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* Immunology Research Group, Department of Physiology and Biophysics, and
Cancer Biology Research Group, University of Calgary, Calgary, Alberta, Canada; and
Center for Inflammatory Diseases, Department of Medicine, Monash University, Clayton, Victoria, Australia
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Abstract |
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Introduction |
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The EAE-enhancing effects of PTX are universal among all species in which EAE can be induced, including mice (9, 10, 11, 12), rats (13), and nonhuman primates (14). It is sufficient to initiate disease in animals that would not normally develop disease. For example, inclusion of PTX in EAE induction protocols can render EAE-resistant strains of mice susceptible to disease (12). Indeed, PTX is commonly used in the induction of all EAE models. The prevailing explanation/speculation concerning the action of PTX is that it induces an increase in permeability across the blood-brain barrier (9, 10). This could allow easier passage for T cells across the endothelium. However, visualization of normal brain microvessels revealed that no basal leukocyte/endothelial interactions occur (15, 16), so that a simple increase in permeability would not be sufficient to recruit cells to the vessel wall. We hypothesized that PTX or environmental factors, such as bacterial infections, may sufficiently activate the brain endothelium to establish a proadhesive environment that becomes permissive for T cell recruitment. In addition, we proposed that pattern recognition receptors responsible for the detection of a wide array of infectious agents would potentially also be responsible for the transition from infection to autoimmune disease. Specifically, we hypothesized that the major LPS receptor, TLR4, was the critical molecule via which PTX induces a proadhesive environment within the brain, leading to the development of EAE.
We visualized the brain microvasculature and report for the first time that TLR4 is central to the EAE-inducing effects of PTX by up-regulating adhesion molecules and initiating leukocyte recruitment, including the recruitment of activated T cells, to the CNS. Although PTX also increased microvascular permeability, it was a result, rather than a cause, of leukocyte recruitment. This study demonstrates an important link between innate immune mechanisms and the initiation of CNS autoimmunity and may lead to important insights into the development of human multiple sclerosis.
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Materials and Methods |
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Whole PTX holotoxin, the PTX B-oligomer and ultrapure LPS were purchased from List Biological Laboratories (Campbell, CA). Ultrapure lipoteichoic acid (LTA) was provided by Dr. T. Hartung (University of Konstanz, Konstanz, Germany). Anti-phospho- and anti-total JNK/stress-activated protein kinase, anti-phospho- and anti-total ERK1/2, and anti-phospho p38 were purchased from Cell Signaling Technologies (Beverly, MA). Anti-total I
B
, anti-total p38, and a rabbit anti-mouse HRP secondary Ab were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-P-selectin and nonbinding control Ab (A110-1) were purchased from BD Pharmingen (Ontario, Canada). Anti-CD4 microbeads were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany), and anti-IL-4 (11B11) was a gift from Dr. C. Weaver (University of Alabama, Birmingham, AL).
Mice
C57BL/6 mice were purchased from Charles River Laboratories (Québec, Canada). P-selectin–/– and CD-14-deficient mice on a C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/10ScCr, a strain of mice with a naturally occurring deletion of the Tlr4 gene (17) (referred to in this study as TLR4-deficient mice) were also purchased from The Jackson Laboratory. Animal protocols were approved by the University of Calgary animal care committee and met the Canadian Guidelines for Animal Research.
Intravital microscopy
Intravital microscopy of the brain microcirculation was performed as described in detail previously (15, 16). Briefly, pial vessels in the brain were exposed by removing a piece of the parietal bone and underlying dura mater. Mice were then administered rhodamine 6G (0.3 mg/kg i.v.; Sigma-Aldrich, Ontario, Canada) to label circulating leukocytes. Leukocyte/endothelial interactions were observed using a microscope (Axioskop; Carl Zeiss Canada, Don Mills, Canada; x10 eyepiece and x25 objective lens) outfitted with a fluorescent light source (epi-illumination at 510–560 nm using a 590-nm emission filter). All experiments were recorded onto videotape for playback analysis. Rolling leukocytes were defined as those cells moving at a velocity less than that of erythrocytes within a given vessel. Leukocytes were considered adherent to the venular endothelium if they remained stationary for 30 s or longer.
Experimental protocol
Mice were injected with 250 ng of PTX in 250 µl of sterile PBS through the tail vein. For some experiments, PTX was heat inactivated by incubation in an 80°C water bath for 20 min. All observations were made 5 h after administration of PTX unless otherwise stated. Twenty micrograms of RB40.34 (blocking anti-P-selectin Ab) in 200 µl of saline was administered i.v.
For experiments using activated T cells, cells were isolated and cultured as described below. Cells were then collected and incubated in 15 ng/ml rhodamine 6G. Intravital microscopy was performed on healthy control mice or mice in the acute stage of EAE. Initial observations were made before administration of labeled T cells to gauge background fluorescence. Cells (1 x 107 in 200 µl of saline) were then administered i.v. and allowed to circulate for 10 min before quantification of leukocyte/endothelial interactions.
Generation of activated T cells
CD4+ T cells were isolated from pooled spleens of DO11.10 mice using mouse anti-CD4 (L3T4) MACS microbeads according to the manufacturer’s protocol (Miltenyi Biotec). FACS analysis revealed that isolations routinely resulted in >90% purity of CD4+ cells. Th1 and Th2 cells were generated in vitro from Ag-naive CD4+ T cells as described previously (18). Th1 cells were cultured in Iscove’s medium (Invitrogen Life Technologies, Burlington, Canada) at 6.25 x 105 cells/ml in the presence of 3 x 106 cells/ml irradiated splenocytes from BALB/c mice, 5 µg/ml OVA peptide 323–339 (New England Peptides, Gardner, MA), antibiotics, and 10% FCS. To drive cells to a Th1 phenotype, 50 U/ml IL-12 (R&D Systems, Abingdon, U.K.) and 10 µg/ml anti-IL-4 (11B11) were added to the culture medium. Cells were cultured for 5–9 days before use.
Western blots for signaling molecules
Western blots were performed as previously described (19). Briefly, Raw 264.7 cells were plated in six-well tissue culture plates and allowed to grow to 75% confluence. The cells were treated with LPS (50 ng/ml), PTX (500 ng/ml), or B oligomer (500 ng/ml) for the indicated times, then lysed using Nonidet P-40 lysis buffer. LTA, which signals through TLR2, was also administered to demonstrate that TLR4-deficient cells were still capable of mediating intracellular signaling events. Insoluble material was removed from the lysates by centrifugation. Samples were prepared using 2x Laemmli’s buffer and were resolved on a 10% SDS-PAGE. After electrophoresis, proteins were transferred to nitrocellulose membranes, which were then blocked by incubation in 5% BSA in TBS plus 0.05% Tween 20 (TBST). The membranes were then incubated at 4°C overnight in the primary Ab, washed for 40 min in TBST, incubated for 1 h in secondary Ab, and washed for another 40 min in TBST. The membranes were developed using an ECL substrate.
For experiments using peritoneal macrophages, cells were isolated by peritoneal lavage with saline from untreated wild-type C57BL/6 or TLR4-deficient mice. Nonadherent cells were removed before treatment with LTA (1 µg/ml) or PTX (500 ng/ml). Western blots were then performed as described above.
Induction of EAE
Nine-week-old C57BL/6 or TLR4-deficient mice were immunized s.c. with 50 µg of a peptide generated from myelin oligodendrocyte glycoprotein (MOG35–55) in CFA (Sigma-Aldrich). A second immunization with MOG peptide in IFA was performed 1 wk later. In some cases mice were immunized with MOG peptide in CFA supplemented with additional Mycobacterium tuberculosis (5 mg/ml total). When indicated, 250 ng of PTX (List Biological Laboratories) was injected i.p. along with the first immunization as well as 2 days later. Disease was monitored daily and was scored as follows: 0, no clinical signs; 1, tail paralysis; 2, tail paralysis and hind limb weakness; 3, hind limb paralysis; and 4, complete hind limb paralysis and front limb weakness.
Blood-brain barrier permeability assay
Permeability across the blood-brain barrier was assayed as previously described (20). Briefly, brain microvessels were exposed as described for intravital microscopy (see above). A stainless steel superfusion chamber was then attached to the skull surrounding the window. Artificial cerebrospinal fluid was continuously superfused through the chamber at a constant rate. Permeability of the blood-brain barrier was assessed by measuring the clearance (nanoliters per second) of 70-kDa FITC-dextran from the pial vessels into the suffusate fluid as previously described (20). Experiments were timed to allow for surgical preparation and equilibration before the 5 h point of either PBS or PTX treatment. Exactly 5 h after treatment, FITC-dextran was administered, and permeability was assayed over the following 30 min.
Dual radiolabeled Ab assay for P-selectin expression
P-selectin expression was quantitatively measured in the brain and other tissues of untreated, 5-h PTX-treated mice, and PTX-treated, P-selectin-deficient mice by the dual radiolabeled Ab assay as previously described (15, 16). Briefly, a mixture of 125I-labeled anti-P-selectin (RB40.34) and 131I-labeled nonbinding Ab (A110–1, anti-keyhole limpet hemocyanin) was injected i.v. The Abs were allowed to circulate for 5 min, at which point circulating blood was cleared by perfusing the whole mouse with bicarbonate-buffered saline. Tissues were harvested and weighed, and 125I and 131I activities were measured. P-selectin expression was calculated per gram of tissue by subtracting accumulated 131I activity (labeled nonbinding Ab) from accumulated 125I activity (labeled anti-P-selectin). Data are presented as the percentage of the injected dose of Ab per gram of tissue.
Statistics
Data in figures and tables are shown as the mean ± SEM unless indicated otherwise. Student’s t test with Bonferroni correction was used for multiple comparisons. Statistical significance was set at p < 0.05.
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Results |
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We used intravital microscopy of the mouse brain to investigate the effects of PTX on leukocyte recruitment in the cerebromicrovasculature. No leukocyte rolling or adhesion (Fig. 1, A, C, and D) was observed in untreated mice or up to 30 min after the administration of PTX. In contrast, there was a significant induction of leukocyte/endothelial interactions 5 h after PTX injection, which remained elevated over the next 48 h (Fig. 1, B–D). Leukocyte adhesion was also induced 
15-fold, but returned to near-control levels within 24 h (Fig. 1D). Vehicle (PBS) had no effect on recruitment (data not shown). A dose of 250 ng of PTX/mouse was chosen because this is a common dose used in the induction of EAE. Because maximal recruitment was observed 5 h after administration of PTX, all subsequent experiments were performed at this time point.
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We recently demonstrated an important role for P-selectin in leukocyte recruitment to the brain in EAE (16). We therefore hypothesized that PTX may activate the cerebrovascular endothelium to express P-selectin. Mice were administered a P-selectin-blocking Ab at the same time as PTX, and leukocyte/endothelial interactions were observed by intravital microscopy 5 h later. Anti-P-selectin completely blocked leukocyte rolling (Fig. 2A) and reduced leukocyte adhesion by 70% (Fig. 2B). Identical results were observed when PTX was administered to P-selectin-deficient mice. Acute treatment with anti-P-selectin (5 min) blocked leukocyte rolling, but could not dislodge already adherent cells, consistent with P-selectin-mediating rolling, but not adhesion (16, 21) (data not shown).
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We used the radiolabeled Ab assay of adhesion molecule expression to measure P-selectin protein in the cerebrovasculature. This quantitative assay is capable of detecting very low levels of P-selectin expression in the CNS that are simply impossible to detect using immunohistochemistry (16). There was no difference in expression between the brains of untreated mice and P-selectin-deficient mice, demonstrating that under basal conditions, P-selectin is not expressed in this tissue (Fig. 2C). In contrast, significant amounts of P-selectin protein were measured 5 h after injection of PTX.
PTX induces recruitment of activated T cells to the CNS
EAE develops when myelin-reactive T cells, once activated in the periphery, gain access to the CNS. Activated, but not naive, T cells express functional P-selectin ligand (22), and it has been demonstrated that only activated cells, regardless of their Ag specificity, can gain entry to the CNS (23). To determine whether PTX facilitates the recruitment of activated T cells to the CNS, T cells were isolated and cultured in vitro in the presence of peptide as described in Materials and Methods. T cells from mice bearing a transgenic TCR for an OVA peptide were used to generate the numbers of cells required for the experiment. Cells were then fluorescently labeled and transferred i.v. to PTX-treated or untreated mice. Because recruitment is independent of Ag specificity (23), this accurately models the entry of disease-inducing T cells to the CNS. Significant rolling and adhesion of the activated Th1 T cells were observed in PTX-treated, but not control untreated, mice (Fig. 3).
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We investigated PTX-induced permeability across the blood-brain barrier. Very little leakage of 70-kDa FITC-dextran was detected in vehicle-treated mice (250 µl of sterile PBS; Fig. 4). In contrast, permeability to 70-kDa FITC-dextran across the blood-brain barrier was >2-fold greater in mice 5 h after i.v. administration of 250 ng of PTX. This is consistent with previous reports that PTX induces increased blood-brain barrier permeability (9, 10). Interestingly, when a blocking anti-P-selectin Ab was administered at the same time as PTX, a treatment that effectively blocks all leukocyte rolling and most adhesion (Fig. 2, A and B, respectively), the increase in blood-brain barrier permeability was completely eliminated. This suggests that the rolling/adhering leukocytes were responsible for the increased blood-brain barrier permeability. Interestingly, the low level of basal permeability was also eliminated with anti-P-selectin, suggesting that this may also be mediated by a very low number of trafficking leukocytes.
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It has been suggested that PTX may share a putative receptor with LPS (24), which we now know includes the GPI-linked protein CD14 and TLR4. To determine whether CD14 or TLR4 plays a role in PTX-induced leukocyte recruitment to the CNS, intravital microscopy of the cerebromicrovasculature was performed on CD14- and TLR4-deficient mice. In the absence of TLR4, PTX was unable to induce leukocyte rolling or adhesion (Fig. 5, A and B, respectively) in the cerebrovasculature. A >60% reduction in leukocyte rolling was observed in CD14-deficient mice compared with controls (Fig. 5A); however, the decrease in adhesion was not significant (Fig. 5B).
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To determine whether the PTX holotoxin was required to induce leukocyte/endothelial interactions in the cerebromicrovasculature, mice were treated with either whole PTX or the B oligomer fragment of PTX, which is thought to bind to the cell surface, but has no enzymatic activity itself (25). In contrast to treatment with the PTX holotoxin, the B oligomer was unable to induce leukocyte recruitment in the CNS (Fig. 5, C and D), implying that the complete toxin was required to activate cerebrovascular endothelium.
PTX induces an intracellular signal through TLR4
Members of the IL-1R family, which includes the TLRs, activate a number of signaling pathways, including the NF-B pathway, the JNK pathway, and the ERK and p38 MAPK pathways (reviewed in Ref.26). To determine whether PTX induced the activation of similar pathways, the presence of phosphorylated JNK, p38, and ERK1/2 and total IB in cell lysates of TLR4+ RAW 264.7 cells was assayed by Western blot. Phosphorylated JNK, p38, and ERK1/2 could not be detected in the lysates of unstimulated RAW 264.7 cells (Fig. 6A). Stimulation with LPS resulted in rapid phosphorylation of p38, ERK1/2, and JNK, peaking within 15 min of stimulation. Similarly, total IB protein degradation was complete at 15 min. PTX induced similar signaling in RAW 264.7 cells, although p38 and ERK1/2 phosphorylation as well as IB degradation were slightly delayed and did not peak until 60 min after treatment. JNK phosphorylation could only be detected at 60 min. The B oligomer of PTX was completely unable to induce intracellular signaling, consistent with the leukocyte recruitment data.
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B protein degradation occurred with similar kinetics (data not shown). Interestingly, although LPS and PTX activated the same signaling events, PTX-mediated signaling was delayed compared with that mediated by LPS in both RAW 264.7 (Fig. 6A) cells and peritoneal macrophages (data not shown).
To determine whether TLR4 is required to mediate PTX-induced intracellular signaling, peritoneal macrophages were isolated from wild-type and TLR4-deficient mice (Fig. 6B). These cells were treated with either PTX or LTA (which signals through a TLR other than TLR4) and were included as a positive control to demonstrate that intracellular signaling was not grossly impaired in TLR4-deficient cells. ERK1/2 phosphorylation as well as IB degradation were entirely dependent on TLR4, because PTX was completely unable to induce these events in peritoneal macrophages isolated from TLR4-deficient mice. Phosphorylation of JNK and p38 still occurred, but to a lesser extent compared with that in wild-type macrophages (Fig. 6B), demonstrating that although the majority of signaling is mediated through TLR4, PTX can induce some events through a TLR4-independent pathway.
Induction of EAE by PTX is dependent on TLR4
To determine whether the disease-inducing activity of PTX was dependent on TLR4, as is the case for its capacity to induce intracellular signaling and activity on leukocyte recruitment, EAE in C57BL/6 wild-type and age-matched TLR4-deficient mice was investigated. Wild-type mice treated with PTX first showed signs of disease 12 days after the first immunization with MOG (Fig. 7). Disease progressed over the next 4–5 days, at which time symptoms stabilized. Mice that did not receive PTX did not develop any sign of disease. Disease incidence was substantially lower in TLR4-deficient mice, in that only 45% of these mice developed disease compared with 95% of wild-type mice (Fig. 7A). For those TLR4-deficient mice that did develop disease, TLR4 deficiency did not have any impact on disease severity or time of onset, implicating TLR4-mediated mechanisms in the initiation, but not the subsequent development, of EAE.
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In a final series of experiments, wild-type mice were immunized with MOG in CFA supplemented with sufficient M. tuberculosis to overcome the requirement for PTX. When the same protocol was applied to TLR4-deficient mice, these animals were capable of developing disease, suggesting that TLR4 is required for PTX-induced mechanisms of disease induction (data not shown).
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Discussion |
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Our studies demonstrate for the first time that TLR4 is a receptor for PTX, and that PTX is largely dependent on TLR4 to induce intracellular signaling events. Indeed, PTX induced the activation of a number of intracellular signaling pathways, including degradation of IB and phosphorylation of JNK, which would, respectively, result in activation and translocation to the nucleus of the transcription factors NF-B and AP-1 (26). This is significant, because these two factors are very important to the transcription and subsequent production of many inflammatory factors, including murine P-selectin (27) and other adhesion molecules. Intracellular signaling was nearly abolished in the absence of TLR4, as was leukocyte recruitment in the CNS as observed by intravital microscopy. Combined, these observations clearly demonstrate an important role for TLR4 in PTX-mediated events both in vitro and in vivo. The fact that the enzymatic subunit of PTX was required to induce these events suggests either that the entire intact molecule is required to activate TLR4 to initiate signaling and/or that the ADP-ribosylation activity of PTX may also be necessary.
The contribution of TLR4 to EAE induction implicates innate immune processes in the development of CNS autoimmune disease. It is well known that the local environment, in particular the presence of infectious agents, can have a tremendous impact on the course of disease. Importantly, this also appears to be the case for human disease, because exposure to various childhood infections has been linked to MS (28), and infections can exacerbate ongoing disease (6). In the animal model, PTX appears to act as a surrogate for environmental conditions by inducing the same mechanisms that are required for disease development. Indeed, Goverman et.al (7) reported that either PTX or a "dirty" environment induced EAE in their anti-myelin TCR transgenic mice. However, our observation that under some circumstances PTX could induce EAE independently of TLR4 suggests that additional factors, probably environmental (or PTX itself), are able to act through other mechanisms such as other TLRs to induce disease. The central role of the innate immune system in the initiation and regulation of a subsequent Ag-specific immunity is becoming increasingly apparent (29, 30). It seems logical that this would also extend to the dysregulation of immunity to an autoantigen. However, to date this has remained largely unaddressed, and investigations of the initiation of autoimmune disease have instead focused nearly exclusively on the T cell.
Leukocytes are recruited from the circulation through a well-characterized cascade of events, where they first tether to and then roll along the blood vessel wall before firmly adhering to and transmigrating across the endothelial layer into the surrounding tissue (31). This process is entirely dependent on the expression of adhesion molecules by endothelial cells lining the lumen of the blood vessel. In the current study, adhesion molecule expression was very low to absent in the microvasculature of the CNS under normal conditions, consistent with previous findings (15, 16). Therefore, for PTX to mediate leukocyte recruitment to the CNS, its primary mechanism would have to be to induce the expression of adhesion molecules. Indeed, P-selectin protein could be detected in the CNS of PTX-treated mice, and leukocyte/endothelial interactions could be blocked with a P-selectin-blocking Ab, clearly demonstrating that PTX acts to activate endothelium.
It has been known for some time that treatment with PTX results in increased permeability across the blood-brain barrier (9, 10). It has since been regularly stated that PTX "opens up" the blood-brain barrier, allowing immune cell access to the CNS and subsequent development of autoimmune disease. In this study we demonstrate that the PTX-mediated increase in permeability across the blood-brain barrier could be completely prevented by blocking leukocyte/endothelial interactions with an adhesion molecule-blocking Ab. Therefore, the effects of PTX on permeability are indirect and secondary to its primary mechanism of leukocyte recruitment, which is through the expression of adhesion molecules. This is not without precedent, because anti-adhesion therapy has been shown to prevent permeability across the blood-brain barrier after ischemia/reperfusion injury (32) and mast cell degranulation (33).
There are two potential mechanisms through which PTX may induce autoimmune disease. The first is to facilitate the entry of anti-myelin T cells to the CNS through endothelial activation. Recent studies have identified P-selectin as critical to mediating the recruitment of disease-inducing T cells to the CNS (34, 35, 36). Our demonstration that PTX induces P-selectin expression and the recruitment of activated Th1 T cells to the CNS implicates PTX in this initial and critical step in the development of EAE. In another study, PTX alone induced encephalopathy characterized by a massive mononuclear inflammation in transgenic mice with CNS-specific expression of the chemokine MCP-1 (37). This demonstrates that PTX can indeed facilitate leukocyte access to the CNS in an Ag-independent model of CNS inflammation. However, it is not clear whether endothelial activation and leukocyte recruitment are the primary mechanisms by which PTX induces EAE.
A second disease-inducing mechanism for PTX has been proposed by studies demonstrating that it is also an adjuvant, acting on dendritic cells to drive T cell responses toward a Th1 phenotype (38, 39). Our demonstration that PTX induces signaling through TLR4 may explain these observations, because TLR4 on dendritic cells has been identified as important for the development of a Th1 response (40). PTX has been demonstrated to induce EAE in a spontaneous disease model in a myelin-specific TCR transgenic mouse (8). In a recent study, in vivo administration of CpG DNA or, to a lesser degree, LPS was also demonstrated to have disease-inducing activity in a similar transgenic model of EAE (41). This was attributed to the activation of dendritic cells through TLR9 and TLR4, respectively, to break self tolerance and activate the autoimmune, anti-myelin T cell response. This study strongly supports our hypothesis that activation of TLRs is an important inducer of autoimmune disease. Indeed, in our model it may be that at least part of the contribution of PTX to disease is simply to bolster the Th1 adjuvant effect of the immunization protocol. However, in the study just described, PTX was by far superior in inducing EAE in their model compared with CpG DNA or LPS (41). Therefore, PTX may act through multiple and potentially cumulative pathways to induce disease.
In conclusion, very little is known about the requirements for the initiation of CNS autoimmune disease. Most work has focused on the activation of autoreactive T cells; however, it is clear that other mechanisms are also required. PTX is able to induce these mechanisms and is therefore very important to the development of EAE. We identify a role for TLR4 in this process and in EAE development, linking innate inflammatory mechanisms to the development of CNS autoimmune disease.
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Footnotes |
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1 This work was supported by a group grant from the Canadian Institute of Health Research and a Multiple Sclerosis Society of Canada grant. P.K. and S.M.R. are Alberta Foundation for Medical Research Scientists and Canada Research Chairs. S.M.K. is the recipient of an Alberta Foundation for Medical Research Studentship. E.M.L. is funded by Natural Sciences and Engineering Research Council. B.M.L. is a Canadian Institute of Health Research-Astra-Zeneca fellow, and G.A. and R.C.O.Z. are Canadian Association of Gastroenterology fellows. 
2 Address correspondence and reprint requests to Dr. Paul Kubes, Immunology Research Group, Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive Northwest, Calgary, Alberta, Canada T2N 4N1. E-mail address: pkubes{at}ucalgary.ca
3 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental autoimmune encephalomyelitis; LTA, lipoteichoic acid; MOG, myelin oligodendrocyte glycoprotein; PTX, pertussis toxin.
Received for publication April 13, 2004. Accepted for publication September 24, 2004.
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A. Nogai, V. Siffrin, K. Bonhagen, C. F. Pfueller, T. Hohnstein, R. Volkmer-Engert, W. Bruck, C. Stadelmann, and T. Kamradt Lipopolysaccharide Injection Induces Relapses of Experimental Autoimmune Encephalomyelitis in Nontransgenic Mice via Bystander Activation of Autoreactive CD4+ Cells J. Immunol., July 15, 2005; 175(2): 959 - 966. [Abstract] [Full Text] [PDF]
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