Hepatic injury and lethality following ricin administration
Because findings may be mouse strain-specific, we first sought to define a range of lethal doses of ricin usable in the C57BL/6 mouse, a strain which our laboratory has utilized in prior published work with A-B toxins (10
). In dose-response studies, intraperitoneal challenge with ricin at 20 μg/kg (400 ng/20g mouse) or more was associated with profound lethargy by 48 hours and lethality within 4 days (). In subsequent studies, we chose to use a dose of ricin (40 μg/kg) which was clearly at or exceeded the LD100
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 (more ...)
The presence of lethargy in our model as well as hypoglycemia reported after exposure to the ricin holotoxin (11
) suggested the liver may be a target organ for this toxin, given that hepatic glycogen is the principal site for carbohydrate storage in the host, and is integral to maintaining glucose homeostasis. Therefore, we examined whether inflammation and/or injury were present in the liver that might contribute to dysfunction of glycogen-containing hepatocytes. Histological assessment of the liver in these mice showed generalized hypercellularity, as well as focal areas of mixed inflammatory cells, particularly near the peri-portal zones (). Further, specialized staining (MSB) revealed progressive fibrin deposition in the liver. Fibrin was simultaneously detected in the peri-portal and centro-lobular areas (, respectively) at 24 hours after challenge with ricin, and by 48 hours involved greater than 30% of microscopic fields studied (p < 0.04) (). In addition, red blood cell congestion of the hepatic parenchyma was observed at 4–6 hours, but was transient, diminishing by 24 hours (). Together, these findings suggest that inflammation and subsequent injury, as indicated by fibrin deposition, is present in the liver after ricin exposure, and may be an important antecedent of lethargy and death in the model.
Nature of hepatic inflammation after ricin challenge
To define the nature of the inflammatory infiltrate in the liver associated with lethal ricin intoxication (), mice were exposed to ricin, euthanized at pre-established times, then examined for the number, distribution and time course of two inflammatory cell types, neutrophils and macrophages, associated with parenchymal injury in other mouse models using binary toxins (10
). Initially, the number of both cell types rose rapidly above baseline after ricin challenge (), with a distribution that was diffuse throughout the liver, involving both the peri-portal and centro-lobular hepatic zones (). However, quantification of each cell type at later time points revealed marked differences. That is, the number of neutrophils present in hepatic parenchyma remained robust (>10/HPF; ), while hepatic macrophage/Kupffer cell numbers peaked at 2–4 hours, then rapidly became nearly undetectable by 8–12 hours and beyond (). These results suggest that powerful and sustained chemotactic stimuli for neutrophil and macrophage invasion of liver arise quickly following systemic exposure to ricin. Nevertheless, hepatic macrophages/Kupffer cells, while numerous at two hours (e.g., 9–17 cells/HPF), receded over the next 10 hours and did not re-accumulate.
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 (more ...)
Chemokine gene activation in the liver after challenge with ricin
To determine the chemotactic and adhesion molecules likely responsible for neutrophil and macrophage entry into the liver, mRNA isolated from hepatic tissue was analyzed by microarray at 0, 2 and 24 hours following lethal challenge with ricin. Biotinylated cRNA made with T7 RNA polymerase was hybridized with oligonucleotides on Affymetrix Gene Chips, and the results analyzed by GeneX VA software (). Among chemotaxins for neutrophils, CXCL1/KC and CXCL2/MIP-2 mRNA demonstrated a 16-and 3-fold increase, respectively, over control values at 2 hours following ricin challenge, and continued to be increased at 24 hours (>40-fold over baseline). Genes for other chemokines able to initiate neutrophil migration (C5a, PAF, LTB4, NAP2, NCFA) were not induced (≤2-fold increased; and data not shown). For chemokines with selectivity for macrophages, four were modestly elevated (>2.9-fold) at 2 hours, but only two of these [CCL2/MCP-1(JE), CCL3/MIP-1α] showed sustained and progressive increases over time (). Among molecules expressed on endothelium and important for adhesion of circulating inflammatory cells before diapedesis, gene products for two ( ICAM-1 and VCAM) were expressed in the liver at two hours after ricin exposure, but only ICAM showed a sustained increase (>21-fold at 24 hours; ).
Microarray Analysis of Liver mRNA: Identification of Genes Up-regulated after in vivo Challenge with Ricin
To independently confirm the microarray findings and to acquire a more complete time course for transcription and translation of specific chemokines, total hepatic RNA and protein homogenates were temporally collected and analyzed by additional techniques (see below) following in vivo challenge with a lethal dose of ricin. Messenger RNA and protein belonging to chemokines relevant to neutrophil migration (CXCL1/KC and CXCL2/MIP-2) were detectible at 2 hours, rose further over 6–12 hours after ricin exposure, and were sustained (). Similarly, mRNA and protein for the macrophage chemokine CCL2/MCP-1 was evident at 2 hours and remained sustained, as did that for MIP-1α ( and data not shown). Together, as determined by RPA, Northern blotting and dual antibody ELISA, these studies suggest that potent chemotactic stimuli for inflammatory cell migration into the liver persist for days in the host after a single parenteral lethal challenge with ricin. Thus, the disappearance of hepatic macrophages/Kupffer cells over 8–12 hours following ricin exposure was not due to the absence of a locally generated relevant chemokine.
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 (more ...)
Glycogen in the liver after ricin challenge
Given the prolonged presence of hepatic inflammation following ricin challenge in vivo, we postulated that important hepatic functions such as synthesis and storage of carbohydrate as glycogen would be disrupted. Therefore, we serially assessed the presence and distribution of liver glycogen as determined by PAS staining of individual hepatocytes in tissue sections following ricin administration (). The number of PAS+ hepatocytes remained at the baseline value of 12–18 cells/field over the first 4–6 hours before it decreased markedly at 8–12 hours, both in peri-portal areas containing the bile ducts, hepatic artery and portal vein ((), and in the centro-lobular areas surrounding the central vein (). Subsequently, hepatocytes did not re-accumulate glycogen over the following 40 hours, and this corresponded with progressive and, ultimately, profound hypoglycemia in the mice (). In contrast, control mice, receiving vehicle (normal saline) without ricin, demonstrated stable blood glucose levels (> 100 mg/dl), and maintained substantial hepatic carbohydrate stores at 24 and 48 hours (> 23 +/− 4 PAS+ cells/field). Taken together with experiments described in & , these studies suggest that lethal challenge with ricin leads to a chemokine-driven mixed inflammatory infiltration of liver by 2 hours, persistent loss of liver glycogen by 8–12 hours, progressive severe hypoglycemia by 12–24 hours, and death by day 4.
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 (more ...)
Monoclonal anti-ricin A chain immunoglobulin diminishes hypoglycemia and prevents lethality
One mechanism to account for events in the liver described above is to postulate that holotoxin in the intra-peritoneal space disseminates intravascularly to reach vital organs including the liver and kidney. This would suggest that hypoglycemia and death in ricin challenged mice could be ameliorated or prevented by the presence in the intravascular space of anti-ricin immunoglobulin capable of immuno-neutralizing toxin. We studied this by administering ricin A chain-specific monoclonal immunoglobulin -- 20 μg each of RAC 17, 18, 23 -- to mice one hour prior to a lethal challenge with ricin (). At 20, 27, and 36 hours following ricin exposure, mice pre-treated with anti-ricin immunoglobulin maintained their blood glucose levels (mean > 100 mg/dl) when, at the same time, controls given irrelevant isotype-matched immunoglobulin in the same amount became progressively hypoglycemic (; p<0.05). Further, when these mice were followed over four days, all (10/10) anti-ricin immunoglobulin-treated mice survived, while 9 of 10 mice receiving irrelevant immunoglobulin died ().
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 (more ...)
Interestingly, the single surviving mouse in the latter group maintained a blood glucose of > 105mg/dl over the first 72 hours. In mice treated with ricin-specific monoclonal immunoglobulin, behavior (activity level, posture) and appearance (hair) remained unaltered, while these parameters progressively deteriorated in 9 of the 10 controls that succumbed (data not shown). In this group, mice that experienced marked hypoglycemia early (mean glucose, 42+/− 7 mg/dl at 27 hours; N=6) were the first to expire, all by 48 hours. The remaining mice had more moderate hypoglycemia (mean glucose, 90+/− 27 mg/dl at 27 hours; N = 4) and lived longer, succumbing at 72–96 hours following ricin challenge. A subsequent experiment confirmed these findings, with survival of all anti-ricin immunoglobulin-treated mice (10 of 10). These data suggest that ricin in the peritoneal cavity first traverses the intravascular space, where it is bound by 160,000 dalton IgG before reaching vital organs, thereby abrogating hypoglycemia and death in the model.
Benefit of post-exposure ricin-specific monoclonal antibody
To determine whether immunoglobulin to ricin A chain would benefit the host after challenge with a lethal dose of ricin, the same regimen of monoclonal antibodies (RAC 17, 18, and 23, at 20 μg/mouse each) was administered intravenously once to mice, at 1, 3, 6, 8, 10 or 14 hours after toxin. Blood sugar levels were determined daily, and mice were monitored for signs of illness. Delay of immunoglobulin administration for 1, 3, or 6 hours after ricin was associated with 100% survival (10/10 animals in each group), while a similar delay in receiving irrelevant immunoglobulin matched for isotype and amount was associated with death of all mice by day 5 (). Compared to saline-injected, ricin-free control mice, mild hypoglycemia was detected on days 1 & 2 when ricin-specific monoclonal antibody was delayed up to three hours after toxin exposure (control and 3 hour delay data shown in ), but blood glucose recovered by day 3. On the other hand, delay in administration of ricin-specific monoclonal antibody for 8 or 10 hours after ricin challenge was associated with minimal mortality (10% by day 5) and more marked hypoglycemia, with full recovery of glucose homeostasis requiring four days (). However, when the same monoclonal antibody regimen was delayed for 14 hours after ricin challenge, profound hypoglycemia was seen on days 1 & 2 where blood glucose decreased to < 50% (e.g., <72 mg/dl) of its pre-toxin level, and this was associated with 70% mortality by day 5 (). Surviving mice regained a blood glucose level to near that of controls on days 4–8, and demonstrated a clear survival advantage over mice receiving irrelevant immunoglobulin (). Overall, these data suggest that 20 μg quantities of each of three monoclonal antibodies to ricin A chain protect 90% of mice when administered up to 10 hours after a lethal challenge with ricin, and that protection is associated with an early (day 1 & 2) blunting of ricin-induced hypoglycemia.
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 (more ...)
Individual monoclonal antibodies to ricin A chain are protective
The studies above were conducted using three well-characterized monoclonal antibodies used in 20 μg amounts each, and compared to equal quantities of isotope-matched immunoglobulins. Because each monoclonal antibody is directed to a known but unique epitope on the A subunit of ricin A chain (data in reference 11
), we sought to define the possible benefit of each individually for post-exposure survival and for normalizing blood glucose. To study this, groups of mice were challenged with ricin, then administered six hours later a 20 μg quantity of RAC 17, 18, 23, or irrelevant IgG. Compared with saline-injected (non-ricin) control mice, all groups experienced marked hypoglycemia with those receiving RAC 23 needing the longest time to regain glucose levels similar to those of controls (). Nevertheless, all groups demonstrated normalization of blood glucose by day 4 except those receiving irrelevant immunoglobulin, all of whom died by day 5 (). Despite recovery of serum glucose to > 100 mg/dl by day 3, mice receiving RAC 23 died on days 6–9 after challenge with ricin (). Together with prior data (11
), we conclude that RAC 17 & 18 monoclonal antibodies binding to specific amino acid sequences on ricin A chain (HAEL and QXXWXXA) promoted long term survival. In contrast, RAC 23 binding to GTXS on ricin A chain allowed short term survival with recovery from hypoglycemia, but was insufficient to retard long-term mortality by itself.
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 (more ...)
Regression of intra-hepatic pathobiology when antibody administration is delayed after toxin exposure
The studies above demonstrated that a standard regimen of monoclonal antibodies (RAC 17, 18, and 23, at 20 μg/mouse each) reduced mortality in 90% of mice, even when administered 10 hours following a lethal ricin challenge. Benefit included not only long-term survival but normalization of blood glucose. To discern the mechanism underlying host recovery, we examined the effect of post-exposure ricin A chain-specific monoclonal antibody on major intra-hepatic events observed during the studies described above in ricin challenged mice ( & ). By 48 hours after ricin exposure, administration of anti-ricin monoclonal antibodies was associated with restoration of carbohydrate stores in hepatocytes (, p<0.001), a return of F4/80+
immuno-reactive macrophages (, p<0.001), but little change in the number of neutrophils migrating to the liver in either the centro-lobular or peri-portal regions (). In contrast, mice receiving irrelevant monoclonal immunoglobulin matched for isotype and quantity demonstrated progressive and persistent loss of hepatic glycogen as well as disappearance of macrophges/Kupffer cells () at 48 and 72 hours after ricin challenge. These data indicate the capacity of post-exposure antibody to abrogate critical elements of ricin-induced intra-hepatic pathobiology associated with host death. While anti-ricin antibody in the intravascular space may limit toxin accumulation in several
organs, its ability to reverse profound and prolonged hypoglycemia, considered the cause of death in the model (11
), points to the centrality of hepatic
events in the pathogenensis of ricin intoxication.
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 (more ...)