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The Journal of Immunology, 2001, 167: 4719-4728.
Copyright © 2001 by The American Association of Immunologists

Apoptosis-Inducing Human-Origin Fc{epsilon}-Bak Chimeric Proteins for Targeted Elimination of Mast Cells and Basophils: A New Approach for Allergy Treatment1

Ruth Belostotsky and Haya Lorberboum-Galski2

Department of Cellular Biochemistry and Human Genetics, Hebrew University, Hadassah Medical School, Jerusalem, Israel


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During the past few years, many chimeric proteins have been developed to specifically target and kill cells expressing specific surface molecules. Generally these molecules carry a bacterial or plant toxin to destroy the unwanted cells. The major obstacle regarding these molecules in their clinical application is the immunogenicity and nonspecific toxicity associated with bacterial or plant toxins. We lately reported a new approach for construction of chimeric proteins: we successfully replaced bacterial or plant toxins with human apoptosis-inducing proteins. The resulting chimeras were shown to specifically induce apoptosis in the target cells. Taking advantage of the human apoptosis inducing proteins Bak and Bax as novel killing components, we have now constructed new chimeric proteins targeted against the human Fc{epsilon}RI, expressed mainly on mast cells and basophils. These cells are the main effectors of the allergic response. Treatment of the target cells with the new chimeric proteins, termed Fc{epsilon}-Bak/Bax, had a dramatic effect on cell survival, causing apoptosis. The effect was specific to cells expressing the Fc{epsilon}RI of both human and, very unexpectedly, also of mouse origin. Moreover, interaction of the chimeric proteins with the mast cells did not cause degranulation. Fc{epsilon}-Bak/Bax are new chimeric proteins of human origin and, as such, are expected to be both less immunogenic and less toxic and, thus, may be specific and efficient reagents for the treatment of allergic diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Our knowledge of the mechanisms of apoptosis induction and the main actors in the well-orchestrated play known as programmed cell death is growing every day. Analysis of the available data convinced us that this complicated mechanism could be the perfect tool for practical applications in medicine for the elimination of specific cell populations such as cancer cells, cells involved in immune diseases, and others. The idea of targeted elimination of specific cell populations was previously addressed by constructing molecules called chimeric proteins. These proteins contain a targeting moiety fused to a bacterial or plant toxin that serves as a killing moiety. By linking these two functions, the chimeric proteins are able to recognize and destroy cells overexpressing specific surface molecules (1). However, the clinical application of such chimeras is restricted, due to the immunogenicity and nonspecific toxicity of the bacterial and plant toxin components. Construction of a new type of chimeric proteins containing a human proapoptotic molecule fused to a targeting moiety might solve the problem, generating highly specific, nonharmful therapeutic reagents.

We recently described the in vitro and in vivo activity of the chimeric protein Fc{epsilon}-PEA, which contains the bacterial Pseudomonas exotoxin A (PEA)3 as a killing moiety (2, 3). The chimera was designed to target mast cells and basophils, the main inducers of allergic responses. Both mast cells and basophils express Fc{epsilon}RI. Interaction of a polyvalent allergen with specific IgEs bound to Fc{epsilon}RI leads to cross-linking of the receptors, triggering degranulation of these cells. This results in the release of mediators provoking the physiological manifestations of allergy. Use of the Fc portion of the mouse IgE in the Fc{epsilon}-PEA protein allowed specific targeting of the chimera to mast cells and basophils, because only these two types of cells exhibit relatively high levels of the Fc{epsilon}RI. It has been shown that treatment with Fc{epsilon}-PEA leads to mast cells elimination in vitro (2) and prevents passive coetaneous anaphylaxis in vivo (3). Moreover, Fc{epsilon}-PEA does not cause degranulation in vitro or in vivo (2, 3). These findings indicated that the targeting of mast cells and basophils by such chimeric proteins is a promising treatment for allergic diseases.

We now describe the construction of new chimeric proteins combining the two approaches: 1) use of Fc{epsilon} as a targeting moiety to direct the killing component to the cell via the Fc{epsilon}RI, and 2) use of human proapoptotic proteins as the killing moiety. We have chosen two of the upstream proteins of the apoptotic cascade, Bak and Bax, to force cells involved in allergic responses to undergo apoptosis. Both proteins are members of the Bcl-2 family, characterized as powerful proapoptotic factors that are activated upon events committing cells to death. Intracellular overexpression of these proteins induces apoptosis (4, 5).

The killing ability of Bax as part of a chimeric protein was recently proved in the IL-2-Bax construct. This chimera has a high proapoptotic effect on IL-2R positive cells (6). Bak counteracts the protection from apoptosis provided by the Bcl-2 and other antiapoptotic members of the family such as Bcl-xL (7). Bak is known to interact with these proteins as well as to produce homodimers (8). It is expressed in a broad range of cells, including terminally differentiated cell types. The role of Bak as the regulator of antiapoptotic proteins and its broad tissue distribution prompted us to introduce it as an alternative killing moiety of the chimeric proteins.

To prove that introduction of human proapoptotic Bcl-2-family proteins as new killing moieties causes apoptotic death of mast cells and basophils, we first constructed chimeric proteins composed of the mouse Fc{epsilon}-targeting moiety by replacing the bacterial toxin in Fc{epsilon}-PEA with the human Bak and Bax proteins. The resulting chimeric proteins, m-Fc{epsilon}-Bak and m-Fc{epsilon}-Bax, showed a strong apoptotic effect that was restricted to Fc{epsilon}RI- positive cells. We next used a human Fc{epsilon}-targeting moiety. Because there are controversial data in the literature regarding the involvement of various Fc domains (C{epsilon}1–C{epsilon}4) in the interaction with Fc{epsilon}RI, we introduced two human Fc{epsilon} sequences. The short variant comprises amino acids 301–437 of human IgE, which corresponds exactly to the mouse sequence, introduced into the Fc{epsilon}-PEA chimeric protein (2). It includes the C{epsilon}2-C{epsilon}3 junction and the C{epsilon}3 domain of the Fc{epsilon}. The long variant comprises amino acids 224–443 of the human IgE and includes the complete C{epsilon}2 and C{epsilon}3 domains and the C{epsilon}3-C{epsilon}4 junction. These chimeric proteins, containing the short and long human Fc{epsilon} portions, were designated h-Fc{epsilon}3-Bak and h-Fc{epsilon}2–4-Bak.

Both Bax and Bak proteins fused to mouse or human Fc{epsilon}-targeting moieties were shown to cause apoptosis of target Fc{epsilon}RI-positive cells, leading to massive cell death. Treatment with the various chimeric proteins did not result in cell degranulation, nor did the Fc{epsilon}-proapoptotic chimeric proteins have cytotoxic effect on nontarget cells. These observations confirm the ability of the new proapoptotic chimeric proteins to effect specific elimination of basophilic and mast cells of human and mouse origin via apoptosis. Fc{epsilon}-proapoptotic chimeric proteins may prove to be powerful agents for the treatment of severe forms of human allergic diseases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of plasmids

Cloning was performed into pAY1 plasmid (6) containing the human Bax sequence from which the IL-2 coding region was removed. The mouse Fc{epsilon} coding sequence was cleaved from pAF2302 plasmid (2) by NdeI and ligated 5' to the above Bax sequence, resulting in the pRBaxM10 plasmid. To construct the pRBakM31 plasmid encoding the Bak protein, we replaced the Bax-coding fragment with the human Bak-coding insert, which was cleaved from the plasmid pYA2 (9). The correctness of the resulting plasmids was proved by sequence analysis.

To construct chimeric proteins containing the constant domains of human IgE, the mouse Fc{epsilon} fragment in pRBakM31 was replaced by human Fc{epsilon} fragments as follows: Total RNA was isolated from fresh human lymphocytes with the TriPure Isolation reagent (Boehringer Mannheim, Mannheim, Germany) and reverse transcribed into first strand cDNA with a reverse transcription system (Promega, Madison, WI). C{epsilon}2-C{epsilon}4 and C{epsilon}3 fragments representing cDNA sequences corresponding to residues 225–443 and 301–437 of the human Fc{epsilon}, respectively, flanked from both sides with NdeI restriction sites, were generated by PCR using synthetic oligonucleotide primers: for the long Fc{epsilon} fragment, 5'-CGGAATTCCATAT GGTCTGCTCCAGGGACTTCACCC-3' (sense) and 5'-CGGAATTCCATATGCGGGGCAGCACGCGGGCCGCT-3' (antisense); for the short Fc{epsilon} fragment, 5'-CGGAATTCCATATGCAGAAGCACTGGCTGTCAGACC-3' (sense) and 5'-CGGAATTCCATATGGCTGGTCT TGGTCGTGGACCGC-3' (antisense). The PCR mixture was incubated in a DNA thermal cycler (MJ Research, Watertown, MA) for 40 cycles. Each cycle consisted of 1 min at 95°C, 1 min at 65°C, and 2 min at 72°C. The resulting PCR products (long and short variants) were cleaved with NdeI and ligated with the vector cleaved from the pRBakM31 plasmid with the same enzyme. Clones with the appropriate orientation were chosen by restriction analysis. The correctness of the resulting plasmids pRBakHL13 and pRBakHS1 was proved by sequence analysis.

Expression and partial purification of the chimeric proteins

Escherichia coli strain BL21-plus ({lambda}DE3) was transformed with one of the four plasmids, pRBakM31, pRBaxM10, pRBakHL13, and pRBakHS1, encoding m-Fc{epsilon}-Bak, m-Fc{epsilon}-Bax, h-Fc{epsilon}2–4-Bak, and h-Fc{epsilon}3-Bak proteins, respectively. Cells were grown in medium (SLB; 16 g/L Tryptone, 10 g/L yeast extract (both from Difco, Detroit, MI) and 5 g/L NaCl) containing 100 µg/ml ampicillin. At OD600 = 1.5, the cultures were supplemented with 1 mM isopropyl-1-thio-{beta}-D-galactopyranoside to induce protein synthesis and were then incubated for 2 h at 37°C. The cells were spun down and the pellet was stored for at least 2 h at -70°C and then suspended in lysis buffer (50 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 0.2 mg/ml lysozyme) followed by sonication and centrifugation at 35,000 x g for 30 min. The pellet thus obtained was sonicated in washing buffer (50 mM Tris-HCl (pH 8.0), 0.25 M NaCl, and 0.5% Triton X-100) and centrifuged at 35,000 x g for 30 min. The resulting pellet was resuspended in 0.5 ml of cold denaturation buffer (6 M guanidinium-HCl, 0.1 M Tris-HCl (pH 8.6), 1 mM EDTA, 50 mM NaCl, and 10 mM DTT) per gram wet E. coli. The supernatant was cleared by centrifugation at 35,000 x g for 30 min. The protein solution was then diluted 50-fold in refolding buffer (20 mM Tris-HCl (pH 8.0), 0.25 M L-arginine, 1 mM EDTA, 0. 25 M NaCl, and 5 mM DTT) and left to refold for 48 h at 4°C, followed by dialysis against PBS at 4°C. Aliquots of the partially purified chimeric proteins m-Fc{epsilon}-Bak, m-Fc{epsilon}-Bax, h-Fc{epsilon}2–4-Bak, and h-Fc{epsilon}3-Bak preparations were kept at -20°C until used.

Cell lines

Fc{epsilon}RI-positive cell lines from different species were used. MC-9, an IL-3-dependent mouse mast cell line of fetal liver origin (10), and Abelson virus-transformed mast cell line of mouse placental origin (11) were a gift of E. Rasin (Hebrew University, Hadassah Medical School, Jerusalem, Israel); C57, a mast cell line of mouse bone marrow origin (12), was provided by Y. A. Mekori (Meir Hospital, Kfar Saba, Israel); human chronic myeloid leukemia cells KU812 (13) and LAMA-84 (14) were purchased from DSMZ (Braunschweig, Germany).

Other cell lines were obtained as follows: mouse myelomonocytic cell line WEHI-3 was purchased from the American Type Culture Collection (Manassas, VA); human mast cells HMC-1 were kindly gifted by J. H. Butterfield (Mayo Clinic, Rochester, MN); T cell lymphoma HUT-102 was kindly provided by T. Waldmann (National Institutes of Health, Bethesda, MD); T cell lymphoma Jurkat was kindly provided by H. Ben-Bassat (Hadassah Hospital, Jerusalem, Israel); and mouse T cell lymphoma 2B4 was kindly provided by R. Gay (Hebrew University).

Cell lines were grown in a humidified atmosphere of 5% CO2 at 37°C in RPMI 1640 medium containing 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FCS. In addition, C57, MC-9, Abelson, and HUT-102 cells were supplemented with 5 mM HEPES buffer solution (pH 7.3), 1 mM sodium pyruvate, 1x MEM amino acids solution, and 5 x 10-5 M 2-ME. The IL-3-dependent line MC-9 was additionally supplemented with a WEHI-3 conditional medium (15). All media and supplements were acquired from Biological Industries (Beit Ha’emek, Israel).

Chimeric protein internalization

Identification of the chimeric proteins within treated cells. Cells (106 cells/ml) were incubated for 2, 6, or 24 h with the various chimeric proteins (10 µg/ml, total protein concentration). The cells were washed twice with PBS, and the proteins were extracted using the TriPure isolation reagent according to the manufacturer’s instructions. The chimeric proteins within the cells were detected by Western blot analysis using anti-Bak and anti-IgE Abs (see below).

Confocal microscopy. The C57 mouse mast cells were incubated with 10 µg/ml (total protein concentration) of h-Fc{epsilon}2–4-Bak chimeric protein at 37°C for various time intervals or alternatively in the presence of 0.2% NaN3 for 3 h. The cells were then collected, washed with PBS, and allowed to adhere to coverslips pretreated with 10% (poly)L-lysine (Sigma, St. Louis, MO). The slides were washed twice with PBS, and the cells were fixed with formaldehyde (3.7% in PBS) for 10 min at 25°C. The slides were then washed twice with PBS and incubated with PBS containing 0.2% Triton X-100 and 0.2% BSA for 4 min at 25°C. After two washings with PBS, the slides were blocked with 1% BSA in PBS for 20 min at 25°C, incubated with rabbit-anti-human Bak Abs (BD PharMingen, San Diego, CA) for 40 min at 25°C, and washed three times with PBS. Second Abs conjugated to Cy5 (Jackson ImmunoResearch Laboratories, West Grove, PA) were used to detect h-Bak. The slides were incubated with second Ab for 40 min and washed four times with PBS. Slides were then treated at the same conditions with sheep-anti-human IgE Abs (Upstate Biotechnology, Lake Placid, NY), followed by incubation with second FITC-conjugated Abs (Vector Laboratories, Burlingame, CA) for the detection of h-Fc{epsilon}2–4. Nuclei were counterstained with 4',6'-diamidino-2-phenylindole (1 µg/ml). Slides were mounted, examined, and screened with a Zeiss LSM 410 confocal laser scanning system attached to a Zeiss Axiovert 135 M inverted microscope with a 63x/1.2 C-apochromat water immersion lens (Zeiss, Oberkochen, Germany). Samples were analyzed using 488 nm excitation for green fluorescence, 633 nm for red excitation, and a 364 Innova Enterprise ion laser (Coherent, Santa Clara, CA) for 4',6'-diamidino-2-phenylindole staining (blue).

Cell viability

Cells were seeded onto 24-well plates (5 x 104 cells in 1 ml culture medium). Chimeric proteins (final concentration, 2–20 µg/ml) or an equal volume of PBS were added, and the cultures were harvested after various incubation periods and counted in a hemocytometer. Viability was assessed by trypan blue exclusion.

{beta}-Hexosaminidase activity

The degree of degranulation was determined by measuring the release of {beta}-hexosaminidase. Degranulation was measured during treatment with the chimeric proteins as well as following triggering with DNP-HSA of cells passively activated with mouse IgE-anti-DNP (clone SPE-7; Sigma). The cells were washed twice with HBSS, resuspended in HBSS with 1% gelatin (5 x 105 cells/ml), seeded onto 96-well plates at 250 µl/well, and incubated with the chimeric proteins or IgE-anti-DNP (1 µg/ml) for 45 min at 37°C. A quantity of 0.1 µg/ml of DNP-HSA was added 15 min after preactivation of the samples with IgE-anti-DNP. Cells were collected 30 min after triggering, centrifuged at 500 x g for 5 min, and aliquots of the supernatant (200 µl) were incubated with an equal volume of 5 mM p-nitrophenyl-N-acetyl-glucosamine (Sigma) in 0.2 M sodium-citrate buffer (pH 4.5). The reaction was conducted at 37°C for 2 h and terminated by the addition 0.6 ml of Tris 1 M (pH 9.0). Degranulation was quantified by absorbtion at 405 nm.

FACS analysis

Cell cycle. Cells were incubated overnight at 37°C (106 cells/5 ml) with the various chimeric proteins. The cells were centrifuged at 450 x g for 6 min., washed with cold PBS, resuspended in 300 µl PBS, fixed with 5 ml methanol at -20°C for 1 h, and centrifuged at 800 x g for 5 min. The cells were then resuspended in 1 ml PBS, incubated for 30 min on ice, centrifuged at 800 x g for 5 min, washed in 1 ml of PBS, and resuspended in 0.5 ml PBS supplemented with 0.5 µg RNase and 50 µg propidium iodide. The cells were then analyzed for DNA content as a function of cell number by a FACScan (BD Biosciences, San Jose, CA) using the LYSYS II program (BD Biosciences).

Analysis of Fc{epsilon}RI expression. Cells (1–2 x 106) were washed twice in 3% FCS/PBS, centrifuged at 300 x g for 5 min, resuspended in 50 µl of the buffer, and incubated for 30 min at 4°C with anti-human Fc{epsilon}-RI (1:150) (Upstate Biotechnology). The cells were washed twice with 3% FCS/PBS, resuspended in 50 µl of the same buffer, and incubated with 1 µl FITC conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) for 20 min at 4°C. The cells were washed twice with 3% FCS/PBS, resuspended in 0.5 ml PBS, and analyzed by FACScan.

Western blot analysis

Various protein preparations were run on 15% polyacrylamide/SDS mini-gels (~7.5 µg protein/lane) and transferred onto a nitrocellulose filter. The membranes were analyzed according to the Amersham ECL system (Amersham, Little Chalfont, Buckinghamshire, U.K.). Abs were purchased as follows: anti-Bak and anti-caspase-3 were purchased from BD PharMingen International, anti-Bax from Santa Cruz Biotechnology (Santa Cruz, CA), anti-FceRI from Upstate Biotechnology, goat-anti-rabbit HRP-conjugated Abs from Jackson ImmunoResearch Laboratories, and anti-human IgE-HRP-conjugated Abs from Serotec (Kidlington, Oxford, U.K.).

DNA laddering

After 24 h incubation with the chimeric proteins, 106 MC-9 cells were collected, washed in PBS, and resuspended in 4 ml of lysing buffer (15 mM Tris (pH 7.4), 3 mM EDTA (pH 8.0), 150 mM NaCl, 0.2% SDS, 100 µg/ml protein kinase K, and 50 µg/ml RNase) and incubated overnight at 37°C. DNA was extracted with an equal volume of phenol plus chloroform (1:1 v/v) and then with chloroform. The final concentration of NaCl was adjusted to 0.5 M, and DNA was precipitated at -70°C by the addition of 2 vol of ethanol for 1 h. The DNA was recovered by centrifugation, washed twice with 70% cold ethanol, air-dried, resuspended in 10 mM Tris and 1 mM EDTA (pH 8.0), and run on 1.5% agarose gel for detection of DNA ladders.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Construction, expression, partial purification, and characterization of new Fc-based proapoptotic chimeric proteins

Our aim was to construct recombinant chimeric proteins able to specifically eliminate populations of mast cells and basophils, using proapoptotic proteins as the killing moiety. We first designed two plasmids, pRBaxM10 and pRBakM31, encoding m-Fc{epsilon}-Bax and m-Fc{epsilon}-Bak proteins, respectively (Fig. 1GoA). These chimeric proteins contain a mouse Fc{epsilon}-fragment as a targeting moiety, used successfully in the Fc{epsilon}-PEA chimeric protein (2), fused to either Bax or Bak human proapoptotic proteins. The second generation of the chimeric proteins (h-Fc{epsilon}2–4-Bak and h-Fc{epsilon}3-Bak) contained human targeting moieties, the C{epsilon}2-C{epsilon}4 (amino acids 224–443) or the C{epsilon}3 (amino acids 301–437) fragments of the constant region of the human IgE (Fc{epsilon} portion) fused to the human Bak protein. For this purpose, the DNA fragment encoding the mouse Fc{epsilon} in plasmid pRBakM31 was replaced by human Fc{epsilon}-encoding fragments of various lengths. The resulting long (C{epsilon}2-C{epsilon}4) and short (C{epsilon}3) plasmids were designated pRBakHL13 and pRBakHS1, respectively. The DNA constructs and related chimeric proteins are schematically represented in Fig. 1GoA. Following transformation of E. coli BL21-plus ({lambda}DE3) cells with one of the four plasmids, expression of the fusion genes was controlled by the bacteriophage T7 late promoter, as described previously (16). SDS-PAGE and Western blot analysis, using anti-Bak and anti-Bax Abs, revealed that the chimeric proteins accumulate mainly in the insoluble subcellular fraction (Fig. 1Go, B–D). These partially purified insoluble protein preparations, after denaturation and refolding, were used in all the experiments.



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  		FIGURE 1. Design and expression of Fc{epsilon}-Bak and Fc{epsilon}-Bax chimeric proteins. A, Schematic representation of Fc{epsilon}-Bak/Bax chimeric proteins, m-Fc{epsilon}-Bak, m-Fc{epsilon}-Bax, h-Fc{epsilon}2–4-Bak, and h-Fc{epsilon}3-Bak; and corresponding plasmids, pRBaxM10, pRBakM31, pRBakHL13, and pRBakHS1. B, SDS-PAGE analysis of subcellular fractions of E. coli-expressing cells. Lanes 1–2, h-Fc{epsilon}2–4-Bak; lanes 3–4, h-Fc{epsilon}3-Bak; and lanes 5–6, m-Fc{epsilon}-Bax. Lanes 1, 3, and 5, Insoluble fractions after denaturing and refolding; and lanes 2, 4, and 6, soluble fractions. C and D, Characterization of Fc{epsilon}-Bak (C) and Fc{epsilon}-Bax (D) chimeric proteins by immunoblotting. The insoluble fractions of E. coli-expressing cells were immunobloted using anti-Bak (C) or anti-Bax (D) Abs. Lane a, m-Fc{epsilon}-Bak; lane b, h-Fc{epsilon}3-Bak; lane c, h-Fc{epsilon}2–4-Bak; and lane d, m-Fc{epsilon}-Bax. For details, see Materials and Methods.

 
Internalization of chimeric proteins into target cells

Proapoptotic proteins of the Bcl-2 family are intracellular molecules, which are not considered to be able to cause apoptosis from outside the cell. Therefore, to induce apoptosis, Bak-based chimeric proteins have to be internalized into the target cells. We investigated the process of chimeric protein internalization by Western blot analysis of the total protein fractions extracted from control and treated cells of various lines. Using anti-Bak Abs (Fig. 2GoA) and anti-IgE Abs (results not shown), the full-length chimeric proteins could be detected within the target cells as soon as 2–6 h following treatment.



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  		FIGURE 2. Internalization of the Fc{epsilon}-based chimeric proteins into target cells. A, Western blot analysis of the total protein fractions extracted from control and treated cells, using anti-Bak Abs. Lanes 1–4, C57 cells; lanes 5–8, LAMA-84 cells; lanes 1 and 5, nontreated cells; lanes 2 and 6, cells treated with h-Fc{epsilon}2–4-Bak for 3 h; lanes 3 and 7, cells treated with h-Fc{epsilon}3-Bak for 3 h; and lanes 4 and 8, cells treated with h-Fc{epsilon}3-Bak for 6 h. Chimeric proteins were added at 10 µg/ml (total protein concentration). B–D, Confocal microscopy of C57 cells exposed to h-Fc{epsilon}2–4-Bak chimeric protein (10 µg/ml, total protein concentration) for different time periods. Anti-Bak was visualized with Cy5 (red), whereas anti-Fc was visualized with FITC (green). Slides were examined by laser fluorescence confocal microscopy as described in Materials and Methods. B, Kinetics of internalization of the chimeric protein into the cells. 1–3, Control untreated cells; 4–6, cells exposed to h-Fc{epsilon}2–4-Bak for 3 h; 7–9, cells exposed to h-Fc{epsilon}2–4-Bak for 6 h. Green fluorescence (1, 4, and 7) represents staining with anti-h-IgE Abs; red fluorescence (2, 5, and 8) represents staining with anti-hBak Abs; lanes 3, 6, and 9, superposition of three colors (blue fluorescence represents nuclear staining). All fields were photographed with a 63 x 1.2 objective lens, with zoom x3. C, Incubation of C57 mast cells with h-Fc{epsilon}2–4-Bak for 3 h: green (1) for visualization with anti-h-IgE Abs; red (2) for visualization with anti-hBak Abs; lane 3, superposition of three colors, blue for nuclear staining. The field was photographed with a 63 x 1.2 objective lens, with zoom x4. D, Incubation of C57 cells with h-Fc{epsilon}2–4-Bak for 3 h in the presence of NaN3 prevents internalization of the chimeric proteins. Staining with anti-h-IgE Abs. The field was photographed with a 63 x 1.2 objective lens, with zoom x4.

 
For more detailed characterization of the process, the cells were subjected to confocal microscopy using Abs specific to both moieties of the chimeric proteins: anti-human IgE and anti-human Bak. The untreated control cells show very slight staining with both types of the Abs (Fig. 2GoB, 1–3), whereas upon treatment of the cells with h-Fc{epsilon}2–4-Bak, augmented amounts of the chimeric protein were detected as a function of incubation time (Fig. 2GoB). An overlay of the two Abs showed that the Fc moiety colocalized with the Bak moiety (indicated by the yellow color) within the cytoplasm of the target cells (Fig. 2GoB, 3, 6, and 9, and C, 3). This signal is stable for at least 24 h (results not shown). The similar results were obtained when following internalization of h-Fc{epsilon}2–4-Bak into KU812 cells (results not shown). Preventing the internalization of the chimeric protein by treating the cells with 0.2% NaN3 results in surface staining of cell membranes (Fig. 2GoD), confirming binding of the chimeric protein to the receptor without internalization.

The effect of Fc{epsilon}-based chimeric proteins on target cells viability

Because we intended to cause apoptosis by specifically introducing the Bax and Bak proteins into the target cells, we closely monitored the viability of cultured cells treated with the new chimeras. The chimeric proteins efficiently inhibited the growth of mouse C57 mast cells, eventually causing cell death. We found that the inhibition of cell proliferation was dose (Fig. 3GoA and results not shown) and time (Fig. 3GoB) dependent for both the long and short variants of the human Fc{epsilon}-Bak chimeric proteins, indicating that the chimeric proteins affect cell viability. Similar results were obtained using the m-Fc{epsilon}-Bak and -Bax chimeric proteins (see below). The activity of the chimeric proteins was also tested on other cell lines from various species known to express the Fc{epsilon}RI. The cell lines were divided into three groups according to their sensitivity to the treatment: 1) highly reactive lines, such as the mouse C57 and MC-9 mast cells that were almost totally eliminated after 48 h of treatment; 2) intermediately reactive mouse Abelson line, whose viability was inhibited by 60–80% following 48 or 72 h of treatment; and 3) lines exhibiting low reactivity, such as KU812 and LAMA-84, both of human origin, whose viability was inhibited by 20–30% at the most (Fig. 3GoA).



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  		FIGURE 3. Effect of Fc{epsilon}-based chimeric proteins on various cell lines. A, Concentration dependency. Cell lines: C57 ({diamondsuit}), MC-9 {blacksquare}, Abelson {blacktriangleup}, LAMA-84 (*) (5 x 104 cells/ml), and Ku-812 {diamond} (105 cells/ml) were incubated with increasing concentrations of h-Fc{epsilon}2–4-Bak for 48 or 72 h (KU-812) and counted in a hemocytometer. The number of cells is presented as the mean number of the triplicates of treated cells compared with the mean number of cells in control samples. Each result is the mean of three separate experiments. B, Time dependency. C57 mouse mast cells (5 x 104 cells/ml) were incubated with 10 µg/ml (total protein concentration) of the various chimeric proteins or with PBS and counted every 24 h. , h-Fc{epsilon}3-Bak; , h-Fc{epsilon}2–4-Bak; , m-Fc{epsilon}-Bak; and {square}, PBS. Each result is the mean of three separate experiments, with each point counted in triplicates.

 
Expression levels of Fc{epsilon}RI on various cell lines

Expression of Fc{epsilon}RI on a cell line is necessary for the specific interaction of the Fc{epsilon}-based chimeras with the cells. The wide variability in the sensitivity of various cell lines toward the chimeric proteins led us to hypothesize that the sensitivity of a particular cell line depends on the expression level of its Fc{epsilon}RI. We determined the level of Fc{epsilon}RI expression on various cell lines by FACS analysis, using anti-human Fc{epsilon}RI Abs. The highest levels of the receptor were observed on mouse mast cells C57 (90% FITC-positive cells) and MC-9 (84% FITC-positive cells) (Fig. 4GoA). Because we used anti-human Fc{epsilon}RI Abs with unknown cross-reactivity with other species, the result implies cross-reactivity of the Abs with mouse Fc{epsilon}RI. In the case of the human LAMA-84 cells, the percentage of FITC-positive cells was only ~60%. The HMC-1 human mast cell line and nonrelevant T cell lymphoid lines HUT-102 and Jurkat were confirmed to be Fc{epsilon}RI-negative cells. Therefore, we can conclude that the expression level of surface Fc{epsilon}RI of the various cell lines corresponds to their sensitivity toward the chimeric proteins (Fig. 3GoA).



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  		FIGURE 4. Characterization of Fc{epsilon}RI expression. A, Levels of Fc{epsilon}RI expression in various cell lines measured by FACS analyses. Cells were incubated with anti-human Fc{epsilon}RI Abs, stained with the second FITC-conjugated Abs, and analyzed by FACS. Standard line, Cells stained with the second Ab alone; bold line, cells treated with both Abs. M1 represents the percentage of Fc{epsilon}RI-positive cells. Cell lines C57, MC-9, and LAMA-84 were Fc{epsilon}RI positive; HMC-1, Jurkat, and HUT-102 were Fc{epsilon}RI negative. Data show one representative experiment of three. B, Degranulation capacity of various cell lines. The degree of degranulation was determined by measuring the activity of {beta}-hexosaminidase released by cells passively activated with increasing concentrations of IgE-anti-DNP, followed by challenge with 0.1 µg/ml of DNP-HSA. The figure shows the percentage of {beta}-hexosaminidase activity in the medium of the activated cells as compared with that of nonactivated cells, as a function of IgE-anti-DNP concentration. Spontaneous release was taken as zero level. The following cell cultures were tested: C57 {diamondsuit}, MC-9 {blacksquare}, Abelson {blacktriangleup}, KU-812 {diamond}, and LAMA-84 (*). Each result is the mean of three separate experiments, with each point measured in duplicates. B, Sensitivity of various cell lines to the cytotoxic activity of Fc{epsilon}-PEA correlates to the level of Fc{epsilon}RI expression by the cells. Cells were treated overnight with increasing concentrations of Fc{epsilon}-PEA. Protein synthesis was measured following [H3]leucine incorporation into total protein as described in Ref. 16 . The following cell cultures were tested: C57 {diamondsuit}, MC-9 {blacksquare}, LAMA-84 (x), and KU-812 {diamond}. Results represent the mean of triplicates of three separate experiments.

 
Because cell degranulation is provided by the interaction of IgE with Fc{epsilon}RI, followed by triggering with Ag, susceptibility to passive degranulation may be another measure of the level of Fc{epsilon}RI expression. We tested the dependence of passive degranulation triggered in the various cell lines on IgE-anti DNP concentration, using a constant DNP-HSA concentration. The degree of degranulation under passive activation was measured as the increase of the activity of {beta}-hexosaminidase, one of the enzymes released by mast cells during degranulation (17). We noticed that triggering of degranulation in the cell lines used shows variation in the levels of {beta}-hexosaminidase activity and in the concentration of IgE-anti DNP sufficient to passive activation (Fig. 4GoB). We found that readily degranulating cell lines are characterized by low concentrations of IgE sufficient to activate cells for degranulation and a high degree of degranulation. C57 and MC-9 cell lines demonstrated an ~4-fold increase in {beta}-hexosaminidase activity at concentrations of IgE as low as 1–5 µg/ml (Fig. 4GoB). These lines were also highly sensitive to treatment with the chimeras (Fig. 3GoA). In contrast, KU812 and LAMA-84 cells were activated only by exposing them to 20–40 µg/ml of IgE before triggering with DNP-HSA. Their degranulation level was also very low, with an increase of only 15–30% in {beta}-hexosaminidase activity (Fig. 4GoB). Correspondingly, only ~20–30% of these cells died following the treatment with the chimeric proteins (Fig. 3GoA). Our observations confirm that the ability of cells to undergo degranulation correlates with their sensitivity to the chimeric proteins.

Target cells’ sensitivity to treatment with our previous chimera, m-Fc{epsilon}-PEA, was measured accurately by following the inhibition of protein synthesis due to the ADP-ribosylation activity of the PE-toxin (18). Because m-Fc{epsilon}-PEA interacts with specific cells through Fc{epsilon}RI, inhibition of protein synthesis during Fc{epsilon}-PEA treatment of various cell lines could be an additional measure for the expression level of Fc{epsilon}RI. We found that in C57 cells treated with 0.4 µg/ml of Fc{epsilon}-PEA, protein synthesis was inhibited by >90%, whereas in the KU812 and LAMA-84 cell lines, it was inhibited by only 15–20% (Fig. 4GoC). These observations indicate the direct correlation between the level of surface Fc{epsilon}RI expression by various cell lines and their sensitivity to the different chimeric proteins (compare Figs. 3GoA and 4, A–C).

Specificity of chimeric proteins activity

Cytotoxic activity was also tested on nontarget cell lines not known to express the Fc{epsilon}RI. We measured the survival of HUT-102, WEHI-3, HMC-1, and 2B4 nontarget cell lines in the presence of the chimeric proteins. In all tested cases, no influence on cell viability was observed (Table IGo). The finding that the chimeric proteins were unable to reduce the viability of nonspecific cells confirms the specific mode of action of the Fc{epsilon}-based proapoptotic proteins. It also corroborates that the partially purified protein preparations of the chimeric proteins used in our experiments do not contain nonspecific toxic impurities.


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  		Table I. Fc{epsilon}2–4-Bak has no effect on nontarget cells

 
To further substantiate that the cytotoxic activity is related exclusively to the chimeric proteins rather then to the impurities in the protein extracts, we used anti-IgE to precipitate the active component of the protein preparations. Immunoprecipitation of h-Fc{epsilon}3–4-Bak protein preparation with anti-human-IgE Abs completely abolished the specific cytotoxic activity on MC-9 cells of the residual supernatant (Fig. 5GoC). In contrast, pretreatment of the same protein preparation of the chimeric protein with unrelated Abs did not reduce the activity of the chimera (Fig. 5GoD). Partially purified preparates of nonrelevant chimeric proteins, such as GnRH-Bik (9) and IL-2-Bax (6), had no cytotoxic effect on C57 and MC-9 mast cell lines (results not shown).



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  		FIGURE 5. Preimmunoprecipitation of h-Fc{epsilon}2–4-Bak with anti-IgE Abs prevents chimeric protein activity against target MC-9 mast cells. A, Nontreated cells; B, cells treated for 24 h with 10 µg/ml (total protein concentration) of h-Fc{epsilon}2–4-Bak. C and D, The h-Fc{epsilon}2–4-Bak chimeric protein was immunoprecipitated with anti-IgE Abs (C) or with unrelated Abs (D). Cells were then treated for 24 h with the resulting supernatant and photographed under a light microscope (magnification, x100).

 
Apoptotic effect of chimeric proteins on various cell lines

The specific cytotoxicity of the new chimeric proteins toward target cells raises the question of the mechanism of cell death in cells exposed to the chimeras. According to our assumption, the cells should undergo apoptosis upon treatment. The effect of the chimeric proteins on various cell lines was tested by FACS analysis of the cell cycle. The increase in the sub-G1 cell population was an indication of the number of cells undergoing an apoptotic process. Fig. 6GoA demonstrates a cell cycle typical of untreated C57 cells, with 16% of the total cells in the sub-G1 phase. After overnight treatment with m-FcBax, the sub-G1 population constituted as much as 67% (Fig. 6GoB), whereas treatment with the nonrelevant chimeric protein GnRH-Bik had no influence on the cell cycle (Fig. 6GoC). Treatment of MC-9 cells with m-Fc{epsilon}Bax, as well as treatment of C57 cells with m-Fc{epsilon}-Bak, h-Fc{epsilon}2–4-Bak, and h-Fc{epsilon}3-Bak, resulted in similar changes in the cell cycle (results not shown). In all cases, a decrease in cell survival following treatment was accompanied by an increase in the sub-G1 population after overnight incubation. Increase in sub-G1 population was concentration dependent (Fig. 6GoF). No effect on nontarget cell lines HUT-102 (a human T cell lymphoma) (Fig. 6Go, D and E), T24A (bladder carcinoma cells), or LAM (human lymphoma cells) was observed (data not shown).



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  		FIGURE 6. Induction of apoptosis in target cells by m-Fc{epsilon}-Bax chimeric protein. A, MC-9 cells incubated with PBS; B, MC-9 cells incubated with m-Fc{epsilon}-Bax; C, MC-9 cells incubated with a nonrelevant chimeric protein, purified under the same protocol; D, nontarget cell line HUT-102 incubated with PBS; E, HUT-102 cells incubated with m-Fc{epsilon}-Bax; and F, M1 dependence on m-Fc{epsilon}-Bax concentrations. M1 represents the percentage of cells in the sub-G1 phase. Cells (106 cells/5 ml) were incubated overnight with PBS (control cells, A) or with 10 µg/ml of chimeric protein (B, C, and E) or with increasing concentrations of m-Fc{epsilon}-Bax (F). Samples were analyzed by FACS for DNA content (x-axis) as a function of cell number (y-axis), as described in Materials and Methods. Data show one representative experiment of three.

 
Following death triggering, proapoptotic members of the Bcl-2 family are known to stimulate a cascade of downstream events, including processing of procaspases to their active forms (19). To investigate whether introduction of Fc{epsilon}-Bak into target cells initiates one of the pathways finally leading to apoptosis, we examined the activation of caspase-3 by Western blot analysis of protein extracts from cells treated with h-Fc{epsilon}2–4-Bak and h-Fc{epsilon}3-Bak. The treatment resulted in the appearance of a 17-kDa protein corresponding to the active form of caspase-3. In some cases, treatment also caused augmentation of the full-length 32-kDa caspase-3 (Fig. 7Go, A–C).



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  		FIGURE 7. Apoptotic changes in target cells following treatment with chimeric proteins. A–C, Treatment with the h-Fc{epsilon}2–4-Bak chimeric protein activates caspase-3 in the target cells. Immunoblotting of protein extracts from target cells using anti-caspase-3 Abs. -, Nontreated cells; +, cells treated with 10 µg/ml of h-Fc{epsilon}2–4-Bak; A, C57 cells, 6 h treatment; B, LAMA-84 cells, 6 h treatment; C, KU-812 cells, 24 h treatment; and D, DNA laddering. MC-9 mouse mast cells (2 x 106 cells/ml) were incubated with h-Fc{epsilon}3-Bak or PBS overnight. DNA extracts were electrophoresed on 1.5% agarose gels. -, Nontreated cells; +, treated cells.

 
Another characteristic of the apoptotic process is DNA laddering. DNA fragmentation during apoptosis leads to a typical pattern upon agarose-gel electrophoresis. We observed the laddering pattern after 48-h treatment of the MC-9 cells (Fig. 7GoD). Apoptosis was also evident when cells were examined for morphological changes under the light microscope. C57 mast cells treated with h-Fc{epsilon}3-Bak exhibited morphological changes typically seen in apoptotic cells, including nucleus condensation and blebbing (20) (Fig. 5Go, B and D).

Effect of chimeric proteins on cell degranulation

One of the major obstacles related to clinical application of Fc{epsilon}RI-target chimeric proteins is their theoretical ability to cause cell degranulation that can be as severe as anaphylaxis and can even culminate in the patient’s death. The bacterial toxin-based chimeric protein Fc{epsilon}-PE did not trigger degranulation in vitro, or in vivo (3). However, because we are using as the killing moiety of the chimeras Bax or Bak–molecules that are able to undergo dimerization–the possibility still exists that, upon binding of the new chimeric protein to target cells, Bak dimerization will result in Fc{epsilon}RI aggregation. This interaction could lead to massive degranulation. The ability of mouse and human Fc{epsilon}-Bak chimeric proteins to trigger degranulation during various periods of incubation was tested by measuring {beta}-hexosaminidase activity. No increase in {beta}-hexosaminidase activity was observed following incubation of C57 mast cells even with very high concentrations of the chimeric proteins (20–30 µg/ml) for 30 min to 18 h (Fig. 8Go, lanes 1–5). A nonrelevant chimeric protein, GnRH-DFF, did not trigger degranulation as well (Fig. 8Go, lanes 6–8). The ability of C57 cells to undergo degranulation was confirmed by activating them with IgE-anti-DNP followed by triggering with DNP-HSA (Fig. 8Go, lane 9). The {beta}-hexosaminidase activity of the passively activated cells increased ~3-fold as compared with that of the control cells.



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  		FIGURE 8. Treatment with the m-Fc{epsilon}-Bak chimeric protein does not trigger degranulation of C-57 mast cells. Cells were incubated with 15 µg/ml of the chimeric proteins (total protein concentration) for various time periods. Degranulation was assessed by comparing the percentage of {beta}-hexosaminidase activity in the medium of the treated cells with that of the nontreated cells. Lane 1, Nontreated cells; lanes 2–5, cells treated with m-Fc{epsilon}-Bak; lanes 6–8, cells treated with a nonrelated protein (GnRH-DFF); and lane 9, cells passively activated with IgE-anti-DNP and triggered with DNP-HSA. Lanes 2 and 6, 30 min of treatment; lane 3, 2 h of treatment; lanes 4 and 7, 4 h of treatment; and lanes 5 and 8, 24 h of treatment. Each result is the mean of three separate experiments, with each point measured in duplicates.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Mast cells are the primary initiating cells of immediate hypersensitivity reactions. Therefore, effective targeted elimination of mast cells and basophils is expected to be a powerful approach to the treatment of allergic diseases, which are major contributors to morbidity, and less frequently but still significantly, to mortality in the developed world. Production of effective apoptosis-inducing chimeric proteins targeted against mast cell and basophils, and composed of proteins of human origin both as the targeting and the killing moieties, was the goal of the present investigation. Our approach was based on targeted delivery of the proapoptotic proteins Bak or Bax into these cells, by fusing them to the constant portion of the IgE molecule (Fc{epsilon}). The basis for the new design was our previous construct, m-Fc{epsilon}-PEA, which exhibited strong cytotoxic activity toward mast cells both in vitro and in vivo (2, 3). We first confirmed that replacement of the PEA portion in the chimeric protein by human Bax or Bak proteins resulted in the production of effective chimeric proteins that caused apoptotic death specifically of Fc{epsilon}RI-positive cells.

Because our aim was to use only human protein-based moieties, we replaced the mouse Fc{epsilon} sequence with the human one. At this point, we were faced with the problem of choosing the human Fc{epsilon}RI-targeting moiety. Fc{epsilon} contains four Ig domains (C{epsilon}1–C{epsilon}4). The significance of each of the domains in Fc{epsilon}RI binding has been extensively investigated (21, 22, 23, 24). It is commonly accepted that the principal determinants are located in the C{epsilon}3 domain of IgE (22, 23). However, there is no agreement on the involvement of the adjoining C{epsilon}2 and C{epsilon}4 regions in the high affinity interaction. The fragment comprising 301–437 amino acids, including the C-terminal part of C{epsilon}2 and the complete C{epsilon}3 domain, was 10–13 times less effective than IgE itself (21). Basu et al. (24) hypothesize that the C{epsilon}2 domain, even if not directly involved in the interaction with Fc{epsilon}RI, may be important to the proper folding of C{epsilon}3, whereas the C{epsilon}4 domain seems to be involved in the binding to Fc{epsilon}RI. To ensure that the fragment chosen as a targeting moiety would posses high affinity for Fc{epsilon}RI, we designed two constructs: the longer one (amino acids 224–443), comprising the whole C{epsilon}2 and C{epsilon}3 domains and six N-terminal residues of the C{epsilon}4 domain; and the shorter one (amino acids 301–437), representing the C{epsilon}2-C{epsilon}3 junction and the C{epsilon}3 domain.

To prove that h-Fc{epsilon}2–4-Bak and h-Fc{epsilon}3-Bak are able to enter the target cells, we examined internalization of these proteins into C57 mast cells by the aid of confocal microscopy (Fig. 2Go, B and C). Staining the cells with anti-human Bak and anti-human IgE confirm internalization of the chimeric protein into the cells. The process begins by binding of the chimeric protein most probably to the Fc{epsilon}RI, demonstrated by membrane localization of the chimeric protein (Fig. 2GoD). Following internalization of the chimeric protein, it could be detected inside the target mast cells, mainly in the cytosol.

We compared the relative activity of the various chimeric proteins containing mouse or human Fc{epsilon}3 portions and the human Fc{epsilon}2–4 portion as the targeting moieties. We found that the various chimeric proteins had a similar effect on the target cells. Our results suggest that the C{epsilon}2 domain and the C{epsilon}3-C{epsilon}4 junction are not involved in the interaction of the Fc{epsilon} portions with Fc{epsilon}RI and that the C{epsilon}3 domain is sufficient for targeting the Fc{epsilon}RI receptor, at least in the context of chimeric proteins. However, this comparison is only a qualitative one, because the chimeric protein preparations we used were partially purified and their exact concentrations could not be determined.

In investigating the ability of the various chimeric proteins to enter the cells (Fig. 2Go, A–D), we found that constructs containing the human Fc{epsilon} portion are able to recognize mouse cells nearly as well as those with a mouse Fc{epsilon} portion. In our experiments, mouse C57 and MC-9 cells were similarly sensitive to the effect of h-Fc{epsilon}-Bak and of m-Fc{epsilon}-Bak (Fig. 3GoA). This observation contradicts the accepted dogma claiming that human IgE does not recognize mouse Fc{epsilon}RI. Nevertheless, our results suggest that at least human and mouse C{epsilon}3 domains posses a similar affinity for the mouse Fc{epsilon}RI. To date, there is no established human cell culture known to express considerable levels of Fc{epsilon}RI. Therefore, our observation is of most importance for future research in the field of allergy.

Delivery of the Fc{epsilon}-Bak chimeric proteins into the cell theoretically causes an imbalance between pro- and antiapoptotic Bcl-2-family proteins, which may induce apoptosis. A similar process is encountered upon overexpression of the Bak protein in transfected cells (5) or as the result of introduction of the BH3 fragment of Bak into HeLa cells via fusion to the Antennapedia internalization sequence (25). The mechanism of Fc{epsilon}-Bak chimeric protein action differs from that of the Fc{epsilon}-PEA chimeric protein, because low amounts of PEA delivered into the cell are enough to inhibit protein synthesis. The amount of Bak or Bax proteins required to change the balance within the cell is hard to calculate, but it is expected to be much higher. This may explain the higher range of Fc{epsilon}-Bak concentrations (Fig. 3GoB) than those of Fc{epsilon}-PEA (Fig. 4GoC) needed to kill the target cells. However, because the h-Fc{epsilon}2–4-Bak and h-Fc{epsilon}3-Bak chimeric proteins are expected to be less immunogenic and less toxic than the Fc{epsilon}-PEA molecules, administration of high concentrations of these chimeras will, most probably, be tolerable when adopted as treatment for humans.

The new chimeric proteins cause death of the target cells via apoptosis, as demonstrated by an increase in the sub-G1 population, caspase-3 activation, morphological changes, and DNA laddering ( Figs. 5–7GoGoGo). The apoptosis-inducing capacity of the Fc{epsilon}-Bak chimeric proteins is interesting in the light of recent observations that conformational changes in the Bak N terminus are critical to its function as an apoptosis inducer (26). Because Fc{epsilon} is fused to the N terminus of the Bak molecule, it is unlikely that the apoptotic activity of the chimeric protein is abolished. However, the strong proapoptotic effect of the chimeric proteins leads us to speculate that addition of the Fc portion N terminus to Bak does not interfere with the ability of Bak to undergo activation via conformational changes, or may even prevent folding into a nonactive conformation, thus augmenting the apoptotic potential of the molecule. An alternative explanation is that abolishment of the ability to undergo conformational changes in the N terminus of Bak does not completely eliminate the apoptotic effect of the molecule. A fragment of the Bak molecule lacking its N terminus and comprising only the BH3 domain possesses activity resembling that of the wild-type Bak protein (27). It was active also when the Antennapedia internalization sequence was coupled to the N terminus of BH3 (25).

Clinical application of the novel chimeric proteins to the treatment of allergic diseases could be considered only if the four following questions are resolved: 1) Do the mast cells and basophils posses some positive vital functions in the human organism? This question is widely discussed in the scientific literature, and most investigators agree that these cells play a beneficial role in our defense against parasites. However, in the modern world, this function becomes less important, and the involvement of the cells in pathological allergic processes has the first priority. Allergy and asthma now represent a far greater threat than parasitic infection. In addition to their involvement in allergic diseases, mast cells can also develop into malignancies and hyperplasias (28). No effective therapy for patients with these diseases is known.

Recent data on the possible role of mast cells in Ag presentation and promotion of anti-bacterial immune response should also be considered (29). However, stem cells do not express the Fc{epsilon}RI; consequently, the chimeric proteins targeted against mast cells have no effect on progenitor cells. Therefore, in vivo usage of chimeric proteins is not expected to damage the bone marrow and will cause only temporary elimination of mast cells.

2) Does the introduction of the chimeric proteins cause damage to the human organism by elimination of other Fc{epsilon}RI carrying cells? This reservation is reasonable because, in humans, the cellular distribution of Fc{epsilon}RI is wider than in rodents. In addition to mast cells and basophils, human Fc{epsilon}RI is expressed also by monocytes, eosinophils, platelets, dendritic cells, and Langerhans cells that have many important functions in immunological responses (30, 31, 32, 33, 34). In human eosinophils, despite a sizable intracellular pool of Fc{epsilon}RI{alpha}, its surface level is undetectable (30, 31, 32), implying that eosinophils will not be sensitive to such treatment. Platelets were shown to express surface Fc{epsilon}RI, but <10% of them are Fc{epsilon}RI{alpha} positive (33). Normal human monocytes express 10–100 times less receptors than basophils. The same holds true for other Fc{epsilon}RI{alpha}-positive cells (34). Although the Fc{epsilon}RI level is up-regulated during allergic diseases, it is still much lower than the levels expressed by mast cells and basophils.

3) Does treatment with Fc{epsilon}-Bak trigger cell degranulation? If cell degranulation is triggered, "cure" with the chimeric proteins may escalate the allergy reaction and be even more dangerous than the disease itself. Our experimental results show that, at least in vitro, no degranulation is observed during treatment of mouse mast cells C57 and MC-9 with the different chimeric proteins for various time periods (Fig. 8Go). As the mouse Fc{epsilon}-PEA chimeric protein did not trigger degranulation in vivo (3), it is most likely that this may be also be true for the new Fc{epsilon}-Bak chimeric proteins.

4) Is h-Fc{epsilon}-Bak immunogenic to the human organism? We designed the apoptosis-inducing chimeric proteins h-Fc{epsilon}2–4-Bak and h-Fc{epsilon}3-Bak in the attempt to reduce their immunogenicity as much as possible. The new chimeric proteins carry, as the killing moiety, the human protein Bak, which is recognized as self by the human immune system. Additional substitution of mouse Fc{epsilon} component by the human one should completely abolish the immunogenicity of the chimeric protein. Nevertheless, these theoretical considerations must be addressed experimentally during the clinical trials.

In our work, we have demonstrated new apoptosis-inducing chimeric proteins targeted against allergy-triggering cells, mast cells and basophils. Application of proapoptotic killing moieties for the treatment of allergic disorders and mastocytosis not only reduces the risk of immunogenic response, which is extremely dangerous in such cases, but also provides an elegant way of death for the target cells. Cells undergoing apoptosis in vivo are recognized and ingested intact by phagocytes, without the release of inflammatory mediators. Thus, apoptotic clearance of mast cells and basophils, whose granules are capable of causing tissue damage, is very advantageous. The strong and highly specific apoptotic effect of h-Fc{epsilon}2–4-Bak and h-Fc{epsilon}3-Bak chimeric proteins on the target Fc{epsilon}RI-positive cells, without triggering degranulation and the immune response, is making these chimeric proteins promising agents for the treatment of severe forms of allergy, asthma and mastocytosis.


   Acknowledgments
 
We would like to thank Rami Aqeilan, Ahmi Ben-Yehudah, and Ronen Abadi for their technical assistance and fruitful review of the manuscript and Mark Tarshish for the help with confocal microscopy.


   Footnotes
 
1 This work was supported by Medical Targeting Recognition Technologies Inc., Jerusalem, Israel. Back

2 Address correspondence and reprint requests to Dr. Haya Loreberboum-Galski, Department of Cellular Biochemistry and Human Genetics, Hebrew University, Hadassah Medical School, Jerusalem, 91120 Israel. E-mail address: hayalg{at}md2.huji.ac.il Back

3 Abbreviation used in this paper: PEA, Pseudomonas exotoxin A. Back

Received for publication February 20, 2001. Accepted for publication August 21, 2001.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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