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Keywords:

  • Bispecific antibody;
  • Chemotaxis immunotherapy;
  • HUVEC;
  • IgA

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. Conflict of interest
  8. References

Neutrophils potently kill tumour cells in the presence of anti-tumour antibodies in vitro. However, for in vivo targeting, the neutrophils need to extravasate from the circulation by passing through endothelial barriers. To study neutrophil migration in the presence of endothelial cells in vitro, we established a three-dimensional collagen culture in which SK-BR-3 tumour colonies were grown in the presence or absence of an endothelial barrier. We demonstrated that — in contrast to targeting FcγR on neutrophils with mAbs — targeting the immunoglobulin A Fc receptor (FcαRI) instead triggered neutrophil migration and degranulation leading to tumour destruction, which coincided with release of the pro-inflammatory cytokines interleukin (IL)-1β and tumour necrosis factor (TNF)-α. Interestingly, neutrophil migration was enhanced in the presence of endothelial cells, which coincided with production of significant levels of the neutrophil chemokine IL-8. This supports the idea that stimulation of neutrophil FcαRI, but not FcγR, initiates cross-talk between neutrophils and endothelial cells, leading to enhanced neutrophil migration towards tumour colonies and subsequent tumour killing.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. Conflict of interest
  8. References

Neutrophils represent the most populous type of cytotoxic effector cells within the blood and their numbers can easily be increased by treatment with granulocyte colony-stimulating factor (G-CSF) [1]. Because depletion of these cells resulted in increased tumour outgrowth in animal models, neutrophils may play a role in tumour rejection in vivo [2-4]. It is also becoming increasingly clear that neutrophils secrete a plethora of cytokines and chemokines that can attract other immune cells, such as monocytes, dendritic cells and T cells [5], which may result in more generalised anti-tumour immune responses. For instance, it was recently demonstrated that neutrophils attract Th17 cells [6], which have been shown to play a role in anti-tumour immunity [7].

Importantly, anti-tumour monoclonal antibodies (mAbs) or bispecific Abs (BsAbs) — which link Fc receptors on immune cells and tumour-associated antigens (TAAs) on tumour cells — enhance neutrophil-mediated tumour cell lysis [8-10]. Initially, the immunoglobulin (Ig) G receptor FcγRI was proposed as a potent target for initiation of neutrophil-induced Ab-mediated tumour cell lysis. In recent years, however, it was demonstrated that targeting the IgA Fc receptor (FcαRI) resulted in more effective neutrophil-mediated Ab-dependent tumour cell lysis [11-19]. Furthermore, killing was initiated through non-apoptotic pathways, which coincided with autophagic characteristics [20]. Moreover, triggering of FcαRI induced recruitment of neutrophils into tumour colonies [9]. We recently demonstrated that IgA induced significant release of the neutrophil chemoattractant leukotriene B4 (LTB4) [21]. Thus, neutrophils represent interesting effector cells for Ab immunotherapy of cancer. However, in order to achieve Ab-mediated lysis of solid tumours in vivo, neutrophils need to extravasate from the circulation into the tumour. Therefore, we now investigated Ab-induced neutrophil migration towards tumour colonies in the presence of an endothelial cell barrier.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. Conflict of interest
  8. References

Targeting FcαRI induces migration, degranulation and release of pro-inflammatory cytokines

Neutrophils were previously demonstrated to induce Ab-dependent killing, which resulted in tumour cell elimination [8, 9, 11-13, 16, 17, 19, 22]. Moreover, FcαRI proved a more potent trigger molecule, as compared with targeting FcγRs [9, 13, 15]. Interestingly, we recently demonstrated that cross-linking of neutrophil FcαRI by IgA resulted in release of LTB4, which is a potent neutrophil chemoattractant [21]. As such, rapid migration of neutrophils was observed towards the site of the IgA-immune complexes. Similarly, when we added an FcαRIxHer-2/neu BsAb to a 3D culture of tumour cells in collagen, we observed massive neutrophil migration towards tumour colonies within 2 h (Fig. 1A). At this time point only minimal degranulation was observed (reflected by lactoferrin release, Fig. 1B). However, neutrophil degranulation increased over time in cultures in which FcαRIxHer-2/neu BsAb had been added.

image

Figure 1. Targeting FcαRI induces migration, degranulation and release of pro-inflammatory cytokines. (A) Neutrophils were added to SK-BR-3 tumour colonies (blue) in collagen, either in the absence (panels I and IV), or presence of an anti-HER-2/neu IgGAb (panels II and V) or an FcαRIxHer-2neu BsAb (panels III and VI), magnification 10×. Representative tumour colonies are shown in inset (40×, scale bar 100 μM). Supernatants of collagen cultures were first harvested and used in (B–E), subsequently cultures were fixed for 2 or 24 h after addition of the neutrophils and slides were stained for CD66b (neutrophils, brown). One representative example out of eight is shown. Neutrophil infiltration/colony/section after 24 h was counted. The graph represents fold increase of neutrophil infiltration/colony/section of three independent experiments (mean ± SEM). The number of neutrophils in the presence of anti-Her-2/neu IgGAb was set at 1. (B–E) Supernatants were collected from various SK-BR-3 collagen gels incubated as described in (A) for indicated time points. (B) Lactoferrin release after the indicated time points was determined. (C) After 24 h of incubation, supernatants from all conditions were tested for chemotactic capacity in a Boyden Chamber assay. (D) Supernatants of FcαRIxHer-2/neu BsAb-treated gels were tested for their chemotactic ability in a Boyden Chamber assay in the absence or presence of an anti-BLTR1 antibody (LTB4 receptor blocking antibody). (E) Twenty-four-hour supernatants of indicated collagen gels were analysed for IL-1β and TNF-α, one representative experiment of three is shown. (F) Neutrophils were added to A431 tumour colonies in collagen, either in the absence (no Ab) or presence of an anti-EGFR-IgA mAb. The collagen cultures were fixed 24 h after the addition of neutrophils and the slides were stained with Mayers’ haematoxylin. Numbers represent neutrophil infiltration/colonies/section (magnification 10×. Insets show magnification (40×, scale bar 100 μM) of one representative tumour colony. (B–E) Data are mean ± SD of triplicates. Statistical differences were determined using two-tailed unpaired Student's t-tests (two groups) or ANOVA (more than two groups), followed by Bonferroni post hoc tests. *p < 0.05; **p < 0.01.

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We previously showed in a 2D culture system that incubation of SK-BR-3 cells and neutrophils in the presence of an FcαRIxHer-2/neu BsAb resulted in tumour cell death [20]. Although we formally cannot show tumour cell killing in our 3D collagen cultures, the integrity of tumour colonies was clearly affected after 24 h incubation with neutrophils and FcαRIxHer-2/neu BsAb (Fig. 1A, panel VI; inset). Chemotactic activity was only observed in the supernatants of cultures in which FcαRIxHer-2/neu BsAb had been added, which was decreased in the presence of a blocking anti-BLTR1 mAb (Fig. 1C and D). This suggested that the observed rapid neutrophil migration was the result of LTB4 release after triggering of FcαRI [21]. Additionally, release of the pro-inflammatory cytokines IL-1β and TNF-α was observed (Fig. 1E). Minimal neutrophil migration and minimal lactoferrin release was observed in the absence of an antibody or in the presence of an anti-HER-2/neu IgG mAb (Fig. 1A and B), even though the experiments were performed with interferon-γ stimulated neutrophils that express FcγRI.

To confirm that tumour colony destruction in the presence of neutrophils and an FcαRIxHer-2/neu BsAb was neither dependent on tumour cell type nor TAA, we also performed experiments with A431 cells. These cells have a high expression of epidermal growth factor receptor (EGFR). No intact tumour colonies were observed after culturing A431 colonies for 24 h in the presence of anti-EGFR IgA mAb (Fig. 1F). Only neutrophils and debris were observed, strongly supporting that tumour cells had been destroyed in our 3D culture system (Fig. 1F, upper panel; inset). Similarly, massive neutrophil migration was observed in 3D collagen assays with SW-948 colon carcinoma tumour colonies in the presence of an anti-EpCAM IgA mAb [23]. Of note, the initial contact of neutrophils with tumour cells was presumably at random. However, when IgA mAbs or FcαRI BsAbs are available, a positive feedback neutrophil migration loop is initiated, which will not occur in the absence of mAbs or in the presence of IgG mAbs [21].

Signalling through either FcαRI or FcγR depends on an association with the FcR γ-chain that bears immunoreceptor tyrosine-based activation motifs (ITAMs) [22, 24]. Tethering the FcαRI and FcR γ-chain into a stable FcαRI–FcR γ-chain complex involves several other aspects, including crucial electrostatic interactions that are absent in FcγRI/FcR γ-chain interactions [9, 22, 24-28]. Furthermore, it was demonstrated that signalling through FcαRI is enhanced as compared with FcγRI [9, 21, 28]. FcγRIIa, which is the major FcγR expressed by unstimulated neutrophils, bears a unique ITAM in its cytoplasmatic tail that initiates signalling pathways [29]. However, the FcγRIIa-ITAM does not mediate cytokine release [29]. As such, signalling through FcγR is either lower as compared with that through FcαRI or induces dissimilar functions, which likely account for the observed differences in neutrophil migration and activation. This presumably also underlies the enhanced tumour cell killing after targeting FcαRI.

Endothelial cells amplify FcαRI-induced neutrophil migration

In vivo, neutrophils need to extravasate from the bloodstream in order to enter tumours. We therefore investigated neutrophil migration in the presence of endothelial cells. HUVECs were grown as confluent monolayers on top of collagen gels that contained SK-BR-3 colonies. The presence of HUVECs increased neutrophil entry into collagen gels in either the absence or presence of antibody (Fig. 2A and B). This was not due to augmented acceleration of neutrophil migration, but the result of increased neutrophil infiltration (Fig. 2B). In the absence of antibody or in the presence of an anti-HER-2/neu IgG mAb, migration was random and no interaction with tumour colonies was observed. Addition of an FcαRIxHER-2/neu BsAb, however, resulted in massive neutrophil infiltration into tumour colonies (Fig. 2A, panel III compared with Fig. 1A panel VI). Based on the results in our 3D collagen culture experiments, we cannot conclude that enhanced neutrophil accumulation into tumour colonies also led to enhanced tumour destruction. However, previous in vitro studies demonstrated that increased effector to target ratios resulted in increased tumour cell killing by neutrophils [8, 10].

image

Figure 2. Endothelial cells amplify FcαRI-induced neutrophil migration. (A) Neutrophils (brown) were added for 24 h to HUVEC-layered collagen gels containing SK-BR-3 tumour colonies (blue), either in the absence (panel I), or presence of an anti-HER-2/neu IgGAb (panel II) or an FcαRIxHer-2neu BsAb (panel III), magnification 10×. Insets show magnification (40×, scale bar 100 μM) of representative tumour colonyies. (B) Chemotactic potential (one representative experiment of three is shown) and (C) IL-8 concentration were determined in supernatants of SK-BR-3 collagen gels, cultured without or with HUVEC monolayers in the absence or presence of the indicated Abs for the indicated time points. IL-8 data are presented as mean ± SD of triplicates, one representative experiment of three is shown. (D) HUVECs were stimulated with supernatants that had been harvested from SK-BR-3 collagen gels, to which neutrophils and an FcαRIxHer-2neu BsAb had been added, after which IL-8 production was measured (black bar). IL-8 concentration in the harvested supernatant, used to stimulate HUVECs, is depicted as the white bar, mean ± SD of triplicates is shown.

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It was demonstrated that TNF-α acts not only as a chemo-attractant for neutrophils, but also induces IL-8 production by endothelial cells, which is the prototypic neutrophil chemokine [5]. We therefore tested IL-8 concentrations in supernatants of the collagen cultures. In the presence of FcαRIxHer-2/neu BsAb, low amounts of IL-8 were detected in the absence of HUVECs (Fig. 2C). However, the IL-8 concentration was profoundly amplified in the presence of HUVECs and an FcαRIxHER-2/neu BsAb, supporting the idea that HUVECs produced IL-8 after activation by neutrophils. No IL-8 was detected in the supernatant of collagen cultures in which an anti-Her-2/neu IgG mAb had been added (data not shown). To confirm IL-8 production by HUVECs in resp-onse to factors that had been secreted by activated neutrophils, we cultured HUVEC monolayers in the presence of supernatant that had been harvested from collagen cultures in which SK-BR-3 colonies had been incubated with neutrophils and an FcαRIxHer-2/neu BsAb (in the absence of HUVECs). Although minimal IL-8 levels were detected in the harvested supernatant, the IL-8 concentration increased when this supernatant was added to HUVEC monolayers, indicating IL-8 production by HUVECs (Fig. 2D). Interestingly, the peak of neutrophil migration was observed after 4 h, at which time hardly any IL-8 release was found (Fig. 2B and C). IL-8 therefore does not appear to play a major role in our in vitro experiments, but migration is likely due to release of LTB4 after targeting FcαRI (Fig. 1D and [21]). LTB4 not only acts as chemoattractant, but also affects the vascular permeability of endothelial cells and transendothelial neutrophil migration [30, 31]. Furthermore, IL-1β and TNF-α (which are also released after FcαRI triggering) are also known to up-regulate BLT receptors on HUVECs with concomitantly enhanced LTB4-mediated responses, such as vascular permeability and transendothelial neutrophil migration [32].

Taken together, targeting FcαRI on neutrophils resulted in release of LTB4, which acted as the major chemoattractant for neutrophil migration. Additionally, release of lactoferrin was observed, reflecting neutrophil degranulation, which resulted in tumour cell killing. IL-8 production was furthermore significantly increased in the presence of endothelial cells, which was due to endothelial cell activation by inflammatory mediators that had been released by neutrophils after activation. Neutrophils can secrete a plethora of different factors, including TNF-α and IL-1β after FcαRI triggering [5]. IL-8 production by HUVECs, which was observed after 24 h, did not, however, contribute to enhanced neutrophil migration in our in vitro cultures, which is likely due to the short half-life of neutrophils in vitro (<24 h). However, IL-8 production by endothelial cells may contribute to amplified migration in vivo, as this is not limited by the short half-life of isolated neutrophils. Thus, in order to recruit neutrophils during antibody immunotherapy of cancer, it is preferable to target FcαRI, as compared with FcγR. Only FcαRI mediates the release of chemoattractants, migration towards tumour colonies and tumour destruction. Moreover, through release of pro-inflammatory mediators, FcαRI may trigger a paracrine amplification loop between neutrophils and endothelial cells, which may contribute to more effective tumour elimination by increased vascular permeability and enhanced numbers of infiltrating neutrophils in vivo (Fig. 3). As such, IgA mAbs that target FcαRI on neutrophils may represent an attractive alternative to IgG therapeutic mAbs.

image

Figure 3. Proposed model for transendothelial neutrophil migration induced by FcαRI. Neutrophils will release LTB4 after binding to tumour cells in the presence of an FcαRIBsAb or an IgA mAb [21], which leads to recruitment of more neutrophils, and tumour cell killing (left panel). Furthermore, endothelial cells will be activated by the inflammatory mediators that are released by neutrophils (such as TNF-α and IL-1β) . Activated endothelial cells will respond by producing IL-8, which may further amplify neutrophil recruitment (right panel). Furthermore, TNF-α and IL-1β influence vascular permeability and neutrophil transmigration [32].

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Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. Conflict of interest
  8. References

Antibodies

Antibodies A77 (mIgG1 anti-FcαRI) and 520C9 (mIgG1 anti-HER-2/neu) were isolated from hybridomas (Medarex, Bloomsbury, NJ, USA). FcαRIxHER-2/neu BsAb (A77×520C9) were produced by chemically cross-linking F(ab′) fragments of 520C9 with F(ab′) fragments of A77 as described [33]. Anti-EGFR IgA mAb was a kind gift of Prof. Dr. T. Valerius (University of Kiel, Germany). Anti-BLTR1 (receptor for LTB4) mAb was obtained from BD Biosciences, Franklin Lakes, NJ, USA.

Cell lines

The mamma carcinoma cell line SK-BR-3 overexpresses the TAA Human Epidermal Growth Factor Receptor 2 (HER-2/neu, also referred to as HER-2 or ErbB-2). Her-2/neu is encoded by the proto-oncogene ERBB2, and is overexpressed in ∼30% of mamma carcinomas. SK-BR-3 cells were cultured in RPMI 1640 medium (Gibco BRL, Paisley, UK), supplemented with 10% FCS and antibiotics and harvested using trypsin-EDTA (Gibco BRL). Human epithelial carcinoma A431 cells were cultured in DMEM (Gibco BRL), supplemented with 10% FCS and antibiotics. The TAA on A431 cells was EGFR (also known as HER-1).

Isolation of human neutrophils

Standard Lymphoprep (Axis-Shield, Rodelokka Oslo, Norway) density gradient centrifugation was used to isolate neutrophils from heparin anti-coagulated peripheral blood samples from healthy volunteers as described [9]. All donors gave informed consent, according to the guidelines of the Medical Ethical Committee of the VUmc (The Netherlands), in agreement with the Declaration of Helsinki.

Isolation of human umbilical vein endothelial cells (HUVECs)

Blood was flushed out of umbilical cords with cordbuffer (containing 0.298 g/L KCL, 8.182 g/L NaCl, 2.621 g/L HEPES and 2.178 g/L D-glucose), after which they were incubated for 20 min at 37°C with 3350 U collagenase (diluted in M199 medium, Gibco BRL). Endothelial cells were harvested, and cultured in endothelial medium (M199 medium, supplemented with 10% pooled human serum, 10% newborn calf serum, 5 ng/mL basic fibroblast growth factor (bFGF), 5 U/mL heparin, glutamin and antibiotics) in 6-well plates (NUNC GmbH, Wiesbaden, Germany) that had been coated for 30 min at 37°C with 1% gelatine. Endothelial cell cultures that had grown confluently were harvested with trypsin-EDTA.

Neutrophil migration assays and immunohistochemistry

Three-dimensional collagen assays and stainings were performed as described [9]. Supernatants were collected for further analyses. For experiments with HUVECs, collagen gels were first cultured for 2 weeks to allow tumour colony formation, after which RPMI/10% supplemented with 10 ng/mL bFGF and 10 U/mL heparin was added for 24 h. HUVECs were added, and formed a confluent layer in 20 h, after which neutrophils and Ab were added.

Chemotaxis assay (Boyden Chamber assay) and ELISA

To measure chemotaxis (specific neutrophil migration) a Boyden Chamber assay was used as described before [34] Fluor-escence was measured in a fluorimeter (excitation wavelength 485 nm/emission wavelength at 520 nm). Lactoferrin ELISA was performed as described [9]. IL-1β, TNF-α and IL-8 ELISA were performed according the manufacture's instructions (Biosource, Camarillo, CA, USA).

Statistical analyses

Data are shown as mean ± standard deviation (SD) or shown as mean ± standard error of the mean (SEM) as indicated. Statistical differences were determined using two-tailed unpaired Student's t-tests (two groups) or ANOVA (more than two groups), followed by Bonferroni post hoc tests. *p < 0.05; **p < 0.01.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. Conflict of interest
  8. References

This work was supported by the Dutch Cancer Society (UU2001-2431), Stichting VUmc Cancer Center Amsterdam and the Netherlands Organization for Scientific Research (VENI 916.36.079, M.A Otten and VIDI 016.086.320, J.E. Bakema).

Conflict of interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. Conflict of interest
  8. References

The authors declare no financial or commercial conflicts of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. Conflict of interest
  8. References
  • 1
    Lieschke, G. J. and Burgess, A. W., Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor (2). N. Engl. J. Med. 1992. 327: 99106.
  • 2
    Lichtenstein, A. and Kahle, J., Anti-tumor effect of inflammatory neutrophils: characteristics of in vivo generation and in vitro tumor cell lysis. Int. J. Cancer 1985. 35: 121127.
  • 3
    Midorikawa, Y., Yamashita, T. and Sendo, F., Modulation of the immune response to transplanted tumors in rats by selective depletion of neutrophils in vivo using a monoclonal antibody: abrogation of specific transplantation resistance to chemical carcinogen-induced syngeneic tumors by selective depletion of neutrophils in vivo. Cancer Res. 1990. 50: 62436247.
  • 4
    Seino, K., Kayagaki, N., Okumura, K. and Yagita, H., Antitumor effect of locally produced CD95 ligand. Nat. Med. 1997. 3: 165170.
  • 5
    Mantovani, A., Cassatella, M. A., Costantini, C. and Jaillon, S., Neutrophils in the activation and regulation of innate and adaptive immunity. Nat. Rev. Immunol. 2011. 11: 519531.
  • 6
    Pelletier, M., Maggi, L., Micheletti, A., Lazzeri, E., Tamassia, N., Costantini, C., Cosmi, L. et al., Evidence for a cross-talk between human neutrophils and Th17 cells. Blood 2010. 115: 335343.
  • 7
    Martin-Orozco, N., Muranski, P., Chung, Y., Yang, X. O., Yamazaki, T., Lu, S., Hwu, P. et al., T helper 17 cells promote cytotoxic T cell activation in tumor immunity. Immunity 2009. 31: 787798.
  • 8
    Valerius, T., Wurflein, D., Stockmeyer, B., Repp, R., Kalden, J. R. and Gramatzki, M., Activated neutrophils as effector cells for bispecific antibodies. Cancer Immunol. Immunother 1997. 45: 142145.
  • 9
    Otten, M. A., Rudolph, E., Dechant, M., Tuk, C. W., Reijmers, R. M., Beelen, R. H., van de Winkel, J. G. et al., Immature neutrophils mediate tumor cell killing via IgA but not IgGFc receptors. J. Immunol. 2005. 174: 54725480.
  • 10
    Stockmeyer, B., Valerius, T., Repp, R., Heijnen, I. A., Buhring, H. J., Deo, Y. M., Kalden, J. R. et al., Preclinical studies with Fc(gamma)R bispecific antibodies and granulocyte colony-stimulating factor-primed neutrophils as effector cells against HER-2/neu overexpressing breast cancer. Cancer Res. 1997. 57: 696701.
  • 11
    Valerius, T., Stockmeyer, B., van Spriel, A. B., Graziano, R. F., van den Herik-Oudijk, I. E., Repp, R., Deo, Y. M. et al., FcalphaRI (CD89) as a novel trigger molecule for bispecific antibody therapy. Blood 1997. 90: 44854492.
  • 12
    Deo, Y. M., Sundarapandiyan, K., Keler, T., Wallace, P. K. and Graziano, R. F., Bispecific molecules directed to the Fc receptor for IgA (Fc alpha RI, CD89) and tumor antigens efficiently promote cell-mediated cytotoxicity of tumor targets in whole blood. J. Immunol. 1998. 160: 16771686.
  • 13
    Stockmeyer, B., Dechant, M., van Egmond, M., Tutt, A. L., Sundarapandiyan, K., Graziano, R. F., Repp, R. et al., Triggering Fc alpha-receptor I (CD89) recruits neutrophils as effector cells for CD20-directed antibody therapy. J. Immunol. 2000. 165: 59545961.
  • 14
    Keler, T., Wallace, P. K., Vitale, L. A., Russoniello, C., Sundarapandiyan, K., Graziano, R. F. and Deo, Y. M., Differential effect of cytokine treatment on Fc alpha receptor I- and Fc gamma receptor I-mediated tumor cytotoxicity by monocyte-derived macrophages. J. Immunol. 2000. 164: 57465752.
  • 15
    Dechant, M. and Valerius, T., IgA antibodies for cancer therapy. Crit. Rev. Oncol. Hematol. 2001. 39: 6977.
  • 16
    Dechant, M., Vidarsson, G., Stockmeyer, B., Repp, R., Glennie, M. J., Gramatzki, M., van De Winkel, J. G. et al., Chimeric IgA antibodies against HLA class II effectively trigger lymphoma cell killing. Blood 2002. 100: 45744580.
  • 17
    Dechant, M., Beyer, T., Schneider-Merck, T., Weisner, W., Peipp, M., van de Winkel, J. G. and Valerius, T., Effector mechanisms of recombinant IgA antibodies against epidermal growth factor receptor. J. Immunol. 2007. 179: 29362943.
  • 18
    Zhao, J., Kuroki, M., Shibaguchi, H., Wang, L., Huo, Q., Takami, N., Tanaka, T. and Kinugasa, T., Recombinant human monoclonal IgA antibody against CEA to recruit neutrophils to CEA-expressing cells. Oncol. Res. 2008. 17: 217222.
  • 19
    Lohse, S., Derer, S., Beyer, T., Klausz, K., Peipp, M., Leusen, J. H., van de Winkel, J. G. et al., Recombinant dimeric IgA antibodies against the epidermal growth factor receptor mediate effective tumor cell killing. J. Immunol. 2011. 186: 37703778.
  • 20
    Bakema, J. E., Ganzevles, S. H., Fluitsma, D. M., Schilham, M. W., Beelen, R. H., Valerius, T., Lohse, S. et al., Targeting Fc{alpha}RI on polymorphonuclear cells induces tumor cell killing through autophagy. J. Immunol. 2011.
  • 21
    van der Steen, L., Tuk, C. W., Bakema, J. E., Kooij, G., Reijerkerk, A., Vidarsson, G., Bouma, G. et al., Immunoglobulin A: Fc(alpha)RI interactions induce neutrophil migration through release of leukotriene B4. Gastroenterology 2009. 137: 20182029, e2011–e2013.
  • 22
    van Egmond, M., van Vuuren, A. J., Morton, H. C., van Spriel, A. B., Shen, L., Hofhuis, F. M., Saito, T., Mayadas et al., Human immunoglobulin A receptor (FcalphaRI, CD89) function in transgenic mice requires both FcR gamma chain and CR3 (CD11b/CD18). Blood 1999. 93: 43874394.
  • 23
    Bakema, J. E. and van Egmond, M., Immunoglobulin A: A next generation of therapeutic antibodies? MAbs 2011. 3: 352361.
  • 24
    Morton, H. C., van den Herik-Oudijk, I. E., Vossebeld, P., Snijders, A., Verhoeven, A. J., Capel, P. J. and van de Winkel, J. G., Functional association between the human myeloid immunoglobulin A Fc receptor (CD89) and FcR gamma chain. Molecular basis for CD89/FcR gamma chain association. J. Biol. Chem. 1995. 270: 2978129787.
  • 25
    Wines, B. D., Trist, H. M., Monteiro, R. C., Van Kooten, C. and Hogarth, P. M., Fc receptor gamma chain residues at the interface of the cytoplasmic and transmembrane domains affect association with FcalphaRI, surface expression, and function. J. Biol. Chem. 2004. 279: 2633926345.
  • 26
    Wines, B. D., Trist, H. M., Ramsland, P. A. and Hogarth, P. M., A common site of the Fc receptor gamma subunit interacts with the unrelated immunoreceptors FcalphaRI and FcepsilonRI. J. Biol. Chem. 2006. 281: 1710817113.
  • 27
    Bakema, J. E., de Haij, S., den Hartog-Jager, C. F., Bakker, J., Vidarsson, G., van Egmond, M., van de Winkel, J. G. et al., Signaling through mutants of the IgA receptor CD89 and consequences for Fc receptor gamma-chain interaction. J. Immunol. 2006. 176: 36033610.
  • 28
    Otten, M. A., Leusen, J. H., Rudolph, E., van der Linden, J. A., Beelen, R. H., van de Winkel, J. G. and van Egmond, M., FcR gamma-chain dependent signaling in immature neutrophils is mediated by FcalphaRI, but not by FcgammaRI. J. Immunol. 2007. 179: 29182924.
  • 29
    Van den Herik-Oudijk, I. E., Capel, P. J., van der Bruggen, T. and Van de Winkel, J. G., Identification of signaling motifs within human Fc gamma RIIa and Fc gamma RIIb isoforms. Blood 1995. 85: 22022211.
  • 30
    Nohgawa, M., Sasada, M., Maeda, A., Asagoe, K., Harakawa, N., Takano, K., Yamamoto, K. et al., Leukotriene B4-activated human endothelial cells promote transendothelial neutrophil migration. J. Leukoc. Biol. 1997. 62: 203209.
  • 31
    Di Gennaro, A., Kenne, E., Wan, M., Soehnlein, O., Lindbom, L. and Haeggstrom, J. Z., Leukotriene B4-induced changes in vascular permeability are mediated by neutrophil release of heparin-binding protein (HBP/CAP37/azurocidin). Faseb. J. 2009. 23: 17501757.
  • 32
    Qiu, H., Johansson, A. S., Sjostrom, M., Wan, M., Schroder, O., Palmblad, J. and Haeggstrom, J. Z., Differential induction of BLT receptor expression on human endothelial cells by lipopolysaccharide, cytokines, and leukotriene B4. Proc. Natl. Acad. Sci. USA 2006. 103: 69136918.
  • 33
    Fanger, M. W., Ball, E. D. and Drakeman, D. L., Comments on the Fourth International Conference on Bispecific Antibodies and Cellular Cytotoxicity, Duck Key, Florida, March 1–5, 1995. J. Hematother. 1995. 4: 345349.
  • 34
    Frevert, C. W., Wong, V. A., Goodman, R. B., Goodwin, R. and Martin, T. R., Rapid fluorescence-based measurement of neutrophil migration in vitro. J. Immunol. Methods 1998. 213: 4152.
Abbreviations
ADCC

Ab-dependent cellular cytotoxicity

BsAb

bi-specific Ab

LTB4

leukotriene B4

TAA

tumour-associated antigen