Mechanism of suppression of human peripheral blood lymphocyte proliferation by platelet activation factor

The influence of the phospholipid platelet activation factor (PAF) was studied on PHA-stimulated proliferation of peripheral blood lymphocytes in patients with bronchial asthma and normal subjects. It was found that influencing on the whole population of lymphocytes PAF suppressed proliferation mainly of T-cells. Besides, the influence of PAF on lymphocyte proliferation seemed to be mediated by monocytes since removal of monocytes from the whole population of mononuclear cells abolished the suppression of lymphocyte proliferation induced by PHA. Pretreatment of lymphocytes with PAF antagonist--BL 8705 almost completely blocked the suppression of lymphocyte proliferation induced by PHA.     

Polynucleotide kinase and aprataxin-like forkhead-associated protein (PALF) acts as both a single-stranded DNA endonuclease and a single-stranded DNA 3' exonuclease and can participate in DNA end joining in a biochemical system

Polynucleotide kinase and aprataxin-like forkhead-associated protein (PALF, also called aprataxin- and PNK-like factor (APLF)) has been shown to have nuclease activity and to use its forkhead-associated domain to bind to x-ray repair complementing defective repair in Chinese hamster cells 4 (XRCC4). Because XRCC4 is a key component of the ligase IV complex that is central to the nonhomologous DNA end joining (NHEJ) pathway, this raises the possibility that PALF might play a role in NHEJ. For this reason, we further studied the nucleolytic properties of PALF, and we searched for any modulation of PALF by NHEJ components. We verified that PALF has 3' exonuclease activity. However, PALF also possesses single-stranded DNA endonuclease activity. This single-stranded DNA endonuclease activity can act at all single-stranded sites except those within four nucleotides 3' of a double-stranded DNA junction, suggesting that PALF minimally requires approximately four nucleotides of single-strandedness. Ku, DNA-dependent protein kinase catalytic subunit, and XRCC4-DNA ligase IV do not modulate PALF nuclease activity on single-stranded DNA or overhangs of duplex substrates. PALF does not open DNA hairpins. However, in a reconstituted end joining assay that includes Ku, XRCC4-DNA ligase IV, and PALF, PALF is able to resect 3' overhanging nucleotides and permit XRCC4-DNA ligase IV to complete the joining process in a manner that is as efficient as Artemis. Reduction of PALF in vivo reduces the joining of incompatible DNA ends. Hence, PALF can function in concert with other NHEJ proteins.     

Anthrax toxins inhibit immune cell chemotaxis by perturbing chemokine receptor signalling

Pathogenic strains of Bacillus anthracis produce two potent toxins, lethal toxin (LT), a metalloprotease that cleaves mitogen-activated protein kinase kinases, and oedema toxin (ET), a calcium/calmodulin-dependent adenylate cyclase. Emerging evidence indicates a role for both toxins in suppressing the initiation of both innate and adaptive immune responses, which are essential to keep the infection under control. Here we show that LT and ET inhibit chemotaxis of T-cells and macrophages by subverting signalling by both CXC and CC chemokine receptors. The data highlight a novel strategy of immunosuppression by B. anthracis based on inhibition of immune cell homing.     

Lethal toxin of Bacillus anthracis causes apoptosis of macrophages

Lethal toxin is a major anthrax virulence factor, causing the rapid death of experimental animals. Lethal toxin can enter most cell types, but only certain macrophages and cell lines are susceptible to toxin-mediated cytolysis. We have shown that in murine RAW 264.7 cells, sublytic amounts of lethal toxin trigger intracellular signaling events typical for apoptosis, including changes in membrane permeability, loss of mitochondrial membrane potential, and DNA fragmentation. The cells were protected from the toxin by specific inhibitors of caspase-1, -2, -3, -4, -6, and -8. Phagocytic activity of macrophages was inhibited by sublytic concentrations of lethal toxin. Infection of cells with anthrax (Sterne) spores impaired their bactericidal capacity, which could be reversed by a lethal toxin inhibitor, bestatin. We suggest that apoptosis rather than direct lysis is biologically relevant to lethal toxin intracellular activity.     

Lethal factor to mice produced by Escherichia coli isolated from chickens with swollen head syndrome

A lethal toxin similar to Bacillus cererus lethal toxin was detected in the culture supernatants of Escherichia coli isolated from chickens with swollen head syndrome. The lethal activity was heat-labile, protease-sensitive and killed mice within 10 min. The light microscopy of the histopathological studies revealed that the principal organ affected by this toxin was the lung but the liver and kidneys also showed lesions. The relevance of this lethal activity from E. coli remains to be determined.     

Monocytes Regulate the Mechanism of T-cell Death by Inducing Fas-Mediated Apoptosis during Bacterial Infection

Monocytes and T-cells are critical to the host response to acute bacterial infection but monocytes are primarily viewed as amplifying the inflammatory signal. The mechanisms of cell death regulating T-cell numbers at sites of infection are incompletely characterized. T-cell death in cultures of peripheral blood mononuclear cells (PBMC) showed ‘classic’ features of apoptosis following exposure to pneumococci. Conversely, purified CD3⁺ T-cells cultured with pneumococci demonstrated necrosis with membrane permeabilization. The death of purified CD3⁺ T-cells was not inhibited by necrostatin, but required the bacterial toxin pneumolysin. Apoptosis of CD3⁺ T-cells in PBMC cultures required ‘classical’ CD14⁺ monocytes, which enhanced T-cell activation. CD3⁺ T-cell death was enhanced in HIV-seropositive individuals. Monocyte-mediated CD3⁺ T-cell apoptotic death was Fas-dependent both in vitro and in vivo. In the early stages of the T-cell dependent host response to pneumococci reduced Fas ligand mediated T-cell apoptosis was associated with decreased bacterial clearance in the lung and increased bacteremia. In summary monocytes converted pathogen-associated necrosis into Fas-dependent apoptosis and regulated levels of activated T-cells at sites of acute bacterial infection. These changes were associated with enhanced bacterial clearance in the lung and reduced levels of invasive pneumococcal disease.     

Role of macrophage oxidative burst in the action of anthrax lethal toxin.

BACKGROUND: Major symptoms and death from systemic Bacillus anthracis infections are mediated by the action of the pathogen's lethal toxin on host macrophages. High levels of the toxin are cytolytic to macrophages, whereas low levels stimulate these cells to produce cytokines (interleukin-1 beta and tumor necrosis factor-alpha), which induce systemic shock and death. MATERIALS AND METHODS: Experiments were performed to assess the possibility that the oxidative burst may be involved in one or both of lethal toxin's effects on macrophages. Toximediated cell lysis, superoxide anion and cytokine production were measured. Effects of antioxidants and macrophage mutations were examined. RESULTS: RAW264.7 murine macrophages treated with high levels of toxin released large amounts of superoxide anion, beginning at about 1 hr, which correlates with the onset of cytolysis. Cytolysis could be blocked with various exogenous antioxidants or with N-acetyl-L-cysteine and methionine, which promote production of the endogenous antioxidant, glutathione. Mutant murine macrophage lines deficient in production of reactive oxygen intermediates (ROIs) were relatively insensitive to the lytic effects of the toxin, whereas a line with increased oxidative burst potential showed elevated sensitivity. Also, cultured blood monocyte-derived macrophages from a patient with Chronic Granulomatous Disease, a disorder in which the phagocyte's oxidative burst is disabled, were totally resistant to toxin, in contrast to control monocytes. CONCLUSIONS: These results imply that the cytolytic effect of the toxin is mediated by ROIs. Additionally, cytokine production and consequent pathologies showed partial dependence on macrophage ROIs. Antioxidants moderately inhibited toxin-induced cytokine production in vitro, and BALB/c mice pretreated with N-acetyl-L-cysteine or mepacrine showed partial protection against lethal toxin. Thus ROIs are involved in both the cytolytic action of anthrax lethal toxin and the overall pathologic process in vivo.     

Purification and biochemical characterization of an 11 000-dalton beta-bungarotoxin

The chromatographic separation and biochemical characterization of a beta-bungarotoxin is described. This toxin is isolated as the most basic eluting protein of Bungarus multicinctus venom when separated by column chromatography on CM-Sephadex C-25. The protein migrated as a single band on pH 4.3 and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight of this toxin was estimated to be 10 000 +/- 1000 by analytical sedimentation analysis. This value was consistent with the electrophoretic mobility of the toxin in SDS-polyacrylamide gels. The amino acid composition of this 11 000-dalton beta-bungarotoxin was similar to that of the 22 000-dalton beta-bungarotoxin previously reported (Lee et al. (1972) J. Chromatogr. 72, 71--82; Kelly, R.B. and Brown, III, F.R. (1974) J. Neurobiol. 5, 135--150; Kondo et al. (1978) J. Biochem. Tokyo 83, 91--99), suggesting that the 11 000-dalton toxin may be one of the polypeptide chains of the larger toxin. The 11 000-dalton beta-bungarotoxin was toxic to mice when injected intravenously. Animals that received lethal doses exhibited hyperexcitability followed by ataxia, convulsions, and death. The minimum lethal dose was 0.12 microgram/g body weight. This beta-bungarotoxin exhibited Ca2+-dependent phospholipase A activity comparable to that of the 22 000-dalton beta-bungarotoxin. The enzyme exhibited phospholipid substrate specificity in the rank order of phosphatidyl-choline, phosphatidylserine, phosphatidylethanolamine, and phosphatidyl-inositol. The enzyme activity was destroyed by boiling for 3 min at pH 8.6. In addition, an enzymatically inactive quantity of the 11 000-dalton toxin, equivalent to five times the minimum lethal dose of enzymatically active toxin, was not lethal when injected into mice. To test whether phospholipase A activity is responsible for lethality, bee venom phospholipase A2 was injected into mice at similar and greater concentrations with no toxic effect. Thus, while phospholipase A activity may be required for the lethal effect of the 11 000-dalton beta-bungarotoxin, the specificity of action of the toxin is not determined by its enzyme activity.     

Implication of HLA class I molecule from monocyte in T-cell activation by CD2 or CD3 monoclonal antibodies

The role of monocytes in human T-cell activation by monoclonal antibodies (mAb) recognizing CD3 molecule or by 2 mAb pairs directed against different epitopes of CD2 "GT2+T11(1)" or "D66+T11(1)" has been studied. It appears that HLA-cl I molecules from monocytes are involved in the early activation phase of T-cells stimulated by CD3 or CD2 when direct contacts between T-cells and monocytes are required. Thus, pretreatment of monocytes with HLA-cl I mAb inhibited IL 2 receptor appearance and IL 2 synthesis on T-cells stimulated by CD3 mAb or CD2 "GT2+T11(1)" mAb pair.     

Colony-stimulating factor 1 activates protein kinase C in human monocytes.

Colony-stimulating factor 1 (CSF-1) is required for the survival, proliferation and differentiation of monocytes. We previously demonstrated that the CSF-1 receptor is linked to a pertussis toxin-sensitive G protein and that the induction of Na+ influx by CSF-1 is a pertussis toxin-sensitive event. The present studies have examined activation of protein kinase C as a potential intracellular signaling event induced by the activated CSF-1 receptor. The results demonstrate that CSF-1 stimulates translocation of protein kinase C activity from the cytosol to membrane fractions. This activation of protein kinase C was sensitive to pretreatment of the monocytes with pertussis toxin. Lipid distribution studies demonstrated that phosphatidylcholine (PC) is the major phospholipid in human monocytes. Moreover, the results indicate that CSF-1 stimulation is associated with decreases in PC, but not in phosphatidylinositol (PI), levels. The absence of an effect of CSF-1 on PI turnover was confirmed by the lack of changes in inositol phosphate production. In contrast, CSF-1 stimulation was associated with increased hydrolysis of PC to phosphorylcholine and diacylglycerol (DAG) in both intact monocytes and cell-free assays. Furthermore, the increase in PC turnover induced by CSF-1 was sensitive to pertussis toxin. The results also demonstrate that the induction of Na+ influx by CSF-1 is inhibited by the protein kinase C inhibitors staurosporine and the isoquinoline derivative H7, but not by HA1004.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tregs Modulate Lymphocyte Proliferation,Activation, and Resident-Memory T-Cell Accumulation within the Brain during MCMV Infection

Accumulation and retention of regulatory T-cells (Tregs) has been reported within post viral-encephalitic brains, however, the full extent to which these cells modulate neuroinflammation is yet to be elucidated. Here, we used Foxp3-DTR (diphtheria toxin receptor) knock-in transgenic mice, which upon administration of low dose diphtheria toxin (DTx) results in specific deletion of Tregs. We investigated the proliferation status of various immune cell subtypes within inflamed central nervous system (CNS) tissue. Depletion of Tregs resulted in increased proliferation of both CD8⁺ and CD4⁺ T-cell subsets within the brain at 14 d post infection (dpi) when compared to Treg-sufficient animals. At 30 dpi, while proliferation of CD8⁺ T-cells was controlled within brains of both Treg-depleted and undepleted mice, proliferation of CD4⁺ T-cells remained significantly enhanced with DTx-treatment. Previous studies have demonstrated that Treg numbers within the brain rebound following DTx treatment to even higher numbers than in untreated animals. Despite this rebound, CD8⁺ and CD4⁺ T-cells proliferated at a higher rate when compared to that of Treg-sufficient mice, thus maintaining sustained neuroinflammation. Furthermore, at 30 dpi we found the majority of CD8⁺ T-cells were CD127^hi KLRG1^- indicating that the cells were long lived memory precursor cells. These cells showed marked elevation of CD103 expression, a marker of tissue resident-memory T-cells (T_RM) in the CNS, in untreated animals when compared to DTx-treated animals suggesting that generation of T_RM is impaired upon Treg depletion. Moreover, the effector function of T_RM as indicated by granzyme B production in response to peptide re-stimulation was found to be more potent in Treg-sufficient animals. Taken together, our findings demonstrate that Tregs limit neuroinflammatory responses to viral infection by controlling cell proliferation and may direct a larger proportion of lymphocytes within the brain to be maintained as T_RM cells.     

Glycerophospholipid:cholesterol acyltransferase complexed with lipopolysaccharide (LPS) is a major lethal exotoxin and cytolysin of Aeromonas salmonicida: LPS stabilizes and enhances toxicity of the enzyme.

An extracellular lethal toxin produced by Aeromonas salmonicida was purified by fast-protein liquid ion-exchange chromatography. The toxin is composed of glycerophospholipid:cholesterol acyltransferase (GCAT) (molecular mass, 25 kilodaltons) aggregated with lipopolysaccharide (LPS), the GCAT/LPS complex having a molecular mass of about 2,000 kilodaltons, estimated by gel filtration chromatography. The toxin is lethal for Atlantic salmon (Salmo salar L.) at a concentration of 0.045 micrograms of protein per g of body weight. The toxin is a hemolysin (T-lysin, active on fish erythrocytes), leukocytolysin, and cytotoxin. Antiserum to the purified toxin neutralized the lethal toxicity of the crude extracellular toxins, indicating this toxin to be the major lethal factor produced by A. salmonicida. In the crude extracellular products, small amounts of free GCAT were also present. This has been purified, and its activities and properties have been compared with those of the GCAT/LPS complex. The presence of LPS did not influence the GCAT activity of the enzyme with egg yolk or phosphatidylcholine (lecithin) as a substrate, but the specific hemolytic activity and lethal toxicity was about eightfold higher in the complexed form. Furthermore, the free GCAT was more susceptible to proteolytic and heat inactivation than was the GCAT/LPS complex. Recombination of LPS (phenol extracted from extracellular products of A. salmonicida) with free GCAT enhanced the hemolytic activity, lethal toxicity, and heat stability of the latter but did not influence its lecithinase activity. In native polyacrylamide gel electrophoresis, the GCAT/LPS complex and the recombined GCAT-LPS both showed a high-molecular-mass band which did not enter the gel, while the free GCAT produced a single band with low molecular mass. In isoelectric focusing gels, the GCAT/LPS and recombined GCAT-LPS produced a nonfocusing smear with pIs from pI 5.0 to 5.8, while the free GCAT produced a single band with pI 4.3. These data show that free GCAT can combine with LPS to produce a high-molecular-mass complex with enhanced toxicity and heat stability compared with those of free GCAT, similar to the preexisting GCAT/LPS complex, and indicate that the LPS moiety of the toxin plays an active role in toxicity.     

Effect of ochratoxin A and ochratoxin C on the monocyte and lymphocyte function

The effect of practically relevant mycotoxin concentrations on functions of immune cells was studied in in vitro experiments. Porcine mononuclear cells were exposed to a crudeAspergillus-ochraceus toxin containing OTA, a HPLC fraction identical with OTC derived from the crude toxin (RE2), as well as pure OTA and OTC in a concentration range from 0.46 to 3000 ng/ml. The influence of mycotoxin exposure on metabolic activity, mitogen induced proliferation, expression of the activation marker CD25 and the cell cycle of lymphocytes and on the formation of free oxygen radicals as well as the production of the cytokines IL-6 and TNF-α by monocytes was determined. Exposure to high concentrations of all mycotoxin preparations lead to non-specific suppression of the immune cell functions, which was related to cytotoxic effects. Low concentrations caused ambivalent reactions, especially on monocyte function. In general, the HPLC fraction RE2 had an up to 100-fold stronger effect than pure OTA. Ochratoxin-induced suppression of lymphocyte proliferation was not abrogated by phenylalanine or aspartame. The results indicate that immunomodulation can be caused by very low mycotoxin concentrations which are not related to clinical symptoms or loss of performance.     

Properties of Immature Myeloid Progenitors with Nitric-Oxide-Dependent Immunosuppressive Activity Isolated from Bone Marrow of Tumor-Free Mice

Myeloid derived suppressor cells (MDSCs) from tumor-bearing mice are important negative regulators of anti-cancer immune responses, but the role for immature myeloid cells (IMCs) in non-tumor-bearing mice in the regulation of immune responses are poorly described. We studied the immune-suppressive activity of IMCs from the bone marrow (BM) of C57Bl/6 mice and the mechanism(s) by which they inhibit T–cell activation and proliferation. IMCs, isolated from BM by high-speed FACS, inhibited mitogen-induced proliferation of CD4⁺ and CD8⁺ T-cells in vitro. Cell-to-cell contact of T-cells with viable IMCs was required for suppression. Neither neutralizing antibodies to TGFβ1, nor genetic disruption of indolamine 2,3-dioxygenase, abrogated IMC-mediated suppressive activity. In contrast, suppression of T-cell proliferation was absent in cultures containing IMCs from interferon-γ (IFN-γ) receptor KO mice or T-cells from IFN-γ KO mice (on the C57Bl/6 background). The addition of NO inhibitors to co-cultures of T-cells and IMC significantly reduced the suppressive activity of IMCs. IFN-γ signaling between T-cells and IMCs induced paracrine Nitric Oxide (NO) release in culture, and the degree of inhibition of T-cell proliferation was proportional to NO levels. The suppressive activity of IMCs from the bone marrow of tumor-free mice was comparable with MDSCs from BALB/c bearing mice 4T1 mammary tumors. These results indicate that IMCs have a role in regulating T-cell activation and proliferation in the BM microenvironment.     

A novel human AP endonuclease with conserved zinc-finger-like motifs involved in DNA strand break responses

DNA damage causes genome instability and cell death, but many of the cellular responses to DNA damage still remain elusive. We here report a human protein, PALF (PNK and APTX-like FHA protein), with an FHA (forkhead-associated) domain and novel zinc-finger-like CYR (cysteine-tyrosine-arginine) motifs that are involved in responses to DNA damage. We found that the CYR motif is widely distributed among DNA repair proteins of higher eukaryotes, and that PALF, as well as a Drosophila protein with tandem CYR motifs, has endo- and exonuclease activities against abasic site and other types of base damage. PALF accumulates rapidly at single-strand breaks in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner in human cells. Indeed, PALF interacts directly with PARP1 and is required for its activation and for cellular resistance to methyl-methane sulfonate. PALF also interacts directly with KU86, LIGASEIV and phosphorylated XRCC4 proteins and possesses endo/exonuclease activity at protruding DNA ends. Various treatments that produce double-strand breaks induce formation of PALF foci, which fully coincide with gammaH2AX foci. Thus, PALF and the CYR motif may play important roles in DNA repair of higher eukaryotes.     

Correlation between In Vitro Cytotoxicity and In Vivo Lethal Activity in Mice of Epsilon Toxin Mutants from Clostridium perfringens

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB.     

Purification and characterization of a lethal toxin from the venom of Heloderma horridum horridum

A lethal toxin was isolated from the venom of Heloderma h. horridum by gel filtration and ion-exchange chromatography. Molecular weight of the purified toxin was determined to be 28 kDa under reducing and nonreducing conditions. Biological activity, assayed by i.v. routes of injection, shows an LD50 for this preparation of 0.135 micrograms/g. Additionally, the toxin possesses an inhibitory effect on direct electrical stimulation of the isolated mouse hemi-diaphragm. However, neither hemorrhagic nor hemolytic activities were detected. Phospholipase A2 activity, proteolytic activity and arginine esterolytic activity were absent. The amino acid composition of the lethal toxin and the NH2-terminal sequence up to residue number 33 were determined. Neither show similarities to other components from H. h. horridum venom.     

Macrophages are sensitive to anthrax lethal toxin through an acid-dependent process

Anthrax lethal toxin, which consists of two proteins, protective antigen and lethal factor, is lethal for experimental animals. This study describes the first in vitro system demonstrating lethality of the toxin. Mouse peritoneal macrophages are killed within 1 h of exposure to the toxin. Neither protein component alone shows any toxic activity. The minimal effective concentration of protective antigen and lethal factor was approximately equal to 10(-2) and approximately equal to 10(-3) micrograms/ml, respectively. None of the several established cell lines examined was killed. Cells could be completely protected from the toxin by pretreatment with agents, such as amines or monensin, which dissipate intracellular proton gradients and raise the pH of intracellular vesicles. This protection was reversible and could be overcome by lowering the intravesicular pH. Antitoxin added after preincubation with amines was unable to protect cells subsequently exposed to low pH treatment. These results suggest that anthrax lethal toxin requires passage through an acidic endocytic vesicle in order to exert its toxic effect within the cytosol.     

Early activation events differentiate the reactivity of two T-cell families to Staphylococcus enterotoxin A

Analysis of early activation events in two SEA responsive T-cell families demonstrated that low doses of SEA induced CD4+Vbeta22 T-cells to down-regulate their TCR and express CD69, considerably earlier than CD4+Vbeta5 T-cells. The rapid down-regulation of Vbeta22 TCR led to its proliferation, whereas even a 10-fold higher dose of toxin induced only a partial down-regulation of Vbeta5 TCR. Stimulation with SEA induced a significantly higher percentage of Vbeta22 T-cells to produce IFN-gamma compared to Vbeta5 T-cells. SEAF47A, a mutant of SEA, known to have a lower binding affinity for the MHC class II molecule, failed to activate Vbeta5 T-cells whereas Vbeta22 T-cell activation was slightly decreased. Hence, early activation events highlighted the differential requirements of T-cell families to respond to SEA.     

Targeted Silencing of Anthrax Toxin Receptors Protects against Anthrax Toxins

Anthrax spores can be aerosolized and dispersed as a bioweapon. Current postexposure treatments are inadequate at later stages of infection, when high levels of anthrax toxins are present. Anthrax toxins enter cells via two identified anthrax toxin receptors: tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2). We hypothesized that host cells would be protected from anthrax toxins if anthrax toxin receptor expression was effectively silenced using RNA interference (RNAi) technology. Thus, anthrax toxin receptors in mouse and human macrophages were silenced using targeted siRNAs or blocked with specific antibody prior to challenge with anthrax lethal toxin. Viability assays were used to assess protection in macrophages treated with specific siRNA or antibody as compared with untreated cells. Silencing CMG2 using targeted siRNAs provided almost complete protection against anthrax lethal toxin-induced cytotoxicity and death in murine and human macrophages. The same results were obtained by prebinding cells with specific antibody prior to treatment with anthrax lethal toxin. In addition, TEM8-targeted siRNAs also offered significant protection against lethal toxin in human macrophage-like cells. Furthermore, silencing CMG2, TEM8, or both receptors in combination was also protective against MEK2 cleavage by lethal toxin or adenylyl cyclase activity by edema toxin in human kidney cells. Thus, anthrax toxin receptor-targeted RNAi has the potential to be developed as a life-saving, postexposure therapy against anthrax.