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Diverse profiles of ricin-cell interactions in the lung following intranasal exposure to ricin.

Sapoznikov A, Falach R, Mazor O, Alcalay R, Gal Y, Seliger N, Sabo T, Kronman C - Toxins (Basel) (2015)

Bottom Line: Neutrophils, which were massively recruited to the intoxicated lung, were refractive to toxin binding.The differential binding and cell-elimination patterns observed may stem from dissimilar accessibility of the toxin to different cells in the lung and may also reflect unequal interactions of the toxin with different cell-surface receptors.The multifaceted interactions observed in this study between ricin and the various cells of the target organ should be considered in the future development of efficient post-exposure countermeasures against ricin intoxication.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 74100, Israel. anitas@iibr.gov.il.

ABSTRACT
Ricin, a plant-derived exotoxin, inhibits protein synthesis by ribosomal inactivation. Due to its wide availability and ease of preparation, ricin is considered a biothreat, foremost by respiratory exposure. We examined the in vivo interactions between ricin and cells of the lungs in mice intranasally exposed to the toxin and revealed multi-phasic cell-type-dependent binding profiles. While macrophages (MΦs) and dendritic cells (DCs) displayed biphasic binding to ricin, monophasic binding patterns were observed for other cell types; epithelial cells displayed early binding, while B cells and endothelial cells bound toxin late after intoxication. Neutrophils, which were massively recruited to the intoxicated lung, were refractive to toxin binding. Although epithelial cells bound ricin as early as MΦs and DCs, their rates of elimination differed considerably; a reduction in epithelial cell counts occurred late after intoxication and was restricted to alveolar type II cells only. The differential binding and cell-elimination patterns observed may stem from dissimilar accessibility of the toxin to different cells in the lung and may also reflect unequal interactions of the toxin with different cell-surface receptors. The multifaceted interactions observed in this study between ricin and the various cells of the target organ should be considered in the future development of efficient post-exposure countermeasures against ricin intoxication.

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Kinetics of ricin binding to individual cell populations in the lungs. Mice were intranasally exposed to ricin-AF488 and lung cells isolated at 3, 6, 12, 18, 24, 48 and 72 h were analyzed by FACS for ricin binding, by detection of AF488+ cells (filled circles) or by staining with anti-ricin RAF5 (open circles) (six mice per group). Quantification by AF488 and RAF5 of ricin-bound MΦs (A), DCs (B), B cells (C), epithelial cells (D) and endothelial cells (E). (F) Mannose receptor expression in various cells as measured by FACS. Values are displayed as mean fluorescence intensity (MFI).
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toxins-07-04817-f002: Kinetics of ricin binding to individual cell populations in the lungs. Mice were intranasally exposed to ricin-AF488 and lung cells isolated at 3, 6, 12, 18, 24, 48 and 72 h were analyzed by FACS for ricin binding, by detection of AF488+ cells (filled circles) or by staining with anti-ricin RAF5 (open circles) (six mice per group). Quantification by AF488 and RAF5 of ricin-bound MΦs (A), DCs (B), B cells (C), epithelial cells (D) and endothelial cells (E). (F) Mannose receptor expression in various cells as measured by FACS. Values are displayed as mean fluorescence intensity (MFI).

Mentions: These findings prompted us to determine the kinetics of ricin binding to individual cell populations of the lung. Mice were intoxicated with fluorescently-labeled ricin, and lung cells isolated at different time points were then stained for anti-ricin antibodies and analyzed for ricin binding. In MΦs and DCs, as gated in Figure S1A, two temporally-distinct peaks were detected by the two techniques (Figure 2A,B). Whilst peak binding at 3–6 h after intoxication was detected by fluorescent toxin, labeling with antibodies identified a later peak, at 18 h after intoxication. The failure of the sensitive antibody-based technique to detect toxin-associated cells at the earlier time point indicated that the peak binding at this time point relates to cells that have already internalized the toxin. However, the ratio between the early and late peaks of toxin association was different in the two cell types. In the case of MΦs (Figure 2A), the greater amount of toxin was internalized at the early time point, while in the case of DCs (Figure 2B), most of the association of toxin with the cells occurred at the late time point, remaining bound to the cell exterior. Early binding of ricin by MΦs preceded that of DCs, while significant binding to MΦs was detected already 1 h after intoxication; binding to DCs was observed at 3 h after exposure (Figure S2A–C). The determination of the binding profile to B cells (Figure S1B) identified a single late peak at 18 h after intoxication by both techniques (Figure 2C), while toxin binding to neutrophils (Figure S2A) could not be detected at all (Figure S2D). Analysis of toxin interactions with parenchymal cells demonstrated that binding to epithelial cells (Figure S1C) is characterized by a single peak detected by anti-ricin antibodies at an early time point of 6 h (Figure 2D). In contrast, ricin binding to endothelial cells (Figure S1C) was discernable only at 24 h after intoxication and did not reach peak levels within the time frame of these experiments (Figure 2E). The spatial localization of the different cells types within the lungs could play a role in the bi-phasic shaping of the ricin binding profile. Following pulmonary intoxication, ricin first encounters the MΦs located in the alveolar lumen and the DCs protruding through the epithelial network, and only then, the toxin has access to the interstitium. However, the fact that a single cell type may display both early and late binding peaks, as in the case of MΦs and DCs, suggests that the composite binding patterns are also an outcome of different mechanisms of binding. The B-chain of ricin is a galactose-specific lectin that binds to glycoproteins or glycolipids at the cell surface [2,3]; yet, in addition to this canonical route, ricin can enter cells through a secondary route, via the mannose receptor that is present on MΦs [12]. This receptor binds to high mannose oligosaccharide chains present on both A and B subunits of the toxin and is more efficient in delivering ricin to the cytoplasm [13]. To evaluate the possible role of this alternative binding pathway, mannose receptor density was measured for different cell types of the lung (Figure 2F). In both MΦs and DCs, the presence of this highly efficient receptor could explain the early peak of ricin binding and the measured difference in receptor density between the two cell types, correlating well with the relative amounts of toxin that were associated with these cells (Figure 2A,B). Epithelial cells were found to express the mannose receptor at levels that did not fall from those of MΦs (Figure 2F) and, indeed, displayed a high early binding peak (Figure 2D), while B cells and endothelial cells, both of which were devoid of the mannose receptor (Figure 2F), did not display an early peak of binding (Figure 2C,E). Toxin association via galactose, which is less efficient than via the mannose receptor, could be responsible for the later peaks of binding observed in the different cell types.


Diverse profiles of ricin-cell interactions in the lung following intranasal exposure to ricin.

Sapoznikov A, Falach R, Mazor O, Alcalay R, Gal Y, Seliger N, Sabo T, Kronman C - Toxins (Basel) (2015)

Kinetics of ricin binding to individual cell populations in the lungs. Mice were intranasally exposed to ricin-AF488 and lung cells isolated at 3, 6, 12, 18, 24, 48 and 72 h were analyzed by FACS for ricin binding, by detection of AF488+ cells (filled circles) or by staining with anti-ricin RAF5 (open circles) (six mice per group). Quantification by AF488 and RAF5 of ricin-bound MΦs (A), DCs (B), B cells (C), epithelial cells (D) and endothelial cells (E). (F) Mannose receptor expression in various cells as measured by FACS. Values are displayed as mean fluorescence intensity (MFI).
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Related In: Results  -  Collection

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toxins-07-04817-f002: Kinetics of ricin binding to individual cell populations in the lungs. Mice were intranasally exposed to ricin-AF488 and lung cells isolated at 3, 6, 12, 18, 24, 48 and 72 h were analyzed by FACS for ricin binding, by detection of AF488+ cells (filled circles) or by staining with anti-ricin RAF5 (open circles) (six mice per group). Quantification by AF488 and RAF5 of ricin-bound MΦs (A), DCs (B), B cells (C), epithelial cells (D) and endothelial cells (E). (F) Mannose receptor expression in various cells as measured by FACS. Values are displayed as mean fluorescence intensity (MFI).
Mentions: These findings prompted us to determine the kinetics of ricin binding to individual cell populations of the lung. Mice were intoxicated with fluorescently-labeled ricin, and lung cells isolated at different time points were then stained for anti-ricin antibodies and analyzed for ricin binding. In MΦs and DCs, as gated in Figure S1A, two temporally-distinct peaks were detected by the two techniques (Figure 2A,B). Whilst peak binding at 3–6 h after intoxication was detected by fluorescent toxin, labeling with antibodies identified a later peak, at 18 h after intoxication. The failure of the sensitive antibody-based technique to detect toxin-associated cells at the earlier time point indicated that the peak binding at this time point relates to cells that have already internalized the toxin. However, the ratio between the early and late peaks of toxin association was different in the two cell types. In the case of MΦs (Figure 2A), the greater amount of toxin was internalized at the early time point, while in the case of DCs (Figure 2B), most of the association of toxin with the cells occurred at the late time point, remaining bound to the cell exterior. Early binding of ricin by MΦs preceded that of DCs, while significant binding to MΦs was detected already 1 h after intoxication; binding to DCs was observed at 3 h after exposure (Figure S2A–C). The determination of the binding profile to B cells (Figure S1B) identified a single late peak at 18 h after intoxication by both techniques (Figure 2C), while toxin binding to neutrophils (Figure S2A) could not be detected at all (Figure S2D). Analysis of toxin interactions with parenchymal cells demonstrated that binding to epithelial cells (Figure S1C) is characterized by a single peak detected by anti-ricin antibodies at an early time point of 6 h (Figure 2D). In contrast, ricin binding to endothelial cells (Figure S1C) was discernable only at 24 h after intoxication and did not reach peak levels within the time frame of these experiments (Figure 2E). The spatial localization of the different cells types within the lungs could play a role in the bi-phasic shaping of the ricin binding profile. Following pulmonary intoxication, ricin first encounters the MΦs located in the alveolar lumen and the DCs protruding through the epithelial network, and only then, the toxin has access to the interstitium. However, the fact that a single cell type may display both early and late binding peaks, as in the case of MΦs and DCs, suggests that the composite binding patterns are also an outcome of different mechanisms of binding. The B-chain of ricin is a galactose-specific lectin that binds to glycoproteins or glycolipids at the cell surface [2,3]; yet, in addition to this canonical route, ricin can enter cells through a secondary route, via the mannose receptor that is present on MΦs [12]. This receptor binds to high mannose oligosaccharide chains present on both A and B subunits of the toxin and is more efficient in delivering ricin to the cytoplasm [13]. To evaluate the possible role of this alternative binding pathway, mannose receptor density was measured for different cell types of the lung (Figure 2F). In both MΦs and DCs, the presence of this highly efficient receptor could explain the early peak of ricin binding and the measured difference in receptor density between the two cell types, correlating well with the relative amounts of toxin that were associated with these cells (Figure 2A,B). Epithelial cells were found to express the mannose receptor at levels that did not fall from those of MΦs (Figure 2F) and, indeed, displayed a high early binding peak (Figure 2D), while B cells and endothelial cells, both of which were devoid of the mannose receptor (Figure 2F), did not display an early peak of binding (Figure 2C,E). Toxin association via galactose, which is less efficient than via the mannose receptor, could be responsible for the later peaks of binding observed in the different cell types.

Bottom Line: Neutrophils, which were massively recruited to the intoxicated lung, were refractive to toxin binding.The differential binding and cell-elimination patterns observed may stem from dissimilar accessibility of the toxin to different cells in the lung and may also reflect unequal interactions of the toxin with different cell-surface receptors.The multifaceted interactions observed in this study between ricin and the various cells of the target organ should be considered in the future development of efficient post-exposure countermeasures against ricin intoxication.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 74100, Israel. anitas@iibr.gov.il.

ABSTRACT
Ricin, a plant-derived exotoxin, inhibits protein synthesis by ribosomal inactivation. Due to its wide availability and ease of preparation, ricin is considered a biothreat, foremost by respiratory exposure. We examined the in vivo interactions between ricin and cells of the lungs in mice intranasally exposed to the toxin and revealed multi-phasic cell-type-dependent binding profiles. While macrophages (MΦs) and dendritic cells (DCs) displayed biphasic binding to ricin, monophasic binding patterns were observed for other cell types; epithelial cells displayed early binding, while B cells and endothelial cells bound toxin late after intoxication. Neutrophils, which were massively recruited to the intoxicated lung, were refractive to toxin binding. Although epithelial cells bound ricin as early as MΦs and DCs, their rates of elimination differed considerably; a reduction in epithelial cell counts occurred late after intoxication and was restricted to alveolar type II cells only. The differential binding and cell-elimination patterns observed may stem from dissimilar accessibility of the toxin to different cells in the lung and may also reflect unequal interactions of the toxin with different cell-surface receptors. The multifaceted interactions observed in this study between ricin and the various cells of the target organ should be considered in the future development of efficient post-exposure countermeasures against ricin intoxication.

Show MeSH
Related in: MedlinePlus