cells. Viability of cells tested by trypan blue exclusion was Ͼ95%.

Allotyping for the R-H131 polymorphism of Fc␥RIIa
Phenotyping for the R131 and H131 alleles of the Fc␥RIIa was performed by quantitative indirect flow cytometry of peripheral blood monocytes (26). Cells were stained with mAb 41H16, which selectively recognizes the Fc␥RIIa-R131 alloform, or by mAb AT10 recognizing both Fc␥RIIa alloforms. ADCC 51 Cr release assays were performed as described elsewhere (27). Briefly, target cells were labeled with 200 Ci 51 Cr for 2 h. After extensive washing with RF10 ϩ , cells were adjusted to 10 5 per milliliter. PMN at the indicated E:T cell ratio, sensitizing Abs at a final concentration of 2 g/ml, and RF10 ϩ were added to round-bottom microtiter plates (Nunc, Roskilde, Denmark). Assays were started by adding 50 l of the target cells, resulting in a final volume of 200 l. After 3 h at 37°C, assays were stopped by centrifugation, and 51 Cr release from triplicates was measured. Percentage of cellular cytotoxicity was calculated using the formula: specific lysis ϭ (experimental cpm Ϫ basal cpm/maximal cpm Ϫ basal cpm) ϫ 100%.
Maximal 51 Cr release was determined by adding perchloric acid to target cells at a final concentration of 3%. Basal release was measured in the absence of sensitizing Abs and effector cells. No Ab-independent cytotoxicity was observed with PMN as effector cells in the absence of targeting Abs.
For analyses of ADAC by flow cytofluorometry, freshly isolated PMN in RF10 ϩ were added to 10 5 SK-BR-3 cells to yield an E:T cell ratio of 10:1 in a final volume of 1 ml. Assays were started by addition of mAb or of bispecific Ab at a final concentration of 2 g/ml, respectively. Assays were incubated for 18 h at 37°C in a humidified atmosphere containing 5% CO 2 .

Immunofluorescence analyses
Phosphatidylserine exposure during apoptosis was detected by FITC-labeled annexin V (Roche, Mannheim, Germany), according to the manufacturer's instruction. Necrotic cells were detected by PI uptake. After a 20-min incubation, annexin V binding was measured with an EPICS Profile flow cytometer (Beckman Coulter, Brea, CA). PMN and target cells were distinguished by forward scatter vs side scatter (FSc-SSc) characteristics. Annexin V-positive and PI-negative cells were considered apoptotic.
Nuclear DNA content was analyzed by PI staining after permeabilization of the cytoplasmic membrane with detergent (28). Two to five volumes of PI-Triton staining solution (0.1% sodium citrate, 0.1% Triton X-100, and 1 mg/ml PI) were added to the cell suspension. The DNA content was measured after an incubation at 4°C in the dark for 24 h. Because the nuclei of the SK-BR-3 target cells were hyperdiploid and displayed different morphology compared to PMN, they could easily be differentiated from the nuclei of the PMN by increased PI staining and by characteristic FSc-SSc.

Fluorescence laser scanning microscopy
The in situ end labeling of DNA strand breaks (TUNEL) generated during apoptosis was performed with the In Situ Cell Death Detection Kit, Fluorescein (Roche), according to the manufacturer's instructions for cytospin preparations. A TUNEL reaction mixture without TdT served as a negative control. After ADCC assay for 18 h at 37°C, PMN and SK-BR-3 cells were centrifuged onto microscope slides. Cells were air-dried and fixed with a solution of 4% paraformaldehyde in PBS at pH 7.4 for 30 min. After flushing with PBS, the slides were incubated in 0.1% Triton X-100 with 0.1% sodium citrate on ice for 2 min. The slides were rinsed twice with PBS and the area around the sample was dried. Fifty microliters of the TUNEL reaction mixture was added and the staining was allowed to proceed for 60 min at 37°C in the dark. The slides were washed three times in PBS and were examined by confocal laser scanning microscopy (Bio-Rad, Munich, Germany).

Statistical analysis
Results are expressed as means Ϯ SEM. The statistical significance was calculated with the Student t test (Excel; Microsoft, Richmond, VA).

High E:T cell ratios are required for lysis of SK-BR-3 cells in 51 Cr release assays
HER-2/neu-positive SK-BR-3 breast cancer cells were incubated in the presence of the murine IgG1 mAb 520C9 (2 g/ml) with increasing PMN to target cell ratios. In 3-h 51 Cr release assays, mAb mediated relevant lysis only at E:T ratios above 40:1. Twenty-hour 51 Cr release assays gave similar results (Fig. 1). No significant lysis was to be observed in the absence of either PMN or 520C9 (data not shown).

PMN induce Ab-dependent apoptosis of SK-BR-3 cells
The mechanism of Ab-mediated cell death induced by PMN remains unclear. Therefore, we analyzed the induction of apoptosis in breast cancer cells during ADCC. Apoptosis of the SK-BR-3 cells was measured by binding of Annexin V FITC to target cells after a 20-h incubation with PMN isolated from healthy donors ( Fig. 2A). The assays were run in triplicates and were repeated at least three times. For analysis, cancer cells were gated according to their FSc-SSc characteristics. PI-permeable necrotic cancer cells were excluded from analysis. In the presence of either mAb 520C9 or a bispecific Ab directed against HER-2/neu (expressed by the cancer cells) and Fc␣RI (CD89) (expressed on the effector cells), PMN significantly triggered apoptosis of cancer cells (Fig. 2B). The induction of apoptosis was dependent on the interaction with FcR on effector cells, as F(abЈ) 2 of 520C9 mediated no cytotoxicity. Importantly, we detected significant apoptosis rates already at a low E:T ratio of 10:1.

ADCC results in apoptotic target cells with sub-G 1 DNA contents
Due to their hyperdiploid karyotype, PI-stained G 0 /G 1 and G 2 nuclei of SK-BR-3 cells could easily be distinguished from euploid PMN nuclei (Fig. 3A). During apoptosis induced by irradiation with UV B, chromatin is degraded and apoptotic cells can be identified by their sub-G 1 DNA content. In contrast, induction of necrosis (e.g., by treatment with methanol, ethanol, or heating for 30 min to 56°C) does not generate nuclei with sub-G 1 DNA content (data not shown). In the presence of conventional HER-2/neu-directed mAb or bispecific Ab (Fc␣RI ϫ HER-2/neu), SK-BR-3 cells underwent apoptosis, reflected by the disappearance of target cell nuclei with G 0 / 1 -S-G 2 DNA content (Fig. 3A). F(abЈ) 2 of HER-2/neu-directed mAb or isotype controls had no effect on SK-BR-3 cell death. The latter results demonstrate the involvement of FcR in the induction of apoptosis. In accordance with our previous data obtained with the chromium release assay (13,29), targeting Fc␣RI as cytotoxic trigger molecule on PMN induced significantly more apoptosis than targeting Fc␥R with conventional mAb (Fig.  3B). Similar results were obtained using the HER-/neu positive breast cancer cell line MDA-MB453 (Table I).
In the presence of the murine IgG1 mAb 520C9, PMN of R/R131 individuals were significantly more cytotoxic than those of H/H131 donors Two allelic forms of Fc␥RIIa are expressed on myeloid cells. Because chromium release induced by murine IgG1 has been demonstrated to be more efficient with PMN of R/R131 individuals (4), we examined whether this is also true for the induction of apoptosis. ADAC by PMN from three donors homozygous for Fc␥RIIa-R/R131 or Fc␥RIIa-H/H131 were compared in the presence of the mAb 520C9. As shown in Fig. 4, the viable nuclei count of the tumor cells, measured by PI staining, was significantly ( p Ͻ 0.001) reduced in the presence of PMN isolated from all donors. However, PMN from R/R131 individuals were significantly more cytotoxic than those from H/H131 donors ( p Ͻ 0.001).

TUNEL confirmed apoptosis of breast cancer cells in ADCC
To further confirm apoptotic cell death of the target cells in ADCC, we used the TUNEL method. PMN and SK-BR-3 cells were coincubated for 20 h in the presence or absence of the mAb 520C9. Subsequently, cells were permeabilized and stained with the TUNEL technique. Nuclear counterstaining was performed with PI. Stained cells were analyzed using laser scanning fluorescence microscopy. In the absence of targeting mAb, we detected only background levels of TUNEL-positive SK-BR-3 nuclei. In contrast, in the presence of the mAb 520C9 we observed numerous apoptotic breast cancer cells (Fig. 2C), thus confirming apoptotic death of the tumor cells. PMN could easily be differentiated from the tumor cells by their nuclear morphology. Apoptotic PMN showed bright TUNEL staining, whereas viable PMN were TUNEL-negative. Few contaminating eosinophilic granulocytes showed unspecific dUTP FITC staining (30).

Cytokeratin-18 cleavage confirmed apoptosis of breast cancer cells in ADCC
SK-BR-3 cells were cocultured with PMN at an E:T ratio of 10:1 for 42 h in the presence or absence of HER-2/neu mAb. Adherent cells were stained for a cytoplasmic neoepitope of cytokeratin-18, which is generated by caspase cleavage during late phase of apoptosis of epithelial cells (31). Apoptotic SK-BR-3 cells were almost exclusively observed in the presence of the HER-2/neu mAb. Cytoplasmic staining of apoptotic SK-BR-3 cells could easily be distinguished from rare PMN showing faint, unspecific surface fluorescence (Fig. 2, D-G).

Discussion
The aim of this study was to investigate the mechanism of Abdependent, PMN-mediated cytotoxicity against breast cancer cells. We used flow cytofluorometry and fluorescence microscopy to investigate changes in target cells indicative for apoptotic vs necrotic cell death. Interestingly, we observed annexin V binding to PInegative, apoptotic tumor cells during PMN-mediated ADCC. In addition, analyses of cellular DNA content, TUNEL staining, and detection of specific caspase-cleaved cytokeratin-18 fragments confirmed PMN-induced ADAC in breast cancer cells. Interaction between FcR on PMN and target cells was required for the induction of apoptosis. Conventional Abs targeting Fc␥RII (4), or bispecific Abs targeting Fc␣RI on PMN initiated apoptosis, whereas isotype controls and F(abЈ) 2 of the HER-2/neu Ab had no apoptosis-inducing effect on breast cancer cells. The F(abЈ) 2 of the mAb 520C9 even increased tumor cell survival, most likely due to stimulation of SK-BR-3 due to cross-linking of HER-2/neu molecules.
The polymorphisms of Fc␥RIIa (32, 33), Fc␥RIIb (34), Fc␥RIIIa (15), and Fc␥RIIIb (35,36) have been demonstrated to have clinical implications and functional relevance. In the presence of the murine IgG1 Ab 520C9, apoptosis induction of SK-BR-3 cells was affected by the genetic polymorphism of Fc␥RIIa. Neutrophils isolated from Fc␥RIIa-R/R131 donors induced significantly higher levels of apoptosis than those isolated from Fc␥RIIa-H/H131 donors (Fig. 4). This Fc␥RIIa allotypic polymorphism has previously been shown to be relevant in several types of assays involving murine IgG1 Abs. These include anti-CD3-induced T FIGURE 2. Induction of apoptosis in breast cancer cells after coculture with PMN in the presence of HER-2/neu-specific mAb. Annexin V binding of breast cancer cells after coculture with PMN in the presence of HER-2/neu-specific mAb. SK-BR-3 cells and isolated PMN were incubated with the indicated Abs for 20 h. In the presence of the mAb against HER-2/neu or the bispecific Ab targeting HER-2/neu and Fc␣RI, PMN mediated apoptosis of breast cancer cell line SK-BR-3 (A). Killing via 520C9 was specific and dependent on the interaction with FcR on effector cells, as a murine isotype control mAb or F(abЈ) 2 of 520C9 mediated no significant apoptosis, respectively (B). One representative of at least three similar experiments is shown; error bars show SD of triplicates of a representative cell proliferation, phagocytosis of IgG-coated erythrocytes (37), and HER-2/neu Ab-dependent 51 Cr release with murine IgG1 Ab (4). Thus, the efficacy of murine IgG1 Abs in tumor therapy may also depend on the Fc␥RIIa allotype of the patients' effector cells. Interestingly, apoptosis was seen with E:T cell ratios as low as 10:1, which may reasonably occur in vivo. Targeting HER-2/neu, invasion of PMN into breast cancer metastases has been demonstrated in a phase I clinical trial (38).
However, the mechanism of apoptosis induction by neutrophils remains elusive. It is still unclear whether PMN start an intrinsic cell death program in the target cells, or whether, in analogy to the killing by cytotoxic T cells and NK cells, the release of the contents of neutrophils into the target cells is required. Recently, complement-mediated apoptosis of malignant B cell lines, opsonized with CD20-directed mAb, has been demonstrated in the absence of effector cells (39). Human neutrophils contain a rapidly mobilizable pool of so-called secretory vesicles, which are distinct from the azurophilic and specific granules. They contain plasma-derived proteins (40), e.g. albumin, and are referred to as "easily mobilizable vesicles." These secretory vesicles presumably also contain complement. Because PMN do not contain perforin, one may speculate that complement molecules released from secretory vesicles may permeabilize the cell membrane of target cells. This may enable the penetration of apoptosis-inducing enzymes into the cy-tosol of the target cells. PMN are continuously in a preapoptotic state (41). Therefore, apoptosis-inducing molecules, e.g., activated caspases, may be transferred into the target cell following cell surface contacts, and cytosolic communication after pore-forming or membrane fusion. Alternatively, an intrinsic cell death program, triggered by the engagement of cell surface molecules of the target cells, may operate.
At high E:T cell ratios, as required for chromium release assays, cells are struck with such a strong impact that energy supply quickly ceases, repair function declines, and necrosis ensues. However, less impact, e.g., at low E:T cell ratios, is sufficient to initiate apoptosis. However, the execution of the energy-consuming death program takes time. We conclude that induction of apoptosis and necrosis both contribute to Ab-dependent killing by PMN of target cells.
In the context of biological tumor therapy, induction of tumor cell apoptosis seems to be profitable for the host. Nevertheless, nonopsonized apoptotic cells have been described to exert potent immunosuppressive effects (42). Mice immunized with apoptotic tumor cells mounted a significantly reduced humoral immune response when compared with animals injected with viable tumor cells (43). Therefore, the generation of huge amounts of apoptotic cells may suppress the immune response of the host, thereby enabling the immune escape of the tumors. However, in these experiments, apoptosis was induced by irradiation with UV B in nonimmunized animals, whereas in the present study, tumor cell apoptosis was induced by the concerted action of PMN and tumorspecific mAb via ADAC. Therefore, opsonization with Abs of tumor cells, may target the dying cells to FcR and complementreceptor mediated phagocytosis by professional APC, e.g., dendritic cells. Additionally, the activation of PMN during target cell apoptosis induction may herald a "danger signal" (44) for the immune system. In accordance with this hypothesis, PMN have been demonstrated to be critical in some animal models of tumor rejection (12). Surprisingly, depletion of T cells in a murine model of tumor therapy with Fc␥RI-directed bispecific Abs recognizing the idiotype of a B cell lymphoma resulted in an complete loss of efficacy (17). In conclusion, we demonstrated that PMN are able to kill tumor cell targets via Ab-dependent apoptosis induction. The exact mechanism of this apoptosis pathway and its role during tumor immune responses remain to be defined. a After 20 h of coculture with PMN in the presence of the indicated Ab, staining with PI was performed as described in Materials and Methods. Her-2/neu mAb was 520C9 (murine IgG1), (Fc␣RI ϫ HER-2) was generated from A77 and Herceptin, respectively. Assays were performed in quadruplicate; one of three similar experiments is shown. p for comparison with no mAb. b The number Ϯ SD of MDA-MB 453 nuclei with G 0 / 1 or G 2 nuclear content is shown.
c The percentage Ϯ SD of MDA-MB 453 nuclei with G 0 / 1 or G 2 nuclear content is shown.

FIGURE 4.
The R/H131 phenotype of the Fc␥RIIa on PMN effector cells influences apoptosis induction via murine IgG1 mAb 520C9 directed against HER-2/neu. The number of viable nuclei as measured by PI staining was significantly reduced in the presence of isolated PMN from donors homozygous for H131 or R131 of Fc␥RIIa on PMN (marked by ‫,ء‬ denoting p Ͻ 0.001). However, the PMN from R131/R131 individuals were significantly more efficient in apoptosis induction than effector cells from H/H131 donors (marked by #, denoting p Ͻ 0.001). Data represent mean Ϯ SEM of triplicates, one representative of three similar experiments is shown (30).