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Antibody treatment against pulmonary exposure to abrin confers significantly higher levels of protection than treatment against ricin intoxication
Toxicology Letters 237 (2015) 72–78
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Antibody treatment against pulmonary exposure to abrin confers significantly higher levels of protection than treatment against ricin intoxication Tamar Sabo, Yoav Gal, Eitan Elhanany, Anita Sapoznikov, Reut Falach, Ohad Mazor, Chanoch Kronman* Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, Israel
H I G H L I G H T S
Pulmonary exposure of mice to abrin leads to neutrophilic inflammation of the lungs. Lung pathology following abrin intoxication resembles that of ricin. Efficient Ab-based protection is achieved following abrin but not ricin exposure. Ab treatment against abrin, but not against ricin, reduces pulmonary IL-6 levels.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 February 2015 Received in revised form 14 April 2015 Accepted 2 June 2015 Available online 5 June 2015
Abrin, a potent plant-derived toxin bearing strong resemblance to ricin, irreversibly inactivates ribosomes by site-specific depurination, thereby precipitating cessation of protein synthesis in cells. Due to its high availability and ease of preparation, abrin is considered a biological threat, especially in context of bioterror warfare. To date, there is no established therapeutic countermeasure against abrin intoxication. In the present study, we examined the progress of pulmonary abrin intoxication in mice, evaluated the protective effect of antibody-based post-exposure therapy, and compared these findings to those observed for ricin intoxication and therapy. Salient features of abrin intoxication were found to be similar to those of ricin and include massive recruitment of neutrophils to the lungs, high levels of proinflammatory markers in the bronchoalveolar lavage fluid and damage of the alveolar-capillary barrier. In contrast, the protective effect of anti-abrin antibody treatment was found to differ significantly from that of anti-ricin treatment. While anti-ricin treatment efficiency was quite limited even at 24 h postexposure (34% protection), administration of polyclonal anti-abrin antibodies even as late as 72 h postexposure, conferred exceedingly high-level protection (>70%). While both anti-toxin antibody treatments caused neutrophil and macrophage levels in the lungs to revert to normal, only anti-abrin treatment brought about a significant decline in the pulmonary levels of the pro-inflammatory cytokine IL-6. The differential ability of the anti-toxin treatments to dampen inflammation caused by the two similar toxins, abrin and ricin, could explain the radically different levels of protection achieved following antibody treatment. ã2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Abrin Ricin Pulmonary Antibodies BALF IL-6
1. Introduction
Abbreviations: ANGII, angiotensin-II; BALF, bronchoalveolar lavage fluid; ChE, cholinesterase; RIP-2, ribosome inactivating protein-2; MTTD, mean time to death; IL-6, interleukin-6; MIP-2, macrophage inflammatory protein-2; PBS, phosphatebuffered saline; TNF-a, tumor necrosis factor-a. * Corresponding author at: 19 Reuven Lerer St., P.O.B. 19 Ness-Ziona 74100, Israel. E-mail address: [email protected] (C. Kronman). http://dx.doi.org/10.1016/j.toxlet.2015.06.003 0378-4274/ ã 2015 Elsevier Ireland Ltd. All rights reserved.
Abrin, a plant toxin derived from rosary peas (Abrus precatorius) belongs to the type 2 ribosome inactivating (RIP-2) group of proteins. The heterodimeric glycoprotein consists of two subunits, subunit A, which carries the enzymatic activity of the toxin and subunit B, which is a lectin that binds to eukaryotic cell surfaces via
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glycoproteins and glycolipids containing b-1,4-linked galactose residues (Olsnes, 2004). Upon entry to the cytosol, the A-subunit exerts its toxic effect by site-specific depurination of adenine 4324 of the 28S rRNA in the 60S ribosomal subunit. This irreversible impairment prevents binding of elongation factor-2 to the ribosome, leading in turn to protein synthesis arrest and cell death (Audi et al., 2005). Due to its high availability, ease of preparation and high toxicity, abrin is considered a potential bioterror agent and is classified as a Category B agent by the US Center for Disease Control and Prevention (CDC). As in the case of the similar RIP-2 toxin, ricin, the toxicity of abrin depends on the route of exposure, inhalatory exposure being considered most hazardous (Audi et al., 2005). Though abrin is considered more toxic than ricin in the case of oral or systemic exposures (Wei et al., 1974), the LCt50 values in rats for inhaled ricin and abrin were found to be quite similar, 4.54–5.96 and 4.54 mg min m3, respectively (Griffiths et al., 2007). To date, there are no established post-exposure therapeutic countermeasures for ricin or abrin exposures. Some studies however, have been carried out to evaluate the potency and timeframe for anti-ricin antibody-based passive immunization following exposure of experimental animals to lethal doses of ricin (Maddaloni et al., 2004; Wang et al., 2006, 2007; Prigent et al., 2011). Recently, we evaluated the efficiency of postexposure treatment of pulmonary ricinosis by polyclonal antiricin antibodies, and demonstrated that survival rates can be increased by combining the antidotal treatment with the antiinflammatory compound, doxycycline (Gal et al., 2014). In contrast to ricin, research of anti-abrin antibody-based treatments is relatively meager. In one study, phage display technology was utilized to select anti-abrin human monoclonal antibodies from a human naïve scFv library (Zhou et al., 2007), however these were not examined in animal models for their therapeutic value. In another study, a monoclonal antibody mapped to abrin A-chain, was found to protect mice from abrin challenge when given 1 h prior to exposure (Surendranath and Karande, 2008). The absence of a substantial body of experimental data regarding abrin toxicology and medical countermeasures against pulmonary exposure to this toxin, prompted us in the present study, to delineate the salient features of pulmonary abrin intoxication and to assess the therapeutic potential of passive immunization in mice. 2. Materials and methods 2.1. Extraction and purification of abrin Crude extract was prepared from A. precatorius seeds (18 gr.) essentially as described previously (Olsnes and Pihl, 1973; Hegde and Podder, 1992). Briefly, seed kernels were soaked in 5% acetic acid/phosphate buffer (Na2HPO4, pH7.4) overnight and homogenized in a Waring blender for 4 sequences of 30 s. Proteins obtained from 80% ammonium sulfate precipitation were centrifuged and dialyzed extensively against PBS. Crude abrin protein concentration was determined as 7.5 mg/ml by 280nm absorption (Nanodrop). For the preparation of pure toxin, crude abrin was loaded on 2 columns in-tandem, the first column contains activated Sepharose which binds and thereby depletes the A. precatorius Agglutinin (APA) and the second column, containing a-lactose (lactamyl) agarose, binds the abrin toxin. Proteins bound to the lactamyl agarose column were eluted with 0.5 M Galactose in PBS. Tryptic digest analysis by MALDI-TOF/MS showed that both a and b isoforms of the abrin toxin are present in the galactose eluate.
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2.2. Anti-abrin antibodies Rabbits were immunized with purified abrin toxin in a stepwise manner, injections I–III containing 4, 16 and 50 mg toxin/rabbit respectively, were on alum (Al(OH)3). Injection i.v. was in Freund’s complete adjuvant (100 mg toxin/rabbit) and subsequent injections were in Freund’s incomplete adjuvant (100 mg toxin/rabbit) at 4-week intervals. Blood samples were collected (3 weeks after injection) to ascertain anti-abrin antibody titer build-up. Immunization was continued until steady high anti-abrin titers were obtained. Anti-abrin antibody titers were determined by ELISA. Microtiter plates (Nunc) were coated with purified abrin (2.5 mg/ml in carbonate buffer pH 9.6, overnight incubation at 4 C), washed 3 times in wash buffer (0.8% NaCl/0.05% Tween 20) and then incubated with blocking buffer (PBS/0.05% Tween 20/2% BSA) for 1 h at 37 C. Rabbit antisera samples were added in 2-fold serial dilutions and incubated at 37 C for 1 h. Plates were then washed 3 times with wash buffer and incubated at 37 C for 1 h with APconjugated goat anti-rabbit immunoglobulin (Sigma, 1:500 in blocking buffer). After washing as above, the microtiter plates were developed with substrate (p-NPP, Sigma) and optical densities were measured at 405 nm using an ELISA reader (Molecular Devices). 2.3. Animal studies Animal experiments were performed in accordance with the Israeli law and were approved by the Ethics Committee for animal experiments at the Israel Institute for Biological Research. Treatment of animals was in accordance with regulations outlined in the USDA Animal Welfare Act and the conditions specified in the Guide for Care and Use of Laboratory Animals (National Institute of Health). All animals in this study were female CD-1 mice (Charles River Laboratories Ltd., UK) weighing 27–32 g. Prior to exposure, animals were habituated to the experimental animal unit for 5 days. All mice were housed in filter-top cages in an environmentally controlled room and maintained at 21 2 C and 55 10% humidity. Lighting was set to mimic a 12/12 h dawn to dusk cycle. Animals had access to food and water ad libitum. For the procedure of intoxication, mice were anesthetized by intraperitoneal (i.p.) injection of ketamine (1.9 mg/mouse) and xylazine (0.19 mg/mouse). Crude abrin (50 ml; 8 mg/kg diluted in PBS) was applied intranasally (i.n., 2 25 ml) and mortality was monitored over 14 days. Preceding these studies, we determined that 4.0 mg crude abrin/kg body weight is approximately equivalent to 1 mouse (i.n.) LD50 (95% confidence intervals of 3.43–4.7 mg/ kg body weight). Antibody treatments were performed on mice anesthetized as above and a volume of 50 ml of anti-abrin antibody preparation was delivered intranasally (2 25 ml) or intravenously at various time points following intoxication. 2.4. Analysis of lung cells by flow cytometry Lungs were harvested, cut into small pieces and digested for 2 h at 37 C with 4 mg/ml collagenase D (Roche) in PBS containing Ca+2 and Mg+2 (Biological Industries). The tissue was then meshed through a 40 mm cell strainer and red blood cells were lysed with ACK lysis buffer (150 mM NH4Cl, and 10 mM KHCO3). Cells were costained for surface markers in a flow cytometry buffer (PBS with 2% FCS, 0.1% sodium azide, and 5 mM EDTA) using the antibodies antiGr-1 (RB6-8C5, BioLegend) and anti-CD11c (N418, eBioscience). Neutrophils were gated as CD11c Gr-1high, macrophages as
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CD11chigh Gr-1low. The analysis was performed using FACSCalibur (BD Biosciences) and FlowJo software (Tree Star). 2.5. Bronchoalveolar lavage fluid (BALF) preparation and analysis BAL was performed by flushing the lungs with 1 ml of PBS using a tracheal cannula. The BAL fluid was centrifuged at 950g at 4 C for 10 min and supernatants were collected and stored at 20 C until use. Levels of IL-1b, IL-6, TNF-a, MIP-2 and ANGII were determined by ELISA using commercial kits purchased from R&D Systems. Cholinesterase (ChE) enzymatic activity was measured according to Ellman et al. (1961), in assays performed in the presence of 0.5 mM acetylthiocholine, 50 mM sodium phosphate buffer pH 8.0, 0.1 mg/ml BSA and 0.3 mM 5,50 -dithiobis-(2-nitrobenzoic acid). The assay was carried out at 27 C and monitored by a Thermomax microplate reader (Molecular Devices). 2.6. Histology Lungs were perfused with PBS, removed from the animal and placed in fresh 4% neutral buffered formalin at room temperature for 2 weeks prior to processing and embedding. Sections (5 mM) from each sample were stained with hematoxylin and eosin (H&E) for histopathological evaluation. 2.7. Statistical analysis Individual groups were compared using unpaired t test analysis. To estimate p values, all statistical analyses were interpreted in a two-tailed manner. Values of p < 0.05 were considered to be statistically significant. Kaplan–Meier analysis was performed for survival curves. All data are presented as means SEM. 3. Results 3.1. Pathogenesis of pulmonary abrin intoxication Intranasal instillation of a lethal dose of crude abrin (2 LD50, 8 mg/kg body weight) to mice, results in the death of 95% of the animals with a mean time to death (MTTD) value of 5.3 1.1 days. Hematological analysis of blood samples from abrin-intoxicated mice revealed that elevated neutrophil counts could be detected in the circulation at 24 h post-exposure (PE, 2 fold increase over control) and further increase in neutrophil levels (>4-fold increase over control) was observed at 72 hPE (Fig. 1). Massive infiltration of neutrophils to the lungs was observed, as exemplified by the histological inspection of the lungs of intoxicated mice at 48 h post-exposure. In addition, eosin-stained edema fluid, most strikingly in the vicinity of the bronchioles and blood vessels, as well as abnormal architecture of the alveoli could be discerned (Fig. 2). It is highly likely from the reported damage that at this stage, gas-exchange has been severely impaired. To further characterize the abrin-induced inflammation, proinflammatory markers were measured in the BALF at early (24 h PE) and late (72 h PE) time-points. IL1-b, TNF-a, MIP-2 and angiotensin II (ANGII) displayed a transient pattern of expression following intranasal exposure to abrin; while BALF levels of these markers were significantly elevated at 24 h PE, at 72 h PE they returned to be at least as low as control levels (Fig. 3A). In addition, different cell type populations were quantified following abrin intoxication and monitored by flow cytometry. In contrast to the transient increase of the pro-inflammatory markers, neutrophil counts continued to rise at least up to 72 h post-exposure. At this time point, the neutrophils account for 50% of the lung associated cells, as opposed to 4% in naïve mice. A decrease of >75% in the number of the macrophages was observed at 72 h post-exposure,
Fig. 1. Neutrophil counts in the peripheral blood of mice intranasally exposed to abrin. Neutrophil counts were determined in peripheral blood samples collected from the tail vein of mice, immediately before i.n. exposure to 2LD50 (8 mg/kg) of abrin or at the indicated time-points thereafter. Data, shown as fold-increase over neutrophil counts at T = 0, represent the mean SEM of 3 mice per group. Significant differences from T = 0 are indicated by an asterisk (*), p < 0.01.
as well. Unlike the other pro-inflammatory markers detailed above, IL-6 levels continued to rise up to the later time-point measured (3-fold increase between 24 and 72 h PE). Hypervascular damage was monitored in the BALF of intoxicated mice as well, by measuring BALF levels of a serum-resident protein, cholinesterase (ChE), which under normal conditions is not found in the lungs. ChE levels at 24 and 72 h PE were respectively 8- and 200-fold higher than in control mice (Fig. 3B). Taken together, these findings show that pulmonary abrin intoxication launches a severe inflammation in the lungs, which in turn probably leads to respiratory insufficiency, the latter being the direct cause for the ensuing death. 3.2. Anti-abrin antibody treatment against intranasal intoxication of mice Previous studies have shown that following intranasal instillation of a lethal dose of ricin, mice were protected by treatment with anti-ricin antibodies and that survival rates correlated inversely to the lapse of time between exposure and treatment (Gal et al., 2014). We now examined whether pulmonary intoxication by the closely-related toxin, abrin, could be similarly alleviated by antibody treatment, utilizing anti-abrin antiserum prepared at our laboratory from hyperimmune rabbits. Characterization of this antibody preparation was performed by ELISA towards purified abrin, and by measuring neutralizing titers in a protein synthesis inhibition assay utilizing an acetylcholinesterase-secreting cellline (Cohen et al., 2014). The anti-abrin antibodies display ELISA and neutralizing titers (2.5 106 and 5 105, respectively) similar to those determined for the anti-ricin antibodies used in the ricin protection experiments (5 106 and 5 105, respectively). Likewise, the apparent affinity of the specific fraction of either antiricin or anti-abrin antibodies that was isolated from the hyperimmune sera were also found to have the same value (KD of 1 nM). The high similarity between the two anti-toxin preparations, allowed us to compare the protection efficiency of anti-abrin antibody-based treatment, to that determined previously for ricin intoxication (Gal et al., 2014). To this end, a lethal dose of abrin (8 mg/kg body weight) was instilled intranasally to groups of mice
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Fig. 2. Pathological evaluation of mice lungs following intranasal exposure to abrin. Histological sections of lungs (H&E staining) harvested from A: control mouse, B: mouse at 48 h PE to abrin. Arrows refer to neutrophils (black), eosin-stained edema fluid (green) and demolished architecture of alveoli (blue). Magnification = 100. Bar = 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Pathological indices in lungs and in BALF of abrin intoxicated mice. Mice (5 mice/group) were intoxicated intranasally with abrin (8 mg/kg) and BALF was collected at 24 or 72 h PE. (A) BALF levels of IL1-b, TNF-a, MIP-2 and ANGII (B) Number of neutrophils and macrophages in the lungs, and levels of IL-6 and ChE in the BALF. Normal levels determined in naïve mice, are indicated as “0”. Data represent the mean SEM.
and at various time points following intoxication, mice were intranasally administered a fixed volume (50 ml) of anti-abrin antiserum prepared at our laboratory from hyperimmune rabbits. Mice were monitored for survival for 14 days following exposure. Administration of anti-abrin antibodies at 6 h PE, conferred protection to 100% of the mice. Treatment with antibodies at a later time point, 24 h PE, also conferred high-level protection; in this case, 85% of the mice survived (Fig. 4). Surprisingly, high survival rates were achieved even when the antibody treatment was administered considerably later. Thus, anti-abrin antibody treatment at 48–72 h PE, protected over 70% of the mice (Fig. 4). These exceedingly high protection rates were not dependent on the intranasal route of antibody administration, since intravenous
application of the antibodies at 48 h PE also gave rise to nearly 80% protection (Fig. 4). 3.3. Effect of anti-toxin antibody treatment on lung cell composition and inflammatory markers In contrast to the highly efficient anti-abrin treatment detailed above, survival profiles determined at our laboratory for antibodybased treatment against ricin intoxication, demonstrate that in the latter case, significantly lower levels of protection are attained (Gal et al., 2014). We therefore set out to examine and compare the effects of the antibody treatment against the two toxins, by quantitative analyses of both lung cell composition and
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and ricin-intoxicated mice. Thus, neutrophil counts in both mice treated against abrin or against ricin were approximately 30% of those observed in the corresponding intoxicated mice that were not subjected to antibody treatment (Fig. 5A). Similarly, in the case of macrophages, where intoxication brings about a significant reduction (>75%) in cell counts, antibody treatment at 24 h PE prompted full recovery of the cell populations, both in abrin- and in ricin- intoxicated mice (Fig. 5B). Likewise, we could not discern any significant difference between the antibody treatments against the two toxins, with regard to their effect on vascular permeability. Quantitation of ChE, in BALF samples, demonstrated that neither the antibody-based treatment against abrin, nor that against ricin conferred any beneficial influence with regard to this vascular hyperpermeability marker; in both cases, ChE levels in the antibody-treated mice (150–200-fold higher than in control mice) were similar to those measured in intoxicated non-treated mice (Fig. 5C). In sharp contrast, a marked difference between the effect of antibody-treatment against abrin and ricin was observed with regard to the pro-inflammatory cytokine, IL-6. While antiricin treatment did not affect the levels of IL-6, antibody treatment following abrin exposure significantly reduced IL-6 levels to 10% of those measured in intoxicated non-treated mice (Fig. 5D). Thus, only levels of this cytokine were found to be differentially affected by anti-abrin and anti-ricin treatment. Fig. 4. Therapeutic window of anti-abrin antibody treatment to abrin-intoxicated mice. Groups of mice were intoxicated intranasally with abrin (8 mg/kg) and treated with anti-abrin antibodies at the indicated time-points. Mode of antibody administration, survival rates, and survivor mice counts are indicated below for each experimental group. The term “Total” in the right column, refers to the number of mice that were subjected to antibody treatment in each experimental group. Abrin-intoxicated mice that were not treated with antibodies served as a control (group 6). For this group, data pooled from several experiments is shown.
proinflammatory markers, following anti-toxin antibody administration to mice exposed to abrin or ricin. Flow cytometric analysis of lung cells harvested 72 h PE from mice treated with anti-abrin or anti-ricin antibodies at 24 h PE to the corresponding toxin, revealed a significant reversion in the hematopoietic cell types, neutrophils and macrophages after both intoxications. Despite the difference in treatment efficacy, the effect of antibody treatment on neutrophil recruitment to the lung was similar in both abrin-
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4. Discussion In the present study, we followed in detail the progress of illness and recovery in mice that were intranasally exposed to a lethal dose of abrin and treated with anti-abrin antibodies. Abrin is phylogenetically non-related to ricin (a Ricinus communis plantderived-toxin), yet the two share a 50% homology in amino acid sequence and possess an identical catalytic activity. Thus, both toxins inactivate ribosomes irreversibly by site-specific depurination of the same adenine nucleotide within the 28S rRNA, thereby precipitating cessation of protein synthesis by the cell. The most prominent clinical manifestation following intranasal exposure of mice to abrin was the onset of a localized, yet severe, edematous inflammation. This inflammation was accompanied by a massive recruitment of neutrophils and the stimulation of a turbulent pro-inflammatory cytokine storm within the organ. In
Fig. 5. Effect of antibody treatment on pathological indices in abrin-intoxicated mice. Mice intranasally exposed to abrin (8 mg/kg) or ricin (7 mg/kg), were treated or not with anti-abrin or anti-ricin antibodies, respectively, at 24 h PE. At 72 h PE, lungs were either removed and neutrophil and macrophage numbers, represented by bars, were determined by flow cytometry or were lavaged and the levels of ChE and IL-6 were determined in the BALF. Control: no exposure; Ricin + Ab = ricin-intoxicated followed by anti-ricin antibody treatment; Abrin + Ab = abrin-intoxicated followed by anti-abrin antibody treatment. Number of mice per group: Control and antibody-treated (Ricin + Ab, Abrin + Ab) groups, n = 10; Intoxicated non-treated (Ricin, Abrin) groups, n = 5–6. Data represent mean SEM. Significant (P < 0.05) differences between antibody-treated mice and corresponding intoxicated non-treated mice are indicated by an asterisk (*).
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the past, we reported that i.n. instillation of a lethal dose of ricin to mice also induced a severe pulmonary inflammation, which in turn led to respiratory failure and death (Gal et al., 2014). Hence, a striking similarity exists between the pathologies ensuing abrinand ricin-intranasal exposures. Surprisingly, despite this resemblance in morbidity, the ability to protect mice against the two intoxications by post-exposure antibody-mediated treatment differs radically. In the case of ricin intoxication at a 2LD50 dose, anti-ricin antibody-based treatment at 24 h PE led to the protection of 34% of the mice (Gal et al., 2014), similar to the protection level attained by others at this time-point (Pratt et al., 2007). When antibody treatment was administered very late after ricin intoxication, e.g., 48 h PE, protection was no more than marginal (<8% survival). In contrast, administration of polyclonal anti-abrin antibodies to mice intranasally exposed to abrin at a dose of 2LD50, led to extraordinarily high survival rates even when the antibodies were applied late after intoxication. Thus, passive immunization of mice with anti-abrin polyclonal antibodies led to >70% protection even when this treatment was applied as late as 72 h PE. When antibody treatment was applied at 96 h PE, survival rates declined to 50%, but even these rates were higher than those observed for ricin-intoxicated mice that were treated at 24 h PE (Gal et al., 2014). It should be noted that when treatment was administered at 72 h PE some of the abrin-exposed mice have already died and these were not considered in the survival rate calculations. The fact that such high levels of protection are reached even at a time-point at which 10% of the mice have already succumbed, intensifies our findings. The sharp divergence in the ability to protect mice against the two toxins is further confounded by the fact that under our experimental conditions in which mice were exposed to ricin or abrin at a dose of 2LD50, the MTTD values for the two toxins are very similar, 4.9 0.8 and 5.3 1.1 days, respectively. Since treatment against abrin or ricin intoxications is based on the use of anti-toxin antibodies, one may argue that the differential protectivity against the two toxins stems from differences in the quality of the antibody preparations. However, this seems unlikely, as we have demonstrated that the two anti-toxin preparations resemble one another by their ELISA and neutralizing endpoint titers, as well as by their apparent affinity towards the corresponding toxins. Abrin and ricin operate at the molecular level, in an identical manner by inactivating the ribosomes of the cell. In vitro protein synthesis inhibition studies carried out at our laboratory allowed us to determine that the catalytic activity of abrin was in fact 9fold more potent than that of ricin (ED50 = 0.7 0.07 and 6.6 0.9 ng/ml for abrin and ricin, respectively, in an in vitro translation inhibition assay), suggesting that the ability of abrin to impair ribosomes in an irreversible manner would be even greater than that of ricin. Similar results, suggesting that catalytic activity of abrin is superior to that of ricin, were reported by others as well (Barbieri et al., 2004). However, one should keep in mind that the catalytic activity, which resides in the A subunit of the toxin, is fully manifested only following the dissociation of its subunits (Tsai et al., 2002; Barbieri et al., 2004). In vivo, subunit dissociation occurs at a relatively late stage, after the holotoxin has entered the endoplasmic reticulum (Roberts and Smith, 2004) and does not determine the overall rate of intoxication. Rather, binding to the cell surface, entry and intracellular trafficking of ricin or abrin through the trans-Golgi and Golgi compartments to the ER, all of which precede subunit dissociation and enzymatic catalysis, comprise the rate-determining steps for in vivo ricin- and abrinintoxication. The fact that the therapeutic window for antibody treatment against abrin-intoxication is so much broader compared to ricin-intoxication, may however suggest that the rate of entry of abrin into target cells is slower, hence, its availability for effective antibody neutralization is higher over a greater period of time. To
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date, the kinetics of in vivo intoxications by ricin and abrin have not been compared. Elaborate studies aimed to measure the cellbinding rates of the two toxins within the lungs of experimental animals, as well as to determine the lung cell types which undergo toxin-mediated damage, are now being carried out at our laboratory. However, catalytic disruption of ribosomes and the ensuing cessation of protein synthesis are only one aspect of ricin- and abrin-mediated intoxication. The clinical manifestations following pulmonary exposure to the two toxins, are those of lung inflammation. How does the inflammatory state relate to the catalytic activity of ricin and abrin? Previous studies expounded the view that the actual damage of ribosomes triggers the onset of inflammation and have coined this phenomenon as “ribotoxic stress response” (Iordanov et al., 1997; Korcheva et al., 2005, 2007). In another study, it was proposed that ricin-mediated translational inhibition fostered the disappearance of labile proteins that normally suppress inflammasome formation, thereby inducing an inflammatory response (Lindauer et al., 2009, 2010). Common to these studies is the assertion that catalytic inactivation of ribosomes is a prerequisite for induction of the inflammatory reaction. Do ricin and abrin differ in their ability to elicit pulmonary inflammation? Although exposure to either abrin or ricin leads to elevated expression of a similar set of cytokines, we note that the measured levels of some of the proinflammatory cytokines, i.e., TNF-a, MIP-2 and ANGII, were consistently higher in the BALF of mice which were intranasally exposed to ricin at 24 h PE. In contrast, measurement at 72 h PE of IL-6 in the BALF of intoxicated mice demonstrated that levels of this cytokine were perhaps even higher in the abrin-intoxicated mice. However, the fate of IL-6 following antibody-treatment was profoundly different in abrin- and ricin-intoxicated mice. While anti-abrin antibody treatment at 24 h PE brought about a 90% reduction in IL-6 levels by 72 h PE, application of anti-ricin antibodies to ricin-intoxicated mice did not affect IL-6 levels whatsoever and these remained at least as high as in intoxicated mice that were not subjected to antibody treatment. We note in this context, that various studies have documented a positive correlation between IL-6 levels and excessive pulmonary inflammation in pre-clinical models of lung injuries (Hubeau et al., 2013; Yu et al., 2002; Zhang et al., 2013). Thus, one may be tempted to draw a line between the superior ability to protect against abrin intoxication and the fact that antibody treatment uniquely reduces IL-6 levels in mice exposed to this toxin, yet this apparent correlation remains at present circumstantial and should be further probed. To further delineate the factors/properties responsible for the differential ability to protect against abrin and ricin, we have now generated two chimeric heterologous toxins utilizing monomeric subunits isolated from abrin and ricin. Protection experiments in mice exposed to a lethal dose of the chimeric toxins, AabrinBricin or AricinBabrin, will allow us to determine which of the subunits of abrin source, A or B, confers the uniquely high-efficient therapeutic response to antibody treatment over long periods of time. Acknowledgments We would like to thank Ron Alcalay and Nehama Seliger for their excellent technical assistance. References Audi, J., Belson, M., Patel, M., Schier, J., Osterloh, J., 2005. Ricin poisoning: a comprehensive review. JAMA 294, 2342–2351. Barbieri, L., Ciani, M., Girbes, T., Liu, W.-Y., Van Damme, E.J.M., Peumans, W.J., Stirpe, F., 2004. Enzymatic activity of toxic and non-toxic type 2 ribosome-inactivating proteins. FEBS Lett. 563, 219–222.
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Cohen, A., Mechaly, A., Sabo, T., Alcalay, R., Aloni-Grinstein, R., Seliger, N., Kronman, C., Mazor, O., 2014. Characterization and epitope mapping of the polyclonal antibody repertoire elicited by ricin-holotoxin based vaccination. Clin. Vaccine Immunol. 21, 1534–1540. Ellman, G.L., Courtney, K.D., Andres, V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95. Gal, Y., Mazor, O., Alcalay, R., Seliger, N., Aftalion, M., Sapoznikov, A., Falach, R., Kronman, C., Sabo, T., 2014. Antibody/doxycycline combined therapy for pulmonary ricinosis: attenuation of inflammation improves survival of ricinintoxicated mice. Toxicol. Rep. 1, 496–504. Griffiths, G.D., Phillips, G.J., Holley, J., 2007. Inhalation toxicology of ricin preparations: animal models, prophylactic and therapeutic approaches to protection. Inhal. Toxicol. 19, 873–887. Hegde, R., Podder, S.K., 1992. Studies on the variants of the protein toxins ricin and abrin. Eur. J. Biochem. 204, 155–164. Hubeau, C., Kubera, J.E., Masek-Hammerman, K., Williams, C.M.M., 2013. Interleukin-6 neutralization alleviates pulmonary inflammation in mice exposed to cigarette smoke and poly(I:C). Clin. Sci. 125, 483–493. Iordanov, M.S., Pribnow, D., Magun, J.L., Dinh, T.-H., Pearson, J.A., Chen, S.L.-Y., Magun, B.E., 1997. Ribotoxic stress response: activation of the stress-activated protein kinase JNK1 by inhibitors of the peptidyl transfer reaction and by sequence-specific RNA damage to the a-sarcin/ricin loop in the 28S rRNA. Mol. Cell Biol. 17, 3373–3381. Korcheva, V., Wong, J., Lindauer, M., Jacoby, D.B., Iordanov, M.S., Magun, B., 2007. Role of apoptotic signaling pathways in regulation of inflammatory responses to ricin in primary murine macrophages. Mol. Immunol. 44, 2761–2771. Lindauer, M.L., Wong, J., Iwakura, Y., Magun, B.E., 2009. Pulmonary inflammation triggered by ricin toxin requires macrophages and IL-1 signaling. J. Immunol. 183, 1419–1426. Lindauer, M., Wong, J., Magun, B.E., 2010. Ricin toxin activates the NALP-3 inflammasome. Toxins 2, 1500–1514. Maddaloni, M., Cooke, C., Wilkinson, R., Stout, A.V., Eng, L., Pincus, S.H., 2004. Immunological characteristics associated with the protective efficacy of antibodies to ricin. J. Immunol. 172, 6221–6228. Olsnes, S., 2004. The history of ricin abrin and relate toxins. Toxicon 44, 361–370.
Olsnes, S., Pihl, A., 1973. Isolation and properties of abrin: a toxic protein inhibiting protein synthesis. Eur. J. Biochem. 35, 179–185. Pratt, T.S., Pincus, S.H., Hale, M.L., Moreira, A.L., Roy, C.J., Tchou-Wong, K.M., 2007. Oropharyngeal aspiration of ricin as a lung challenge model for evaluation of the therapeutic index of antibodies against ricin A-chain for post-exposure treatment. Exp. Lung Res. 33, 459–481. Prigent, J., Panigai, L., Lamourette, P., Sauvaire, D., Devilliers, K., Plaisance, M., Volland, H., Creminon, C., Simon, S., 2011. Neutralising antibodies against ricin toxin. PLoS One 6, e20166. Roberts, L.M., Smith, D.C., 2004. Ricin: the endoplasmic reticulum connection. Toxins 44, 469–472. Surendranath, K., Karande, A.A., 2008. A neutralizing antibody to the A chain of abrin inhibits abrin toxicity both in vitro and in vivo. Clin. Vaccine Immunol. 15, 737–743. Tsai, B., Ye, Y., Rapoport, T.A., 2002. Retro-translocation of proteins from the endoplasmic reticulum into the cytosol. Nat. Rev. Cell Biol. 3, 246–255. Wang, Y., Feng, J., Guo, J., Guo, L., Li, Y., Sun, Y., Qin, W., Hu, M., Han, G., Shen, B., 2006. A novel designed single domain antibody on 3-D structure of ricin A chain remarkably blocked ricin-induced cytotoxicity. Mol. Immunol. 43, 1912–1919. Wang, Y., Guo, L., Zhao, K., Chen, J., Feng, J., Sun, Y., Li, Y., Shen, B., 2007. Novel chimeric anti-ricin antibody c4C13 with neutralizing activity against ricin toxicity. Biotechnol. Lett. 29, 1811–1816. Wei, C.H., Pfuderer, P., Yang, W.C., 1974. Purification and characterization of the two major toxic proteins from seeds of Abrus precatorius. J. Biol. Chem. 249, 3061– 3067. Yu, M., Zheng, X., Witschi, H., Pinkerton, K.E., 2002. The role of interleukin-6 in pulmonary inflammation and injury induced by exposure to environmental air pollutants. Toxicol. Sci. 68, 488–497. Zhang, H., Neuhofer, P., Song, L., Rabe, B., Lesina, M., Kurkowski, M.U., Treiber, M., Wartmann, T., Regner, S., Thorlacius, H., Saur, D., Weirich, G., Yoshimura, A., Halangk, W., Mizerd, J.P., Schmid, R.M., Rose-John, S., Algul, H., 2013. IL-6 transsignaling promotes pacreatitis-associated lung injury and lethality. J. Clin. Invest. 123, 1019–1031. Zhou, H., Zhou, B., Ma, H., Carney, C., Janda, K.D., 2007. Selection and characterization of human monoclonal antibodies against abrin by phage display. Bioorg. Med. Chem. Lett 17, 5690–5692.
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Antibody treatment against pulmonary exposure to abrin confers significantly higher levels of protection than treatment against ricin intoxication Tamar Sabo, Yoav Gal, Eitan Elhanany, Anita Sapoznikov, Reut Falach, Ohad Mazor, Chanoch Kronman* Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, Israel
H I G H L I G H T S
Pulmonary exposure of mice to abrin leads to neutrophilic inflammation of the lungs. Lung pathology following abrin intoxication resembles that of ricin. Efficient Ab-based protection is achieved following abrin but not ricin exposure. Ab treatment against abrin, but not against ricin, reduces pulmonary IL-6 levels.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 February 2015 Received in revised form 14 April 2015 Accepted 2 June 2015 Available online 5 June 2015
Abrin, a potent plant-derived toxin bearing strong resemblance to ricin, irreversibly inactivates ribosomes by site-specific depurination, thereby precipitating cessation of protein synthesis in cells. Due to its high availability and ease of preparation, abrin is considered a biological threat, especially in context of bioterror warfare. To date, there is no established therapeutic countermeasure against abrin intoxication. In the present study, we examined the progress of pulmonary abrin intoxication in mice, evaluated the protective effect of antibody-based post-exposure therapy, and compared these findings to those observed for ricin intoxication and therapy. Salient features of abrin intoxication were found to be similar to those of ricin and include massive recruitment of neutrophils to the lungs, high levels of proinflammatory markers in the bronchoalveolar lavage fluid and damage of the alveolar-capillary barrier. In contrast, the protective effect of anti-abrin antibody treatment was found to differ significantly from that of anti-ricin treatment. While anti-ricin treatment efficiency was quite limited even at 24 h postexposure (34% protection), administration of polyclonal anti-abrin antibodies even as late as 72 h postexposure, conferred exceedingly high-level protection (>70%). While both anti-toxin antibody treatments caused neutrophil and macrophage levels in the lungs to revert to normal, only anti-abrin treatment brought about a significant decline in the pulmonary levels of the pro-inflammatory cytokine IL-6. The differential ability of the anti-toxin treatments to dampen inflammation caused by the two similar toxins, abrin and ricin, could explain the radically different levels of protection achieved following antibody treatment. ã2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Abrin Ricin Pulmonary Antibodies BALF IL-6
1. Introduction
Abbreviations: ANGII, angiotensin-II; BALF, bronchoalveolar lavage fluid; ChE, cholinesterase; RIP-2, ribosome inactivating protein-2; MTTD, mean time to death; IL-6, interleukin-6; MIP-2, macrophage inflammatory protein-2; PBS, phosphatebuffered saline; TNF-a, tumor necrosis factor-a. * Corresponding author at: 19 Reuven Lerer St., P.O.B. 19 Ness-Ziona 74100, Israel. E-mail address: [email protected] (C. Kronman). http://dx.doi.org/10.1016/j.toxlet.2015.06.003 0378-4274/ ã 2015 Elsevier Ireland Ltd. All rights reserved.
Abrin, a plant toxin derived from rosary peas (Abrus precatorius) belongs to the type 2 ribosome inactivating (RIP-2) group of proteins. The heterodimeric glycoprotein consists of two subunits, subunit A, which carries the enzymatic activity of the toxin and subunit B, which is a lectin that binds to eukaryotic cell surfaces via
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glycoproteins and glycolipids containing b-1,4-linked galactose residues (Olsnes, 2004). Upon entry to the cytosol, the A-subunit exerts its toxic effect by site-specific depurination of adenine 4324 of the 28S rRNA in the 60S ribosomal subunit. This irreversible impairment prevents binding of elongation factor-2 to the ribosome, leading in turn to protein synthesis arrest and cell death (Audi et al., 2005). Due to its high availability, ease of preparation and high toxicity, abrin is considered a potential bioterror agent and is classified as a Category B agent by the US Center for Disease Control and Prevention (CDC). As in the case of the similar RIP-2 toxin, ricin, the toxicity of abrin depends on the route of exposure, inhalatory exposure being considered most hazardous (Audi et al., 2005). Though abrin is considered more toxic than ricin in the case of oral or systemic exposures (Wei et al., 1974), the LCt50 values in rats for inhaled ricin and abrin were found to be quite similar, 4.54–5.96 and 4.54 mg min m3, respectively (Griffiths et al., 2007). To date, there are no established post-exposure therapeutic countermeasures for ricin or abrin exposures. Some studies however, have been carried out to evaluate the potency and timeframe for anti-ricin antibody-based passive immunization following exposure of experimental animals to lethal doses of ricin (Maddaloni et al., 2004; Wang et al., 2006, 2007; Prigent et al., 2011). Recently, we evaluated the efficiency of postexposure treatment of pulmonary ricinosis by polyclonal antiricin antibodies, and demonstrated that survival rates can be increased by combining the antidotal treatment with the antiinflammatory compound, doxycycline (Gal et al., 2014). In contrast to ricin, research of anti-abrin antibody-based treatments is relatively meager. In one study, phage display technology was utilized to select anti-abrin human monoclonal antibodies from a human naïve scFv library (Zhou et al., 2007), however these were not examined in animal models for their therapeutic value. In another study, a monoclonal antibody mapped to abrin A-chain, was found to protect mice from abrin challenge when given 1 h prior to exposure (Surendranath and Karande, 2008). The absence of a substantial body of experimental data regarding abrin toxicology and medical countermeasures against pulmonary exposure to this toxin, prompted us in the present study, to delineate the salient features of pulmonary abrin intoxication and to assess the therapeutic potential of passive immunization in mice. 2. Materials and methods 2.1. Extraction and purification of abrin Crude extract was prepared from A. precatorius seeds (18 gr.) essentially as described previously (Olsnes and Pihl, 1973; Hegde and Podder, 1992). Briefly, seed kernels were soaked in 5% acetic acid/phosphate buffer (Na2HPO4, pH7.4) overnight and homogenized in a Waring blender for 4 sequences of 30 s. Proteins obtained from 80% ammonium sulfate precipitation were centrifuged and dialyzed extensively against PBS. Crude abrin protein concentration was determined as 7.5 mg/ml by 280nm absorption (Nanodrop). For the preparation of pure toxin, crude abrin was loaded on 2 columns in-tandem, the first column contains activated Sepharose which binds and thereby depletes the A. precatorius Agglutinin (APA) and the second column, containing a-lactose (lactamyl) agarose, binds the abrin toxin. Proteins bound to the lactamyl agarose column were eluted with 0.5 M Galactose in PBS. Tryptic digest analysis by MALDI-TOF/MS showed that both a and b isoforms of the abrin toxin are present in the galactose eluate.
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2.2. Anti-abrin antibodies Rabbits were immunized with purified abrin toxin in a stepwise manner, injections I–III containing 4, 16 and 50 mg toxin/rabbit respectively, were on alum (Al(OH)3). Injection i.v. was in Freund’s complete adjuvant (100 mg toxin/rabbit) and subsequent injections were in Freund’s incomplete adjuvant (100 mg toxin/rabbit) at 4-week intervals. Blood samples were collected (3 weeks after injection) to ascertain anti-abrin antibody titer build-up. Immunization was continued until steady high anti-abrin titers were obtained. Anti-abrin antibody titers were determined by ELISA. Microtiter plates (Nunc) were coated with purified abrin (2.5 mg/ml in carbonate buffer pH 9.6, overnight incubation at 4 C), washed 3 times in wash buffer (0.8% NaCl/0.05% Tween 20) and then incubated with blocking buffer (PBS/0.05% Tween 20/2% BSA) for 1 h at 37 C. Rabbit antisera samples were added in 2-fold serial dilutions and incubated at 37 C for 1 h. Plates were then washed 3 times with wash buffer and incubated at 37 C for 1 h with APconjugated goat anti-rabbit immunoglobulin (Sigma, 1:500 in blocking buffer). After washing as above, the microtiter plates were developed with substrate (p-NPP, Sigma) and optical densities were measured at 405 nm using an ELISA reader (Molecular Devices). 2.3. Animal studies Animal experiments were performed in accordance with the Israeli law and were approved by the Ethics Committee for animal experiments at the Israel Institute for Biological Research. Treatment of animals was in accordance with regulations outlined in the USDA Animal Welfare Act and the conditions specified in the Guide for Care and Use of Laboratory Animals (National Institute of Health). All animals in this study were female CD-1 mice (Charles River Laboratories Ltd., UK) weighing 27–32 g. Prior to exposure, animals were habituated to the experimental animal unit for 5 days. All mice were housed in filter-top cages in an environmentally controlled room and maintained at 21 2 C and 55 10% humidity. Lighting was set to mimic a 12/12 h dawn to dusk cycle. Animals had access to food and water ad libitum. For the procedure of intoxication, mice were anesthetized by intraperitoneal (i.p.) injection of ketamine (1.9 mg/mouse) and xylazine (0.19 mg/mouse). Crude abrin (50 ml; 8 mg/kg diluted in PBS) was applied intranasally (i.n., 2 25 ml) and mortality was monitored over 14 days. Preceding these studies, we determined that 4.0 mg crude abrin/kg body weight is approximately equivalent to 1 mouse (i.n.) LD50 (95% confidence intervals of 3.43–4.7 mg/ kg body weight). Antibody treatments were performed on mice anesthetized as above and a volume of 50 ml of anti-abrin antibody preparation was delivered intranasally (2 25 ml) or intravenously at various time points following intoxication. 2.4. Analysis of lung cells by flow cytometry Lungs were harvested, cut into small pieces and digested for 2 h at 37 C with 4 mg/ml collagenase D (Roche) in PBS containing Ca+2 and Mg+2 (Biological Industries). The tissue was then meshed through a 40 mm cell strainer and red blood cells were lysed with ACK lysis buffer (150 mM NH4Cl, and 10 mM KHCO3). Cells were costained for surface markers in a flow cytometry buffer (PBS with 2% FCS, 0.1% sodium azide, and 5 mM EDTA) using the antibodies antiGr-1 (RB6-8C5, BioLegend) and anti-CD11c (N418, eBioscience). Neutrophils were gated as CD11c Gr-1high, macrophages as
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CD11chigh Gr-1low. The analysis was performed using FACSCalibur (BD Biosciences) and FlowJo software (Tree Star). 2.5. Bronchoalveolar lavage fluid (BALF) preparation and analysis BAL was performed by flushing the lungs with 1 ml of PBS using a tracheal cannula. The BAL fluid was centrifuged at 950g at 4 C for 10 min and supernatants were collected and stored at 20 C until use. Levels of IL-1b, IL-6, TNF-a, MIP-2 and ANGII were determined by ELISA using commercial kits purchased from R&D Systems. Cholinesterase (ChE) enzymatic activity was measured according to Ellman et al. (1961), in assays performed in the presence of 0.5 mM acetylthiocholine, 50 mM sodium phosphate buffer pH 8.0, 0.1 mg/ml BSA and 0.3 mM 5,50 -dithiobis-(2-nitrobenzoic acid). The assay was carried out at 27 C and monitored by a Thermomax microplate reader (Molecular Devices). 2.6. Histology Lungs were perfused with PBS, removed from the animal and placed in fresh 4% neutral buffered formalin at room temperature for 2 weeks prior to processing and embedding. Sections (5 mM) from each sample were stained with hematoxylin and eosin (H&E) for histopathological evaluation. 2.7. Statistical analysis Individual groups were compared using unpaired t test analysis. To estimate p values, all statistical analyses were interpreted in a two-tailed manner. Values of p < 0.05 were considered to be statistically significant. Kaplan–Meier analysis was performed for survival curves. All data are presented as means SEM. 3. Results 3.1. Pathogenesis of pulmonary abrin intoxication Intranasal instillation of a lethal dose of crude abrin (2 LD50, 8 mg/kg body weight) to mice, results in the death of 95% of the animals with a mean time to death (MTTD) value of 5.3 1.1 days. Hematological analysis of blood samples from abrin-intoxicated mice revealed that elevated neutrophil counts could be detected in the circulation at 24 h post-exposure (PE, 2 fold increase over control) and further increase in neutrophil levels (>4-fold increase over control) was observed at 72 hPE (Fig. 1). Massive infiltration of neutrophils to the lungs was observed, as exemplified by the histological inspection of the lungs of intoxicated mice at 48 h post-exposure. In addition, eosin-stained edema fluid, most strikingly in the vicinity of the bronchioles and blood vessels, as well as abnormal architecture of the alveoli could be discerned (Fig. 2). It is highly likely from the reported damage that at this stage, gas-exchange has been severely impaired. To further characterize the abrin-induced inflammation, proinflammatory markers were measured in the BALF at early (24 h PE) and late (72 h PE) time-points. IL1-b, TNF-a, MIP-2 and angiotensin II (ANGII) displayed a transient pattern of expression following intranasal exposure to abrin; while BALF levels of these markers were significantly elevated at 24 h PE, at 72 h PE they returned to be at least as low as control levels (Fig. 3A). In addition, different cell type populations were quantified following abrin intoxication and monitored by flow cytometry. In contrast to the transient increase of the pro-inflammatory markers, neutrophil counts continued to rise at least up to 72 h post-exposure. At this time point, the neutrophils account for 50% of the lung associated cells, as opposed to 4% in naïve mice. A decrease of >75% in the number of the macrophages was observed at 72 h post-exposure,
Fig. 1. Neutrophil counts in the peripheral blood of mice intranasally exposed to abrin. Neutrophil counts were determined in peripheral blood samples collected from the tail vein of mice, immediately before i.n. exposure to 2LD50 (8 mg/kg) of abrin or at the indicated time-points thereafter. Data, shown as fold-increase over neutrophil counts at T = 0, represent the mean SEM of 3 mice per group. Significant differences from T = 0 are indicated by an asterisk (*), p < 0.01.
as well. Unlike the other pro-inflammatory markers detailed above, IL-6 levels continued to rise up to the later time-point measured (3-fold increase between 24 and 72 h PE). Hypervascular damage was monitored in the BALF of intoxicated mice as well, by measuring BALF levels of a serum-resident protein, cholinesterase (ChE), which under normal conditions is not found in the lungs. ChE levels at 24 and 72 h PE were respectively 8- and 200-fold higher than in control mice (Fig. 3B). Taken together, these findings show that pulmonary abrin intoxication launches a severe inflammation in the lungs, which in turn probably leads to respiratory insufficiency, the latter being the direct cause for the ensuing death. 3.2. Anti-abrin antibody treatment against intranasal intoxication of mice Previous studies have shown that following intranasal instillation of a lethal dose of ricin, mice were protected by treatment with anti-ricin antibodies and that survival rates correlated inversely to the lapse of time between exposure and treatment (Gal et al., 2014). We now examined whether pulmonary intoxication by the closely-related toxin, abrin, could be similarly alleviated by antibody treatment, utilizing anti-abrin antiserum prepared at our laboratory from hyperimmune rabbits. Characterization of this antibody preparation was performed by ELISA towards purified abrin, and by measuring neutralizing titers in a protein synthesis inhibition assay utilizing an acetylcholinesterase-secreting cellline (Cohen et al., 2014). The anti-abrin antibodies display ELISA and neutralizing titers (2.5 106 and 5 105, respectively) similar to those determined for the anti-ricin antibodies used in the ricin protection experiments (5 106 and 5 105, respectively). Likewise, the apparent affinity of the specific fraction of either antiricin or anti-abrin antibodies that was isolated from the hyperimmune sera were also found to have the same value (KD of 1 nM). The high similarity between the two anti-toxin preparations, allowed us to compare the protection efficiency of anti-abrin antibody-based treatment, to that determined previously for ricin intoxication (Gal et al., 2014). To this end, a lethal dose of abrin (8 mg/kg body weight) was instilled intranasally to groups of mice
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Fig. 2. Pathological evaluation of mice lungs following intranasal exposure to abrin. Histological sections of lungs (H&E staining) harvested from A: control mouse, B: mouse at 48 h PE to abrin. Arrows refer to neutrophils (black), eosin-stained edema fluid (green) and demolished architecture of alveoli (blue). Magnification = 100. Bar = 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Pathological indices in lungs and in BALF of abrin intoxicated mice. Mice (5 mice/group) were intoxicated intranasally with abrin (8 mg/kg) and BALF was collected at 24 or 72 h PE. (A) BALF levels of IL1-b, TNF-a, MIP-2 and ANGII (B) Number of neutrophils and macrophages in the lungs, and levels of IL-6 and ChE in the BALF. Normal levels determined in naïve mice, are indicated as “0”. Data represent the mean SEM.
and at various time points following intoxication, mice were intranasally administered a fixed volume (50 ml) of anti-abrin antiserum prepared at our laboratory from hyperimmune rabbits. Mice were monitored for survival for 14 days following exposure. Administration of anti-abrin antibodies at 6 h PE, conferred protection to 100% of the mice. Treatment with antibodies at a later time point, 24 h PE, also conferred high-level protection; in this case, 85% of the mice survived (Fig. 4). Surprisingly, high survival rates were achieved even when the antibody treatment was administered considerably later. Thus, anti-abrin antibody treatment at 48–72 h PE, protected over 70% of the mice (Fig. 4). These exceedingly high protection rates were not dependent on the intranasal route of antibody administration, since intravenous
application of the antibodies at 48 h PE also gave rise to nearly 80% protection (Fig. 4). 3.3. Effect of anti-toxin antibody treatment on lung cell composition and inflammatory markers In contrast to the highly efficient anti-abrin treatment detailed above, survival profiles determined at our laboratory for antibodybased treatment against ricin intoxication, demonstrate that in the latter case, significantly lower levels of protection are attained (Gal et al., 2014). We therefore set out to examine and compare the effects of the antibody treatment against the two toxins, by quantitative analyses of both lung cell composition and
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and ricin-intoxicated mice. Thus, neutrophil counts in both mice treated against abrin or against ricin were approximately 30% of those observed in the corresponding intoxicated mice that were not subjected to antibody treatment (Fig. 5A). Similarly, in the case of macrophages, where intoxication brings about a significant reduction (>75%) in cell counts, antibody treatment at 24 h PE prompted full recovery of the cell populations, both in abrin- and in ricin- intoxicated mice (Fig. 5B). Likewise, we could not discern any significant difference between the antibody treatments against the two toxins, with regard to their effect on vascular permeability. Quantitation of ChE, in BALF samples, demonstrated that neither the antibody-based treatment against abrin, nor that against ricin conferred any beneficial influence with regard to this vascular hyperpermeability marker; in both cases, ChE levels in the antibody-treated mice (150–200-fold higher than in control mice) were similar to those measured in intoxicated non-treated mice (Fig. 5C). In sharp contrast, a marked difference between the effect of antibody-treatment against abrin and ricin was observed with regard to the pro-inflammatory cytokine, IL-6. While antiricin treatment did not affect the levels of IL-6, antibody treatment following abrin exposure significantly reduced IL-6 levels to 10% of those measured in intoxicated non-treated mice (Fig. 5D). Thus, only levels of this cytokine were found to be differentially affected by anti-abrin and anti-ricin treatment. Fig. 4. Therapeutic window of anti-abrin antibody treatment to abrin-intoxicated mice. Groups of mice were intoxicated intranasally with abrin (8 mg/kg) and treated with anti-abrin antibodies at the indicated time-points. Mode of antibody administration, survival rates, and survivor mice counts are indicated below for each experimental group. The term “Total” in the right column, refers to the number of mice that were subjected to antibody treatment in each experimental group. Abrin-intoxicated mice that were not treated with antibodies served as a control (group 6). For this group, data pooled from several experiments is shown.
proinflammatory markers, following anti-toxin antibody administration to mice exposed to abrin or ricin. Flow cytometric analysis of lung cells harvested 72 h PE from mice treated with anti-abrin or anti-ricin antibodies at 24 h PE to the corresponding toxin, revealed a significant reversion in the hematopoietic cell types, neutrophils and macrophages after both intoxications. Despite the difference in treatment efficacy, the effect of antibody treatment on neutrophil recruitment to the lung was similar in both abrin-
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4. Discussion In the present study, we followed in detail the progress of illness and recovery in mice that were intranasally exposed to a lethal dose of abrin and treated with anti-abrin antibodies. Abrin is phylogenetically non-related to ricin (a Ricinus communis plantderived-toxin), yet the two share a 50% homology in amino acid sequence and possess an identical catalytic activity. Thus, both toxins inactivate ribosomes irreversibly by site-specific depurination of the same adenine nucleotide within the 28S rRNA, thereby precipitating cessation of protein synthesis by the cell. The most prominent clinical manifestation following intranasal exposure of mice to abrin was the onset of a localized, yet severe, edematous inflammation. This inflammation was accompanied by a massive recruitment of neutrophils and the stimulation of a turbulent pro-inflammatory cytokine storm within the organ. In
Fig. 5. Effect of antibody treatment on pathological indices in abrin-intoxicated mice. Mice intranasally exposed to abrin (8 mg/kg) or ricin (7 mg/kg), were treated or not with anti-abrin or anti-ricin antibodies, respectively, at 24 h PE. At 72 h PE, lungs were either removed and neutrophil and macrophage numbers, represented by bars, were determined by flow cytometry or were lavaged and the levels of ChE and IL-6 were determined in the BALF. Control: no exposure; Ricin + Ab = ricin-intoxicated followed by anti-ricin antibody treatment; Abrin + Ab = abrin-intoxicated followed by anti-abrin antibody treatment. Number of mice per group: Control and antibody-treated (Ricin + Ab, Abrin + Ab) groups, n = 10; Intoxicated non-treated (Ricin, Abrin) groups, n = 5–6. Data represent mean SEM. Significant (P < 0.05) differences between antibody-treated mice and corresponding intoxicated non-treated mice are indicated by an asterisk (*).
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the past, we reported that i.n. instillation of a lethal dose of ricin to mice also induced a severe pulmonary inflammation, which in turn led to respiratory failure and death (Gal et al., 2014). Hence, a striking similarity exists between the pathologies ensuing abrinand ricin-intranasal exposures. Surprisingly, despite this resemblance in morbidity, the ability to protect mice against the two intoxications by post-exposure antibody-mediated treatment differs radically. In the case of ricin intoxication at a 2LD50 dose, anti-ricin antibody-based treatment at 24 h PE led to the protection of 34% of the mice (Gal et al., 2014), similar to the protection level attained by others at this time-point (Pratt et al., 2007). When antibody treatment was administered very late after ricin intoxication, e.g., 48 h PE, protection was no more than marginal (<8% survival). In contrast, administration of polyclonal anti-abrin antibodies to mice intranasally exposed to abrin at a dose of 2LD50, led to extraordinarily high survival rates even when the antibodies were applied late after intoxication. Thus, passive immunization of mice with anti-abrin polyclonal antibodies led to >70% protection even when this treatment was applied as late as 72 h PE. When antibody treatment was applied at 96 h PE, survival rates declined to 50%, but even these rates were higher than those observed for ricin-intoxicated mice that were treated at 24 h PE (Gal et al., 2014). It should be noted that when treatment was administered at 72 h PE some of the abrin-exposed mice have already died and these were not considered in the survival rate calculations. The fact that such high levels of protection are reached even at a time-point at which 10% of the mice have already succumbed, intensifies our findings. The sharp divergence in the ability to protect mice against the two toxins is further confounded by the fact that under our experimental conditions in which mice were exposed to ricin or abrin at a dose of 2LD50, the MTTD values for the two toxins are very similar, 4.9 0.8 and 5.3 1.1 days, respectively. Since treatment against abrin or ricin intoxications is based on the use of anti-toxin antibodies, one may argue that the differential protectivity against the two toxins stems from differences in the quality of the antibody preparations. However, this seems unlikely, as we have demonstrated that the two anti-toxin preparations resemble one another by their ELISA and neutralizing endpoint titers, as well as by their apparent affinity towards the corresponding toxins. Abrin and ricin operate at the molecular level, in an identical manner by inactivating the ribosomes of the cell. In vitro protein synthesis inhibition studies carried out at our laboratory allowed us to determine that the catalytic activity of abrin was in fact 9fold more potent than that of ricin (ED50 = 0.7 0.07 and 6.6 0.9 ng/ml for abrin and ricin, respectively, in an in vitro translation inhibition assay), suggesting that the ability of abrin to impair ribosomes in an irreversible manner would be even greater than that of ricin. Similar results, suggesting that catalytic activity of abrin is superior to that of ricin, were reported by others as well (Barbieri et al., 2004). However, one should keep in mind that the catalytic activity, which resides in the A subunit of the toxin, is fully manifested only following the dissociation of its subunits (Tsai et al., 2002; Barbieri et al., 2004). In vivo, subunit dissociation occurs at a relatively late stage, after the holotoxin has entered the endoplasmic reticulum (Roberts and Smith, 2004) and does not determine the overall rate of intoxication. Rather, binding to the cell surface, entry and intracellular trafficking of ricin or abrin through the trans-Golgi and Golgi compartments to the ER, all of which precede subunit dissociation and enzymatic catalysis, comprise the rate-determining steps for in vivo ricin- and abrinintoxication. The fact that the therapeutic window for antibody treatment against abrin-intoxication is so much broader compared to ricin-intoxication, may however suggest that the rate of entry of abrin into target cells is slower, hence, its availability for effective antibody neutralization is higher over a greater period of time. To
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date, the kinetics of in vivo intoxications by ricin and abrin have not been compared. Elaborate studies aimed to measure the cellbinding rates of the two toxins within the lungs of experimental animals, as well as to determine the lung cell types which undergo toxin-mediated damage, are now being carried out at our laboratory. However, catalytic disruption of ribosomes and the ensuing cessation of protein synthesis are only one aspect of ricin- and abrin-mediated intoxication. The clinical manifestations following pulmonary exposure to the two toxins, are those of lung inflammation. How does the inflammatory state relate to the catalytic activity of ricin and abrin? Previous studies expounded the view that the actual damage of ribosomes triggers the onset of inflammation and have coined this phenomenon as “ribotoxic stress response” (Iordanov et al., 1997; Korcheva et al., 2005, 2007). In another study, it was proposed that ricin-mediated translational inhibition fostered the disappearance of labile proteins that normally suppress inflammasome formation, thereby inducing an inflammatory response (Lindauer et al., 2009, 2010). Common to these studies is the assertion that catalytic inactivation of ribosomes is a prerequisite for induction of the inflammatory reaction. Do ricin and abrin differ in their ability to elicit pulmonary inflammation? Although exposure to either abrin or ricin leads to elevated expression of a similar set of cytokines, we note that the measured levels of some of the proinflammatory cytokines, i.e., TNF-a, MIP-2 and ANGII, were consistently higher in the BALF of mice which were intranasally exposed to ricin at 24 h PE. In contrast, measurement at 72 h PE of IL-6 in the BALF of intoxicated mice demonstrated that levels of this cytokine were perhaps even higher in the abrin-intoxicated mice. However, the fate of IL-6 following antibody-treatment was profoundly different in abrin- and ricin-intoxicated mice. While anti-abrin antibody treatment at 24 h PE brought about a 90% reduction in IL-6 levels by 72 h PE, application of anti-ricin antibodies to ricin-intoxicated mice did not affect IL-6 levels whatsoever and these remained at least as high as in intoxicated mice that were not subjected to antibody treatment. We note in this context, that various studies have documented a positive correlation between IL-6 levels and excessive pulmonary inflammation in pre-clinical models of lung injuries (Hubeau et al., 2013; Yu et al., 2002; Zhang et al., 2013). Thus, one may be tempted to draw a line between the superior ability to protect against abrin intoxication and the fact that antibody treatment uniquely reduces IL-6 levels in mice exposed to this toxin, yet this apparent correlation remains at present circumstantial and should be further probed. To further delineate the factors/properties responsible for the differential ability to protect against abrin and ricin, we have now generated two chimeric heterologous toxins utilizing monomeric subunits isolated from abrin and ricin. Protection experiments in mice exposed to a lethal dose of the chimeric toxins, AabrinBricin or AricinBabrin, will allow us to determine which of the subunits of abrin source, A or B, confers the uniquely high-efficient therapeutic response to antibody treatment over long periods of time. Acknowledgments We would like to thank Ron Alcalay and Nehama Seliger for their excellent technical assistance. References Audi, J., Belson, M., Patel, M., Schier, J., Osterloh, J., 2005. Ricin poisoning: a comprehensive review. JAMA 294, 2342–2351. Barbieri, L., Ciani, M., Girbes, T., Liu, W.-Y., Van Damme, E.J.M., Peumans, W.J., Stirpe, F., 2004. Enzymatic activity of toxic and non-toxic type 2 ribosome-inactivating proteins. FEBS Lett. 563, 219–222.
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