Background: Anthrax lethal toxin (LeTx) is a major virulence factor in anthrax infection but compelling evidence suggests that it exerts a strong inhibitory effect on macrophage cytokine production. This leaves no definitive explanation as to the etiology of many of the early signs and symptoms, seen in inhalational anthrax infection.
Methods: The responses of histamine, bradykinin and serotonin release to challenge by various concentrations of B. anthracis cell wall (CW) components, lethal toxin (LeTx), and lipopolysaccharide (LPS) were measured in vitro using human blood cells and murine mast cells. An in vivo testing using C57BL/6 mice was also performed to further verify the in vitro observations.
Results: In both human and murine cells, the group stimulated with LeTx released significant amounts of histamine, bradykinin and serotonin when compared to the group stimulated with CWs. We also demonstrated that LeTx stimulated the in vivo release of large quantities of histamine and serotonin when injected into a C57BL/6 mouse model.
Conclusions: These results newly implicate these heretofore insufficiently investigated mediators as possible participants in anthrax pathogenesis. This may help our understanding of this often fatal disease, and, may also affect future adjunctive treatment choices in anthrax infection.
Inhalational anthrax is a serious, usually fatal infectious disease produced by the gram-positive, spore-forming Bacillus anthracis. Its role as a potent biological weapon was clearly demonstrated by an accidental release in the city of Sverdlovsk in the former Soviet Union in 1979, and the terrorist attack in the eastern United States in 2001.
The clinical manifestations and pathogenesis of inhalational anthrax infection have been well described [Mayer et al 2001; Jernigan et al 2001; Inglesby et al 2002]. Generally accepted, early clinical signs and symptoms of anthrax infection include fever, chills, fatigue, a non-productive cough, nausea and/or vomiting, dyspnea, hypoxemia, chest pain, myalgias, headache and confusion. Following the 2001 US anthrax attacks, patients, even in the early stages of infection, were also found to have mediastinal widening due to adenopathy, pleural effusions, pulmonary infiltrates, tachycardia, and pericardial effusions. Autopsy victims of the Sverdlovsk outbreak had prominent pulmonary edema [Abramova et al 1993]. A recent re-analysis of the original pulmonary autopsy slides noted an absence of obvious infectious pneumonitis, thus confirming that inhalational anthrax is not primarily a pulmonary infection, even with the hematogenous spread back to the lungs, as well as to other organs, that has been demonstrated in late anthrax [Grinberg et al 2001]. This leaves us to wonder what might be the mechanism for the prominent pulmonary, gastrointestinal and neurological symptomatolgy noted during the early phase of anthrax infection, before the occurrence of terminal bacteremia, and prior to the anthrax exotoxins reaching the critical threshold required for the progressive cascade of immunological dysregulation that quickly leads to cardiovascular collapse and death.
The anthrax toxins are composed of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). At the target cell membrane, PA combines with either LF to form LeTx or with EF to become edema toxin (EdTx) [Brossier and Mock 2001; Liddington 2002] and then transports these toxins across the host cell membrane into the cytoplasm where they exert their actions. The toxins acting in combination are presently believed to produce the majority of clinical symptoms of anthrax. Neither LF nor EF has been shown to be individually toxic when not in the presence of PA [Smith 2002]. EdTx is known to impair neutrophil function in vivo and also acts to produce edema [Crawford et al 2006]. LeTx, which is a zinc metalloprotease whose enzymatic activity on mitogen-activated protein kinase kinase (MAPKK) may account for its cytolytic effects [Baldari et al 2006; Duesbery et al 1998; Nassi et al 2002; Vitale et al 1998], is currently believed to be the most clinically important of the toxins, but our current understanding of LeTx does not adequately explain many of the early signs and symptoms of anthrax, nor does it explain some of the prominent late clinical presentations, such as massive hemorrhage, hemolytic anemia, neurological symptoms and sudden death. To compound the problem, some of the research data on mediators of septic shock induced by anthrax toxin are contradictory [Klimpel et al 1992; Mock and Fouet 2001; Erwin et al 2001; Hanna et al 1994].
We propose here that anthrax LeTx may induce the release of additional immunologically active mediators such as histamine, serotonin, and bradykinin, which may account for many of the unexplained manifestations of inhalational anthrax. Histamine, serotonin, and bradykinin have broad and complex physiological effects on several organ systems, and, may play important roles in causing severe inflammatory reactions and septic shock. To test this hypothesis we compared the in vitro effects of LeTx, B. anthracis CW components and LPS in their abilities to induce the release of histamine, bradykinin and serotonin from murine mast cells, human basophils where histamine is stored, and platelets where serotonin is stored. There can be many factors involved in inducing a release of these immunogenic components; we were primarily concerned with whether LeTx exposure induced these components and therefore decided that the differences between the releases from CW and LPS were not significant to make any conclusions. We also looked at in vivo release of histamine and serotonin, at various time intervals, using female C57BL/6 mice injected with LeTx.
MATERIALS AND METHODS
Human cell preparation:
Human blood was obtained from the American Red Cross (Rockville, MD). The Department of Health and Human Services/NIH guidelines on protection of human subjects were followed, with the authorization of the George Mason University Institutional Review Board. The blood was collected from different healthy individuals and processed separately. PBMCs were isolated by centrifugation for 30 min at 900 g on Fico/Lite-LymphoH (Atlanta Biologicals, Atlanta, GA). Basophils were purified from the PBMCs population by negative selection using MACS cell isolator (Miltenyi Biotec, Germany) after staining with antibodies against CD3, CD7, CD14, CD15, CD16, CD36, CD45RA and HLA-DR. The purity was determined under a microscope following Giemsa staining, with over 90% purity being achieved. Platelets were obtained by using high-speed centrifugation (12,000 x g for 15 min) of the plasma. The time between obtaining the blood (including purifying cells) and initiation of the culture was less than 2 hours. Less than one million basophils were obtained from 100 mL of blood.
Mast cell preparation:
Murine mast cells were derived from bone marrow cells of C57BL/6 mice. The experimental animal protocol was approved by the Committee on Animal Research of the Biomedical Research Institute (Rockville, MD) and George Mason University. Female C57BL/6 mice (9-10 weeks) were obtained from Charles River Laboratory (Wilmington, MA) and maintained in a temperature-controlled room with standard laboratory food and water freely available. Mice were sacrificed under anesthesia by neck dislocation. The femurs and tibias were flushed with phosphate buffer slain (PBS), and mononuclear cells were isolated by using Fico/Lite (D=1.086). The marrow cells were adjusted to 106 per mL in a mixture of DMEM-12 medium (Invitrogen, Carlsbad, CA) and conditioned medium (see below). The cells were cultured at 37° C in the presence of stem cell factor (15 ng/mL) and IL-3 (10 ng/mL) for 2 weeks, with medium change twice a week. The conditioned medium was prepared from mouse splenocytes. The splenocytes were adjusted to 5 x 106 per mL in DMEM-12 medium supplemented with 2 μg/mL of Concanavalin A (Sigma, St. Louis, MO). The cells were incubated at 37°C for 45 hours. Supernatants were harvested and saved as conditioned medium. The purity of mast cells was checked under a microscope following Giemsa staining and indicated that 80% purity was achieved.
Cell wall preparations, LeTx and LPS:
An attenuated B. anthracis strain SNKE-FF-E308D was used for CW preparation. CW preparations were obtained by mixing whole lyophilized bacteria with equal volumes of 8% sodium dodecyl sulfate (SDS) and boiling for 30 min to denature any residual proteins. After overnight incubation at room temperature with agitation, the suspension was centrifuged (30,000 x g for 15 min), and the pellet was extracted twice by boiling with 4% SDS and washed by centrifugation at 20°C four times with water, twice with 2N NaCl, and again with water. Amino acid composition of complete CWs was carried out by gas chromatography-mass spectrometry after hydrolysis with 6N HCl, 150°C, 1 h and derivatization to volatile N-heptafluorobutyryl isobutyl esters of amino acids [MacKenzie 1987], using Hewlett-Packard apparatus (model HP 6890) with a type HP-5 glass capillary column (0.32 mm by 30 m) and temperature programming at 8°C/min, from 125-250°C in the electron ionization (106 eV) mode. Standards of amino acids were hydrolyzed, derivatized and analyzed in the same way. CW sugars were analyzed by gas chromatography-mass spectrometry as alditol acetates after hydrolysis with 10N HCl, 80°C, 30 min under the condition described above. Phosphorus was estimated by the Chen’s method [Chen et al 1956]. The CW preparations were checked for aerobic and anaerobic bacterial contamination. Bacterial contamination was checked using various growth media. Endotoxin was tested using a Limulus amoebocyte lysate (LAL) test kit (E-TOXATE, Sigma) to make sure that there was no any residual endotoxin left over in the preparations.
PA and LF were purchased from List Biological Laboratories Inc (Campbell, CA). The activity of LeTx was determined by its cytolytic effect on murine macrophage cells RAW264.7. E. coli LPS was purchased from Sigma.
Histamine, serotonin and bradykinin release assay:
Basophils, mast cells, platelets, and whole blood were treated with bacterial CW (100, 1000, and 5000 ng/mL) and LeTx (PA 500 ng/mL and LF 100 ng/mL, for a ratio of 5:1) for 30 min. This amount of LeTx was selected as the optimal dose to lyse RAW264.7 murine macrophages, and, was the minimum concentration required for inducing apoptosis of PBMCs in our preliminary studies [Popov et al 2002]. The amount of CW was tested from 10 to 5000 ng/mL in order to achieve maximum release of histamine from human basophils. LPS (100 ng/mL) was used as a positive control, as this amount had been previously shown in our preliminary studies to induce maximal histamine release from murine mast cells. Several concentrations of bacterial CW were tested in order to determine if the histamine release is dose-dependent. To further test for any synergistic effects of CW and LeTx, some cells were treated with both. All supernatants were harvested and submitted for determination of histamine, serotonin, and bradykinin.
Quantification of histamine, serotonin, and bradykinin:
The concentrations of histamine, serotonin, and bradykinin in the supernatants and plasma were determined by using ELISA assays. ELISA kits for testing histamine and serotonin were obtained from ICN Biomedicals, Inc. (Costa Mesa, CA). These kits were designed and manufactured for clinical applications. A bradykinin detection kit was purchased from Phoenix Peptide, Inc. (Belmont, CA). Experiments were performed following the manufacturer’s instruction. Six repeats were performed for each determination. Triplicates were used for each test and a standard curve was plotted each time to ensure the assay worked properly. The results were calculated using semi-logarithmic graph paper.
In vivo studies of histamine and serotonin release:
Murine macrophages derived from different strains have shown different susceptibilities to anthrax LeTx [Muehlbauer et al 2007]. In order to test the dynamic release of the immunological mediators from the cells after encountering LeTx, an immediate cytolysis should be avoided. Thus, a nonsusceptible mouse strain C57BL/6 was selected for this study.
Female C57BL/6 mice (9—10 weeks) were injected through the tail vein with LeTx at a dose of PA:LF = 300 μg:60 μg per animal. Blood was harvested by cardio-puncture after injection of the toxin for 2, 4, 8, and 24 h under anesthesia with isoflurane. Serum was assayed to determine the concentration of histamine and serotonin using ELISA as described above. Cohort mice usually died within 4 days following the injection of this LeTx dose.
Each of the stimulators was tested independently using an analysis of variance (ANOVA). Differences in the stimulator types (CW, LPS, and LeTx) were tested using Tukey’s multiple comparison method to control the overall significant level at 0.05 for each concentration of histamine, serotonin, and bradykinin. Student’s t test was used when it was applicable. The concentration of each element represents a mean of six individual repeats, and standard error was calculated.
Chemical analysis of the Cell Wall: The CW of B. anthracis contains alanine, glutamic acid and diaminopimelic acid as the major amino acids in the molar ratio of 1.6:1.0:1.0, which is in agreement with published results [Ratney 1965]. Sugar constituents of the CW suggest the presence of teichoic acid-like polymers containing glycerol phosphate chains, whereas sugar constituents of B. anthracis CW are characterized by low content of phosphate and high content of galactose and glucosamine. In B. anthracis, capsular polysaccharides and teichoic acids are covalently and non-covalently associated with peptidoglycan. These molecules are responsible for bacteria-host interactions and virulence [Choudhury et al 2006]. The similar CW polysaccharide has also been found by others in B. anthracis Sterne strain [Ekwunife et al 1991]. No bacterial contamination was found after 24 h culture of the CW preparation at 37°C. LAL test did not shown traceable endotoxin in the preparations.
HISTAMINE RELEASE FROM DIFFERENT CELLS IN RESPONSE TO LeTx AND CELL WALL
The ability of anthrax LeTx and CW components to induce histamine release in murine cells was examined. Because the CW was purified from B. anthracis, great attention was paid to eliminate any possible contaminations with LeTx components. In addition, bacterial contamination of the CW preparation was repeatedly tested using different culture media for both aerobic and anaerobic bacteria. Murine mast cells were prepared as described in the method section and the release of histamines was measured. The well-known histamine inducer LPS was used as a positive control [Kim et al 2005].
The histamines were measured for each challenge, CW, LeTx and LPS. Among the tested compounds [Figure 1], LeTx induced the strongest histamine release, producing a 3.3-fold increase over control cells (p=0.014). B. anthracis CW components, CW, also induced a dose dependent histamine release by as much as a 1.8-fold increase over control cells at the highest CW concentration (p=0.026). The amount of histamine released in the CW 5000 ng/mL group was twice that of the CW 100 ng/mL groups [Figure 2A]. However, additional increases in the CW dose to above 5000 ng/mL did not further increase histamine release in the preliminary experiments, indicating that the maximal dose-response effect had been achieved. LPS, a potent inducer of proinflammatory cytokines, produced a 2.1-fold histamine increase over the control (p=0.022) and was not as strong an inducer as was LeTx in this study. The control in [Figure 1] was the measurement of histamines in murine mast cells without a challenge.
Although precursors of mast cells are formed in the bone marrow by hematopoiesis, they differentiate into mature stage in tissues. Therefore, mast cells can be easily found in many tissues but are rarely seen in the circulating blood. The majority of histamine in blood is stored in the basophils. Thus, we purified basophils from human blood and tested histamine storage in the basophilic cells using the same procedure as that used for the murine mast cells. A similar release pattern was observed from human basophils [Figure 3A] as was seen from the murine mast cells, with CW producing a 1.9-fold increase (p=0.025), LPS producing a 4.2-fold (p=0.006) increase, and LeTx producing a 5.3-fold increase in histamine release compared to control cells (p<0.001).
We also treated fresh blood with the same reagents and measured the amount of histamine released into the plasma [Figure 3B]. As expected, the actual amounts of histamine measured were less than those seen in the purified basophilic cell culture. LeTx induced the most significant release of histamine into blood plasma compared to that produced by LPS or CW components. LeTx produced a 6.6-fold increase in histamine release over the control (p<0.001), compared to the 6.5-fold increase seen with LPS and a 5.2-fold increase seen with CW (p=0.001).
Serotonin release from different cells after treatment with cell wall and LeTx: Serotonin is primarily located in the enterochromaffin cells of the intestine and serotonergic neurons of the brain. The majority of the serotonin in the circulating blood is concentrated in platelets, with smaller amounts in basophils. Therefore, platelets and basophils were purified, and treated with CW, LPS, and LeTx. As was performed for the histamine release assays [Figure 2A], plasma from the fresh whole blood was harvested and serotonin release from platelets, basophils, and whole blood was assayed after treatment with CW at different dosages [Figure 2B]. The maximum release was achieved when the concentration of CW reached to 5000 ng/mL.
The platelets enriched from 10 mL of fresh blood were resuspended with 1 mL of PBS in the presence of 10% autologous serum. The greatest amount of serotonin was detected in the supernatants when LeTx was introduced [Figure 4A]. LeTx was able to induce a 2.1 fold increase (p=0.022) over the control compared to the 1.9-fold increase induced by LP (p=0.025). CW components induced the smallest rise in serotonin at 1.6-fold over the control (p=0.03).
Only small amounts of serotonin were released from the basophils [Figure 4B], but the relative increases among the treatment groups were more significant compared to the data from platelet groups. CW, LPS and LeTx were able to induce 11.1, 17.3, and 38-fold increases of serotonin release, respectively, from the basophils compared to the control (p<0.001).
Serotonin detected in fresh blood plasma [Figure 4C] was released by both platelets and basophils. Again, LeTx exhibited the strongest inducer, and resulted in 2.5-fold (p=0.019) increase of serotonin compared to non-treated controls. LPS produced a 2.1-fold increase (p=0.022) and CW a 1.7-fold increase (0.027) over the control.
To investigate the effects of LeTx and CW on bradykinin release, human whole blood was cultured for 30 minutes in the presence of the stimulating agents. As shown in [Figure 5], the mean concentrations of bradykinin in the LeTx and CW treated groups were 60 ng/mL and 18 ng/mL, whereas only 9.8 ng/mL was determined in LPS treated group. LeTx treatment caused a 20-fold increase (p<0.001) in bradykinin release compared to the control. CW produced 5.1-fold increase (p=0.001) and LPS produced an insignificant 3.3-fold increase (p=0.014) compared to control cells.
Both the CW and LeTx can induce the release of histamine, serotonin and bradykinin. Interestingly, no significant synergistic effects between CW and LeTx were noted in the trials (data not shown). The reason could be that LeTx alone was able to deprive the intracellular stockpiled mediators.
We demonstrated that LeTx is a potent inducer for histamine, serotonin, and bradykinin release in vitro. To confirm the in vitro findings, an in vivo murine model was used as human surrogate. All animals died within four days after intravenous administration of the LeTx at the dosage described above. Blood was harvested at various time points after LeTx challenge and assayed to determine histamine and serotonin in the serum [Figures 6A and 6B]. Our data indicate that LeTx was able to induce histamine and serotonin release in vivo with a behavior similar to that observed in the in vitro studies. Maximal release of both histamine (17-fold increase over baseline, p<0.001) and serotonin (3.2-fold increase over baseline, p=0.015) was found in the 2-hour samples. The serum concentration of serotonin rapidly declined two hours after the challenge to a less than 2-fold increase by eight hours. In contrast, the histamine concentration was sustained at more than 2-fold increase, over baseline, even 24 hours after the LeTx injection. Using the same assay as applied for in vitro study, the bradykinin was not detectable in murine serum even after treatment with LeTx.
Histamine, serotonin, and bradykinin have broad and complex physiological effects on several organ systems, and may play important roles in causing severe inflammatory reactions and septic shock. They may also play pivotal roles in the pathogenesis of anthrax and may account for a variety of the clinical signs and symptoms in the presentation of inhalational anthrax infection (Frankel et al, 2009).
We have shown that both LeTx and CW components are able to induce histamine, serotonin and bradykinin release. However, the mechanisms of these actions might be completely different. LeTx is introduced into the target cells through two cell surface receptors: anthrax toxin receptor 1 (ANTXR1) and ANTXR2 [Young and Collier 2007]. ANTXR1 is a tumor endothelial marker-8 whereas ANTXR2 is a capillary morphogenesis protein 2. B. anthracis CW components are composed of heterogeneous molecules that may exert action through toll-like receptors, which recognize pathogen-associated molecular patterns shared by broad spectrum of microorganisms [Hughes et al 2005].
Bronchoconstriction is one of the notable properties of histamine and is mediated by the H1 receptor [Takahashi et al 1995; Casale et al 1985; Ekici et al 2006]. It constricts both central and peripheral bronchial smooth muscle in vitro, with a greater effect on the peripheral airways. These effects are amplified by both serotonin and bradykinin [Funayama et al 2001; Saxena et al 1995]. In addition, histamine induces hypersecretion of mucus in the trachea and the bronchi which can further exaggerate the resistance of the airway. Importantly, histamine can also produce a vasodilatory response (flare in the skin), and an increased permeability of post-capillary venules. This leads to extravasation of fluids, plasma proteins and cells into the extracellular space, and, can cause both edema and hypotension [Takahashi et al 1995]. Bradykinin and serotonin both produce similar effects on the microvascular permeability, and in addition, bradykinin can cause cough [Howl and Payne 2003]. Although we are not postulating that these proinflammatory molecules are the only mediators involved in the clinical presentations of inhalational anthrax, the combined effects of bronchoconstriction, increased airway mucous secretion, and extravasation of intravascular fluids into the tissues would all be sufficient to explain the pulmonary edema, pleural effusions, cough, dyspnea, hypoxia, cyanosis, chest pain, respiratory failure and shock seen in this disease.
The H1 receptor also plays a role in stimulation of the gastrointestinal system and can induce nausea and vomiting [Golembiewski et al 2005]. Through the H3 receptors, broadly expressed in central and peripheral nervous systems, histamine is probably involved in regulation of neural function [Brown et al 2001]. These effects, alone or combined with the various CNS effects of serotonin, could help to explain symptoms such as confusion often noted in anthrax victims. As members of an inflammatory complex network, these small molecules may play important roles in the development of the immunological dysregulation that leads to septic shock and death. As such, interventions which focus on these target molecules, such as H1 blockers, and some experimental H3 agonists and bradykinin antagonists, could be useful in the management of this disease and deserve further investigation.
This study demonstrates that anthrax LeTx induces the elaboration of increased amounts of histamine, bradykinin and serotonin. The data from this study support our hypothesis that anthrax LeTx can induce the release of additional immunologically active mediators, which may be responsible for causing many unexplained manifestations of inhalational anthrax. Those mediators may represent another possible mode of action for LeTx, which strongly correlates the molecular actions of the toxin to the clinical presentations observed in anthrax. This finding, in conjunction with the compelling recent evidence that, overall, LeTx inhibits macrophage cytokine production [Ervine et al 2001] rather than enhancing it as previously thought [Hanna et al 1994], will assist us in understanding the etiology of the immunological dysregulation and septic shock seen in inhalational anthrax infection. The ability of LeTx to induce the release of histamine, serotonin and bradykinin also implies that an adjuvant therapeutic modality with blockers against these immunologically active mediators could help to alleviate certain symptoms associated with anthrax infection.
Based on this finding, we presume that H1 blockers would be able to reduce the airway resistance and avert hypoxemia in inhalation anthrax. H1 blockers could also be applied to improve the symptoms, such as nausea, vomiting and diarrhea, which are commonly observed in gastrointestinal anthrax. Blockers against H3 receptor may help to ameliorate the neurological symptoms associated with anthrax infection.
This work was supported by contracts between the United States Army Medical Research and Materiel Command, the Defense Advanced Research Projects Agency, and AFG Biosolutions, Inc.
Figure 1: Histamine production by murine mast cells in response to different stimuli: The mast cells were derived from BALB/c mice bone marrow in the presence of stem cell factor and IL-3. The cells were treated with CW, LeTx, and LPS for 30 minutes. The supernatants were harvested for ELISA assay to determine the concentration of histamine (for details, see methods). The data in the graph represent 6 repeats and data are expressed as mean ± standard error. *CW = anthrax cell wall at 5000 ng/mL. [go to text reference]
Figure 2: Dose dependent release of (A) histamine and (B) serotonin when cells were treated with different dosages of CW. Cells from various sources were treated with CW for 30 min. Supernatants or plasma were harvested and assayed for concentration of histamine and serotonin using ELISA as described in the text. Data in the figure represent 6 repeats. [go to text reference]
Figure 3: Determination of histamine in human whole blood and basophils. (A) Basophils were purified by magnetic negative selection. The Basophils and (B) blood were treated with bacterial CW of B anthracis, LeTx, and LPS for 30 minutes at 37°C. The concentrations of histamine in the supernatants and blood plasma were detected by ELISA assay. Six repeats were performed, and data are expressed as mean ± standard error. *CW = CW of 5000 ng/mL. [go to text reference]
Figure 4: Serotonin concentrations in human plasma from (C) whole blood, and supernatants from (A) basophil and (B) platelet cultures. Blood and cells were treated with LeTx, LPS, and anthrax CW components for 30 minutes at 37°C. Six repeats were performed, and data are expressed as mean ± standard error. *CW = CW of 5000 ng/mL. [go to text reference]
Figure 5: Determination of bradykinin in human whole blood. Human blood was drawn from healthy volunteers and treated with LeTx, anthrax CW, and LPS for 30 minutes at 37°C. Plasma was harvested, and the concentration of bradykinin was determined by ELISA assays. Data in this figure represent samples from 6 different donors, and data are expressed as mean ± standard error. *CW = CW at 5000 ng/mL. [go to text reference]
Figure 6: Dynamic change of (A) histamine and (B) serotonin in C57BL/6 mouse serum after administration of LeTx. Blood was collected at time 0 and 2, 4, 8, and 24 h after the challenge. Serum was assayed by ELISA to determine the concentration of histamine and serotonin. Mean data in this figure are derived from 10 mice. [go to text reference]
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