Post-exposure targeting of specific epitopes on ricin toxin abrogates toxin-induced hypoglycemia, hepatic injury, and lethality in a mouse model

Abstract

Effects in the liver of fatal intoxication with the binary toxin ricin are unclear. We report a robust neutrophil influx into the liver of C57BL/6 mice after lethal parenteral ricin challenge, occurring in peri-portal and centro-lobular hepatic areas within 2 h, followed by the abrupt disappearance of hepatic macrophages/Kupffer cells. Chemokine profiles determined by microarray, ribonuclease protection assays, northern blotting, and enzyme-linked immunosorbent assays showed rapid (2 h) upregulation and persistence of those for neutrophils (CXCL1/KC, CXCL2/MIP-2) and monocytes (CCL2/MCP-1). Red blood cell pooling (8-12 h), loss of hepatocyte glycogen (8-48 h) associated with progressive hypoglycemia, fibrin deposition (24-48 h), and death (72-96 h) followed. Monoclonal antibody to ricin A chain, administered intravenously, blunted hypoglycemia, and abrogated death. This outcome was observed when anti-ricin antibody was given before toxin exposure as well as when administered approximately 10 h after toxin exposure. Targeting antibody to specific amino-acid sequences on the ricin A chain (HAEL and QXXWXXA) was critical to the therapeutic effect. Re-emergence of liver macrophages/Kupffer cells and replenishment of glycogen in previously depleted hepatocytes preceded full recovery of the host. These data identify critical events for liver injury and healing in ricin intoxication, as well as a new means and specific targets for post-exposure therapeutic intervention.

Figure 1

Specific binding of monoclonal antibodies RAC 17, 18, and 23 to ricin A chain. Ricin holotoxin (1.25 μg/lane) was electrophoresed on a 10% SDS polyacrylamide gel, then stained for protein with a Commassie stain (column 2) or blotted onto nitrocellulose for subsequent exposure to a 1:10,000 dilution of the indicated hybridoma immunoglobulin. Each immunoglobulin bound the 32,000 Dalton A subunit of ricin, but was minimally reactive with the 34,000 Dalton B subunit (columns 3–5). Ctl IgG is a control mouse monoclonal IgG also used at 1:10,000 dilution (column 6). MW standards are shown in column 1. Lower arrow, 32,000 Dalton subunit; upper arrow, 34,000 Dalton subunit.

Figure 2

Hepatic injury and lethality in a mouse model of ricin intoxication. A. H&E stain of peri-portal liver 48 hours following ricin challenge, showing nests of leukocytes (arrows) adjacent to portal tracts. B. Lethality dose-response after ricin administration i.p. at the doses indicated, with 10 mice per group. C. Fibrin in the peri-portal area of liver 48 hours after ricin challenge (40 μg/kg). Deposition of fibrin (arrows) occurred between hepatocytes and was detectable by MSB staining (see Materials and Methods). D. Time course of fibrin deposition in the peri-portal liver after ricin challenge. This is shown as the percent of high-powered (400×) microscopic fields showing fibrin deposits around hepatocytes. E&F, same as C&D, except that the centro-lobular liver was studied. RBC pooling (arrowhead in panel E) refers to aggregates of red blood cells two or more times larger than present in normal liver sinusoids, and scored as the percent of high-powered fields positive for the finding. For D & F, findings are representative of two independent studies, with a total of 4 mice per time point. *, p< 0.04, comparing number of fields showing fibrin deposition in centro-lobular and peri-portal areas at 24 and 48 hours with that at 0–4 hours. **, p<0.05, comparing number of fields showing RBC congestion at 0 and at 8 hours. Magnification: A, C, and E, 400×.

Figure 3

Migration of leukocytes to liver after ricin challenge in vivo. A & C. Immuno-reactive neutrophils in peri-portal ( panel A) and centro-lobular ( panel C) areas of liver 48 hours after toxin administration. B. Quantification of neutrophils as number of 7/4⁺ immuno-reactive cells per high-powered field 0–48 hours after toxin was given. D & F. Immuno-reactive macrophages in peri-portal (panel D) and centro-lobular (panel F) hepatic areas 2 hours after toxin challenge. E. Number of F4/80⁺ immuno-reactive cells per HPF of liver 0–48 hours after administration of toxin. Repeat studies showed loss of stained cells occurred over 8–12 hours. Findings are representative of two independent studies with a total of 4 mice per time point. Magnification: 400× (A, C); 200× (D, E). *, p < 0.01, number of leukocytes per HPF at the designated hours compared with that at 0 hours. **, p < 0.02, number of macrophages per HPF at 0 hours compared with that at 2 hours in the peri-portal area.

Figure 4

Chemokine mRNA and protein in the liver over 0–48 hours after a single injection of ricin (40 μg/kg) in C57BL/6 mice. A,C,E. Messenger RNA for CXCL1/KC, CXCL2/MIP-2, and CCL2/MCP-1(JE) is shown as a ratio of specific message/value of a house-keeping gene (18S or L32), determined by Northern blotting (CXCL1/KC) or by ribonuclease protection assay (CXCL2/MIP-2, CCL2/MCP-1). Specific mRNA for these chemokines was not detected at 0 hours. Two mice were analyzed per time point. B,D,E. Chemokine protein in liver homogenates after ricin challenge, quantified by paired antibody enzyme-linked immuno-absorbent assay, described in Materials and Methods. Shown is the mean +/− 1 SD. Homogenates were analyzed in duplicate from each of two mice per time point (14 mice total per cytokine). *, p< 0.05, comparing values at the indicted hours with that at 0 hours.

Figure 5

Hepatic glycogen and hypoglycemia following ricin challenge (40 μg/kg) in the C57BL/6 mouse. A. Time course for the presence of peri-portal glycogen-containing hepatocytes, displayed as the number per high power field, detected as periodic acid Schiff (PAS⁺) -positive cells. B. PAS staining of peri-portal hepatic parenchyma at 0 and 8 hours (B, left and right panels, respectively) after ricin exposure. Arrows, individual PAS⁺ hepatocytes. C. Number of PAS⁺ cells per HPF in the centro-lobular liver from 0 to 48 hours following ricin. D. Examples of PAS⁺ hepatocytes at 0 and 8 hours (D, left and right panels, respectively) following toxin. For A & C, results are representative of two independent studies, each with 2 mice per time point (total of 4 mice per time point, or 32 mice total). Complete disappearance of glycogen took up to 12 hours in repeat studies. PAS⁺ cell number at 8–48 hours was significantly different from that at 0 hours (p <0.001). E. Blood glucose in mice following ricin challenge, as determined by glucometer on tail vein blood. F. Comparison of blood glucose levels in saline (control)- and ricin-challenged mice at selected times before and after ricin challenge. *, p < 0.02, comparing ricin-treated versus vehicle-treated mice at the indicated time points (14, 36, and 48 hours). C, blood glucose prior to challenge with ricin or vehicle. Magnification: B, D (400×).

Figure 6

Blood glucose and lethality after pre-treatment with monoclonal immunoglobulin to ricin A chain. Mice were injected intravenously with monoclonal immunoglobulin to ricin A chain (RAC 17, 18 and 23; 20 μg/mouse each; N=10) or with irrelevant immunoglobulin matched for isotype and quantity (N=10) one hour prior to challenge with the ricin holotoxin (40μg/kg). A. Blood glucose levels (mean +/− 1 SD) at three intervals after ricin injection. *, p < 0.02, comparing RAC- versus irrelevant antibody-injected mice at 20, 27 and 36 hours following ricin challenge. B. Percentage of mice pre-treated with immunoglobulin which survived lethal ricin exposure. Ig(ricin) mice received 20 μg each of RAC 17, 18, and 23; Ig(irrel) mice were administered monoclonal IgG₁ and IgG_2A in the same quantity and in the same proportion as was received by the experimental group.

Figure 7

Effect of anti-ricin monoclonal immunoglobulin administered after ricin challenge. At designated time intervals after injection of ricin (40 μg/kg), C57BL/6 mice were administered 20 μg each of RAC 17, 18, and 23 i.v., or the same quantity of isotype-matched monoclonal immunoglobulin; assessed every 24 hours for blood glucose,; and monitored for survival. A. Blood glucose of surviving mice by treatment group (N=10/group). One group did not receive ricin (non-ricin ctl) and one group was administered ricin then irrelevant immunoglobulin (ctl IgG). B. Survival shown as the percentage of mice alive in each treatment group (N=10/group). Note that 100 % of mice receiving irrelevant immunoglobulin succumbed by day 4, while 30% of mice administered specific immunoglobulin 14 hours after ricin exposure survived. Survival curves overlap for mice receiving anti-ricin antibody 8 and 10 hours after ricin challenge.

Figure 8

Maintenance of glucose homeostasis and host survival after administration of individual monoclonal antibodies to ricin A chain. Six hours following ricin challenge (40 μg/kg), mice were intravenously given 20 μg of a single immunoglobulin (RAC 17, 18, 23, or irrelevant IgG), then monitored for blood glucose every 24 hours and followed for survival. The data are representative of two independent studies. A. Blood glucose by treatment group (N=5/group) and shown as the mean +/− 1 SD. Glucose values in all groups of ricin-challenged mice diminished on day 1 before normalizing by day 3–4. * indicates p < 0.05, comparing ricin and antibody treated mice with non-ricin controls. Mortality in the irrelevant IgG group after day 2 was large, and the group mean blood glucose was not calculated. B. Survival among mice administered a single monoclonal immunoglobulin. Groups of mice were followed, with early deaths occurring among mice receiving control IgG, and a later loss of mice in those administered RAC 23. Mice not challenged with ricin are shown as ‘non-ricin controls.’

Figure 9

Intra-hepatic findings following 10 hour delay in immunoglobulin administration. Mice were administered i.v. 60 μg of immunoglobulin 10 hours following a lethal ricin challenge (40μg/kg), then euthanized at 48 or 72 hours subsequent to toxin exposure. Control mice were studied just before immunoglobulin administration; peri-portal and centro-lobular regions were studied. A. Number of PAS⁺ (carbohydrate-containing) cells per field. *, p<0.001, comparing mice receiving anti-ricin monoclonal antibody (equal quantities of RAC 17, 18, and 23), designated Ig(R), with those administered irrelevant antibody matched for quantity and isotype, denoted as Ig(I). #, no centro-lobular PAS⁺ hepatocytes observed at 48 hours in mice receiving Ig(I). B. Number of F4/80⁺ immuno-reactive macrophages per field. *, p<0.001, comparing the same groups as in A. C. Number of 7/4⁺ immuno-reactive neutrophils per field. No significant differences were found when mice receiving anti-ricin monoclonal immunoglobulin were compared to those receiving irrelevant immunoglobulin. For A, B, and C, results are representative of two independent studies, each with 2 mice per time point (total of 4 mice per time point).