Multiple linked quantitative trait loci within the Tmevd2/Eae3 interval control the severity of experimental allergic encephalomyelitis in DBA/2J mice.

Theiler's murine encephalomyelitis virus-induced demyelination (TMEVD) and experimental allergic encephalomyelitis (EAE) are the principal animal models of multiple sclerosis (MS). Previously, we provided evidence that Tmevd2 and Eae3 may represent either a shared susceptibility locus or members of a gene complex controlling susceptibility to central nervous system inflammatory demyelinating disease. To explore the genetic relationship between Tmevd2 and Eae3, we generated a D2.C-Tmevd2 interval-specific congenic (ISC) line and three overlapping interval-specific recombinant congenic (ISRC) lines in which the Tmevd2-resistant allele from BALB/cByJ was introgressed onto the TMEVD-susceptible DBA/2J background. These mice, all H2(d), were studied for susceptibility to EAE elicited by immunization with proteolipid protein peptide 180-199. Compared with DBA/2J mice, D2.C-Tmevd2 mice developed a significantly less severe clinical disease course and EAE pathology in the spinal cord, confirming the existence of Eae3 and its linkage to Tmevd2 in this strain combination. Compared with DBA/2J and D2.C-Tmevd2, all three ISRC lines exhibited clinical disease courses of intermediate severity. Neither differences in ex vivo antigen-specific cytokine nor proliferative responses uniquely cosegregated with differences in disease severity. These results indicate that multiple quantitative trait loci (QTLs) within the Tmevd2/Eae3 interval influence EAE severity, one of which includes a homology region for a QTL found in MS by admixture mapping.

The neuropathological and behavioral consequences of intraspinal microglial/macrophage activation.

Activated microglia and macrophages (CNS macrophages) have been implicated in the secondary or "bystander" pathology (e.g. axon injury, demyelination) that accompanies traumatic or autoimmune injury to the brain and spinal cord. These cells also can provide neurotrophic support and promote axonal regeneration. Studying the divergent functional potential of CNS macrophages in trauma models is especially difficult due to the various degradative mechanisms that are initiated prior to or concomitant with microglial/macrophage activation (e.g. hemorrhage, edema, excitotoxicity, lipid peroxidation). To study the potential impact of activated CNS macrophages on the spinal cord parenchyma, we have characterized an in vivo model of non-traumatic spinal cord neuroinflammation. Specifically, focal activation of CNS macrophages was achieved using stereotaxic microinjections of zymosan. Although microinjection does not cause direct mechanical trauma, localized activation of macrophages with zymosan acts as an "inflammatory scalpel" causing tissue injury at and nearby the injection site. The present data reveal that activation of CNS macrophages in vivo can result in permanent axonal injury and demyelination. Moreover, the pathology can be graded and localized to specific white matter tracts to produce quantifiable behavioral deficits. Further development of this model will help to clarify the biological potential of microglia and macrophages and the molecular signals that control their function within the spinal cord.

Rapid response of identified resident endoneurial macrophages to nerve injury.

Macrophages play a central role in the pathogenesis of peripheral neuropathy but the role of resident endoneurial macrophages is undefined because no discriminating markers exist to distinguish them from infiltrating hematogenous macrophages. We identified and characterized resident endoneurial macrophages during Wallerian degeneration in radiation bone marrow chimeric rats created by transplanting wild-type Lewis rat bone marrow into irradiated TK-tsa transgenic Lewis rats. In such animals, resident cells carry the transgene, whereas hematogenous cells do not. As early as 2 days after sciatic nerve crush and before the influx of hematogenous macrophages, resident transgene-positive endoneurial macrophages underwent morphological and immunophenotypic signs of activation. At the same time, resident macrophages phagocytosing myelin were found, and proliferation was detected by bromodeoxyuridine incorporation. Continuous bromodeoxyuridine feeding revealed that resident endoneurial macrophages sequentially retracted their processes, proliferated, and expressed the ED1 antigen, rendering them morphologically indistinguishable from hematogenous macrophages. Resident endoneurial macrophages thus play an early and active role in the cellular events after nerve lesion before hematogenous macrophages enter the nerve. They may thus be critically involved in the pathogenesis of peripheral neuropathy particularly at early stages of the disease and may act as sensors of pathology much like their central nervous system counterparts, the microglial cells.

Diminished virulence of an alpha-toxin mutant of Staphylococcus aureus in experimental brain abscesses.

Staphylococcus aureus is one of the major etiologic agents of brain abscesses in humans, occasionally leading to focal neurological deficits and even death. The objective of the present study was to identify key virulence determinants contributing to the pathogenesis of S. aureus in the brain using a murine brain abscess model. The importance of virulence factor production in disease development was demonstrated by the inability of heat-inactivated S. aureus to induce proinflammatory cytokine or chemokine expression or brain abscess formation in vivo. To directly address the contribution of virulence determinants in brain abscess development, the abilities of S. aureus strains with mutations in the global regulatory loci sarA and agr were examined. An S. aureus sarA agr double mutant exhibited reduced virulence in vivo, as demonstrated by attenuated proinflammatory cytokine and chemokine expression and bacterial replication. Subsequent studies focused on the expression of factors that are altered in the sarA agr double mutant. Evaluation of an alpha-toxin mutant revealed a phenotype similar to that of the sarA agr mutant in vivo, as evidenced by lower bacterial burdens and attenuation of cytokine and chemokine expression in the brain. This suggested that alpha-toxin is a central virulence determinant in brain abscess development. Another virulence mechanism utilized by staphylococci is intracellular survival. Cells recovered from brain abscesses were shown to harbor S. aureus intracellularly, providing a means by which the organism may establish chronic infections in the brain. Together, these data identify alpha-toxin as a key virulence determinant for the survival of S. aureus in the brain.

Basic principles of immunological surveillance of the normal central nervous system.

Unlike most bodily organs, the central nervous system (CNS) exists behind a blood-tissue barrier designed to minimize the passage of cells and macromolecules into the neural parenchyma. Yet, the CNS is routinely and effectively surveyed by the immune system. This review examines the mechanisms and participants in this immunological surveillance mechanism. The nature of the healthy blood-brain barrier, factors modifying it, and its central position in determining the number and nature of leukocytes permitted to enter, are considered. In addition the role in surveillance played by lymphatic drainage, migrating T and B lymphocytes, and elements of the monocyte/macrophage/microglia family are considered. While all these participants are known to be important in responding to a CNS antigen and/or establishing a site of inflammation in the nervous system, they also are major elements in maintaining the homeostasis of the CNS and permitting the necessary immunological surveillance of that organ.

Bone marrow chimeric rats reveal the unique distribution of resident and recruited macrophages in the contused rat spinal cord.

Brain and spinal cord inflammation that develops after traumatic injury is believed to differentially influence the structural and/or physiological integrity of surviving neurons and glia. It is possible that the functional dichotomy of CNS inflammation results from the activity of a heterogeneous macrophage population elicited by trauma. Indeed, unique functions have been attributed to macrophages derived from resident microglia versus those originating from infiltrating monocytes. Thus, whether progressive tissue injury or repair is favored could be explained by the disproportionate contributions of one macrophage subset relative to the other. Descriptive neuroanatomical studies are a reasonable first approach to revealing a relationship between microglia, recruited blood monocytes/macrophages, and regions of tissue degeneration and/or repair. Unfortunately, it is not possible to differentiate between CNS macrophage subsets using conventional immunohistochemical approaches. In the present study, we have used radiation bone marrow chimeric rats to definitively characterize the macrophage reaction elicited by experimental spinal contusion injury. In chimeric animals, antibodies raised against unique cell surface molecules expressed on bone marrow-derived cells (BMCs) were used to distinguish infiltrating BMCs from resident microglial-derived macrophages. Our findings indicate that the onset and plateau of macrophage activation (previously shown to be 3 and 7 days postinjury, respectively) is dominated initially by microglial-derived macrophages and then is supplanted by hematogenous cells. While resident macrophages are ubiquitously distributed throughout the injury site, leukocyte-derived monocytes exclusively infiltrate the gray matter and to a lesser extent subpial white matter. Generally, monocyte foci in white matter remain associated with the lumen or abluminal surface of blood vessels, i.e. few cells actually infiltrate the parenchyma. If functional differences exist between CNS macrophage subsets, differences in the time-dependent accumulation and distribution of these cell types could differentially influence the survival of surrounding neurons and glia.

The clinical course of experimental autoimmune encephalomyelitis and inflammation is controlled by the expression of CD40 within the central nervous system.

Although it is clear that the function of CD40 on peripheral hematopoietic cells is pivotal to the development of autoimmunity, the function of CD40 in autoimmune disease outside this compartment is unresolved. In a model of experimental autoimmune encephalomyelitis (EAE), evidence is presented that CD40-CD154 interactions within the central nervous system (CNS) are critical determinants of disease development and progression. Using bone marrow (BM) chimeric mice, the data suggest that the lack of expression of CD40 by CNS-resident cells diminishes the intensity and duration of myelin oligodendrocyte glycoprotein (MOG)-induced EAE and also reduces the degree of inflammatory cell infiltrates into the CNS. Although CNS inflammation is compromised in the CD40(+/+)-->CD40(-/-) BM chimeric mice, the restricted CD40 expression had no impact on peripheral T cell priming or recall responses. Analysis of RNA expression levels within the CNS demonstrated that encephalitogenic T cells, which entered a CNS environment in which CD40 was absent from parenchymal microglia, could not elicit the expression of chemokines within the CNS. These data provide evidence that CD40 functions outside of the systemic immune compartment to amplify organ-specific autoimmunity.

CXC chemokine receptor-2 ligands are required for neutrophil-mediated host defense in experimental brain abscesses.

We have developed a mouse brain abscess model by using Staphylococcus aureus, one of the main etiologic agents of brain abscesses in humans. Direct damage to the blood-brain barrier was observed from 24 h to 7 days after S. aureus exposure as demonstrated by the accumulation of serum IgG in the brain parenchyma. Evaluation of brain abscesses by immunohistochemistry and flow cytometry revealed a prominent neutrophil infiltrate. To address the importance of neutrophils in the early containment of S. aureus infection in the brain, mice were transiently depleted of neutrophils before implantation of bacteria-laden beads. Neutrophil-depleted animals consistently demonstrated more severe brain abscesses and higher CNS bacterial burdens compared with control animals. S. aureus led to the induction of numerous chemokines in the brain, including macrophage-inflammatory protein (MIP)-1alpha/CCL3, MIP-1beta/CCL4, MIP-2/CXCL1, monocyte chemoattractant protein-1/CCL2, and TCA-3/CCL1, within 6 h after bacterial exposure. These chemokines also were expressed by both primary cultures of neonatal mouse microglia and astrocytes exposed to heat-inactivated S. aureus in vitro. Because neutrophils constitute the majority of the cellular infiltrate in early brain abscess development, subsequent analysis focused on MIP-2 and KC/CXCL1, two neutrophil-attracting CXC chemokines. Both MIP-2 and KC protein levels were significantly elevated in the brain after S. aureus exposure. Neutrophil extravasation into the brain parenchyma was impaired in CXCR2 knockout mice and was associated with increased bacterial burdens. These studies demonstrate the importance of the CXCR2 ligands MIP-2 and KC and neutrophils in the acute host response to S. aureus in the brain.

Co-localization of multiple antigens and specific DNA. A novel method using methyl methacrylate-embedded semithin serial sections and catalyzed reporter deposition.

Co-localization of proteins and nucleic acid sequences by in situ hybridization and immunohistochemistry is frequently difficult as the process necessary to detect the target structure of one technique may negatively affect the target of the other. Morphological impairment may also limit the application of the two techniques on sensitive tissue. To overcome these problems we developed a method to perform in situ hybridization and immunohistochemistry on semithin sections of methyl methacrylate-embedded tissue. Microwave-stimulated antigen retrieval, signal amplification by catalyzed reporter deposition, and fluorescent dyes were used for both techniques, yielding high sensitivity and excellent morphological preservation compared to conventional paraffin sections. Co-localization of in situ hybridization and immunohistochemistry signals with high morphological resolution was achieved on single sections as well as on adjacent multiple serial sections, using computerized image processing. The latter allowed for the co-localization of multiple antigens and a specific DNA sequence at the same tissue level. The method was successfully applied to radiation bone marrow chimeric rats created by transplanting wild-type Lewis rat bone marrow into TK-tsa transgenic Lewis rats, in an attempt to trace and characterize TK-tsa transgenic cells. It also proved useful in the co-localization of multiple antigens in peripheral nerve biopsies.

Identification and characterization of the antigen presenting cell in rat autoimmune myocarditis: evidence of bone marrow derivation and non-requirement for MHC class I compatibility with pathogenic T cells.

In the rat, autoimmune myocarditis can be produced by the infusion of activated myosin peptide specific, CD4(+), class II restricted, effector T cells. Whether antigen presenting cells (APCs), which interact with these effector T cells in the heart, are a fixed population of cells (resident dendritic, macrophage, or endothelial cells), or a dynamic bone marrow derived population has not yet been demonstrated in vivo. To study this question, bone marrow chimeras were generated using inbred Brown Norway (BN) rats, which are resistant to autoimmune myocarditis, and transplanting them after lethal irradiation with (LewisxBN) F1 bone marrow. BN rats differ at both MHC loci from the susceptible inbred Lewis rats. Two months after bone marrow transplantation, chimeric animals received Lewis T cells specific for a myocarditogenic peptide antigen. To characterize the cardiac APCs, immunohistochemistry using a battery of antibodies including Lewis-specific and broadly reactive antibodies for both MHC class I and class II, was performed on chimeric hearts, with and without infused Lewis T cells, and non-transplanted BN control hearts.All chimeric rats infused with allogeneic (Lewis), anti-cardiac myosin peptide effector T cells displayed the lesions of myocarditis. Myocarditis was not present in non-transplanted BN controls given either Lewis or F1 derived myocarditogenic T cells, nor in chimeric animals which did not receive myocarditogenic T cells, thus excluding graft vs host disease as the explanation for the inflammation in chimeric hearts with myocarditis. Marrow derived cells expressing both Lewis class I and class II MHC molecules were demonstrated on perivascular cells in the myocardium of all chimeric animals, and on infiltrating cells in chimeric animals with myocarditis. Cells expressing Lewis-specific MHC antigens were not detected in the non-transplanted BN controls. Furthermore, immunohistochemistry using broadly reactive antibodies demonstrated MHC class II on perivascular cells with a dendritic morphology in all hearts but not on endothelial cells or cardiac myocytes. These results support the hypothesis that in vivo, cardiac APCs which result in MHC class II restricted, T cell induced myocarditis are a dynamic bone marrow derived population and not a fixed population. In order to address the potential requirement of MHC class I for the initiation of autoimmune myocarditis, myocarditogenic T cells derived from either Lewis or DA(RP) rats were infused into a member of the other strain. These strains share common MHC class II genes but differ at the MHC class I loci. Myocarditis identical to that produced in the syngeneic animal was successfully transferred by the MHC class I mismatched T cells, but only after the recipient animal's native immune system was mildly suppressed. These results further support the primary role for professional antigen presentation via MHC class II restriction to the effector T cells at the initiation of autoimmune myocarditis in the heart.Together, these experiments confirm that activated effector T cells, in order to produce myocarditis, require MHC class II compatible APCs in the heart, that these APCs are bone marrow derived, and will endogenously take up and present local antigens in the target organ after bone marrow reconstitution.

Chronic myocarditis induced by T cells reactive to a single cardiac myosin peptide: persistent inflammation, cardiac dilatation, myocardial scarring and continuous myocyte apoptosis.

Recent recognition that an autoimmune myocarditis may precede, and result in, dilated cardiomyopathy has focused attention on immune mechanisms of myocardial injury. In this paper, we describe a model of chronic autoimmune myocarditis in the Lewis rat. The production of myocarditis has been previously described by this group and in brief is accomplished by a single tail vein infusion of activated T cells specific for a 17-amino acid peptide from rat cardiac myosin. In this report, animals were followed for approximately 6 months post-T-cell infusion. Hearts from animals which received cardiac myosin specific T cells all showed extensive fibrosis associated with ongoing inflammation. Apoptosis, identified by TdT-mediated dUTP nick end labelling (TUNEL), was identified as a mode of myocyte death in hearts with acute and chronic myocarditis but not in age- and sex-matched controls. Immunohistochemistry was used to characterize the immune infiltrate and adhesion molecules in hearts with chronic myocarditis and these findings were compared to hearts with acute myocarditis. We propose that this rat model of chronic myocarditis mimics human disease, since inflammation results in ventricular dilatation and myocyte hypertrophy reminiscent of dilated cardiomyopathy. This model offers potential for further investigation of immune, functional and possible therapeutic aspects of autoimmune related cardiomyopathies.

Proinflammatory cytokine, chemokine, and cellular adhesion molecule expression during the acute phase of experimental brain abscess development.

Brain abscess represents the infectious disease sequelae associated with the influx of inflammatory cells and activation of resident parenchymal cells in the central nervous system. However, the immune response leading to the establishment of a brain abscess remains poorly defined. In this study, we have characterized cytokine and chemokine expression in an experimental brain abscess model in the rat during the acute stage of abscess development. RNase protection assay revealed the induction of the proinflammatory cytokines interleukin (IL)-1alpha, IL-1beta, IL-6, and tumor necrosis factor-alpha as early as 1 to 6 hours after Staphylococcus aureus exposure. Evaluation of chemokine expression by reverse transcription-polymerase chain reaction demonstrated enhanced levels of the CXC chemokine KC 24 hours after bacterial exposure, which correlated with the appearance of neutrophils in the abscess. In addition, two CC chemokines, monocyte chemoattractant protein-1 and macrophage inflammatory protein-1alpha were induced within 24 hours after S. aureus exposure and preceded the influx of macrophages and lymphocytes into the brain. Analysis of abscess lesions by in situ hybridization identified CD11b+ cells as the source of IL-1beta in response to S. aureus. Both intercellular adhesion molecule-1 and platelet endothelial cell adhesion molecule expression were enhanced on microvessels in S. aureus but not sterile bead-implanted tissues at 24 and 48 hours after treatment. These results characterize proinflammatory cytokine and chemokine expression during the early response to S. aureus in the brain and provide the foundation to assess the functional significance of these mediators in brain abscess pathogenesis.

Trauma and inflammation modulate lymphocyte localization in vivo: quantitation of tissue entry and retention using indium-111-labeled lymphocytes.

OBJECTIVE: Determine the in vivo localization pattern of indium-111-labeled lymphocytes after a standardized extremity injury or standardized laparotomy and after sterile inflammation of the central nervous system. DESIGN: Prospective animal study with concurrent controls. SETTING: Animal research laboratory. SUBJECTS: Male Lewis rats weighing 150-175 g. INTERVENTIONS: Indium-111-labeled splenic lymphocytes were injected into animals after a standardized hind limb trauma or laparotomy and after induction of sterile central nervous system inflammation. MEASUREMENTS AND MAIN RESULTS: Lymphoid and non-lymphoid organs were removed at fixed intervals after lymphocyte injection and the proportion of injected lymphocytes/gram of tissue was determined using a quantitative radionuclide calculation. Results from treated animals were compared with results from untreated control animals. Muscle injury caused early localization of lymphocytes to injured hind limbs, liver, and spleen compared with controls, whereas laparotomy decreased lymphocyte localization in the thymus and colon. Encephalitis increased localization to the central nervous system with no effect on other tissues. CONCLUSIONS: These results identify a sensitive method to track in vivo leukocyte localization and specifically demonstrate that lymphocyte localization is altered in both traumatic and nontraumatic models of inflammation.

Microglial activation and neuronal apoptosis in Bornavirus infected neonatal Lewis rats.

Lewis rats neonatally infected with Borna disease virus have a behavioral syndrome characterized by hyperactivity, movement disorders, and abnormal social interactions. Virus is widely distributed in brain; however, neuropathology is focused in dentate gyrus, cerebellum, and neocortex where granule cells, Purkinje cells and pyramidal cells are lost through apoptosis. Although a transient immune response is present, its distribution does not correlate with sites of damage. Neuropathology is instead colocalized with microglial proliferation and expression of MHC class I and class II, ICAM, CD4 and CD8 molecules. Targeted pathogenesis in this system appears to be linked to microglial activation and susceptibility of specific neuronal populations to apoptosis rather than viral tropism or virus-specific immune responses.

Inhibition of experimental allergic encephalomyelitis with an antibody that recognizes a novel antigen expressed on lymphocytes, endothelial cells, and microglia.

Experimental allergic encephalomyelitis (EAE) is a frequently employed animal model of the human disease multiple sclerosis. EAE can be induced by adoptive transfer of CD4+ T cells that are specific for central nervous system (CNS) antigens, typically myelin proteins. Although the pathogenic mechanism or mechanisms responsible for the clinical signs and histological changes in EAE and multiple sclerosis are not fully defined, the entry of T lymphocytes and antigen recognition within the CNS are required. The present study describes the participation of a novel cell surface molecule with properties suggesting a role in cell-cell adhesion or co-stimulation, or both, in the development of EAE in the rat. The molecule is defined by the unique monoclonal antibody (mAb) TLD-4A2. The TLD-4A2 antigen is present on resting and activated T lymphocytes, activated CNS endothelial cells, and microglia. The antigen is normally distributed in many tissues including lymph node, thymus, and spleen, as well as in the inflamed CNS. Both its pattern of tissue distribution and immunoprecipitation and immunoblotting studies suggest that the TLD-4A2 antigen is a novel molecule. Treatment of rats with the purified 4A2 mAb resulted in the inhibition of the clinical signs of EAE and also decreased the number T cells and macrophages accumulating in the CNS parenchyma. TLD-4A2 antibody did not seem to directly interfere with T cell viability in vivo, as demonstrated by the ability to recover and stimulate CD4+ encephalitogenic T cells from cervical lymph nodes of 4A2-treated animals. In vitro, the antibody partially blocked T cell proliferation assays. These data suggest that the TLD-4A2 mAb recognizes a novel molecule expressed on lymphocytes, endothelial cells, and macrophages that may play a role in hematogenous cell traffic and the initiation of CNS inflammation.

Inflammatory thoughts about glioma gene therapy.

Gene therapy for treatment of glioma often involves delivery of herpes simplex virus-1 thymidine kinase gene. A new study shows that this approach can induce chronic inflammation, and raises important questions about current adenoviral-based clinical trials (pages 1256-1263).

The pathology of multiple sclerosis: a historical perspective.

In the century and a half since multiple sclerosis (MS) was first recognized, the pathology of the condition has been defined with increasing detail. From the recognition and definition of MS as a clinical phenomenon, studies of the diseased brain tissue have progressed in a manner dependent on the science of the time. Through multiple generations, the increasingly detailed analysis of the MS lesion itself has lead to an increasingly sophisticated understanding of a complex, apparently diverse, immunopathological process. During this evolution, many hypotheses concerning the pathogenesis of MS have been overturned, and the interpretation of some clearly delineated gross and histological findings have been reversed. This review plots the progress and highlights current theories and emerging concepts regarding one of the most enigmatic of neurological diseases.

Fatal poisoning from liquid dimethylmercury: a neuropathologic study.

Since ancient times, mercury has been recognized as a toxic substance. Dimethylmercury, a volatile liquid organic mercury compound, is used by a small number of chemistry laboratories as a reference material in nuclear magnetic resonance spectroscopy. To our knowledge, dimethylmercury has been reported in only three cases of human poisoning, each proving fatal. Very small amounts of this highly toxic chemical can result in devastating neurological damage and death. We report the neuropathologic findings in a fatal case of dimethylmercury intoxication occurring in a laboratory researcher that resulted from a small accidental spill. We compare these findings to those reported in one previously reported fatal case of dimethylmercury poisoning, and to earlier reports of monomethylmercury poisoning, and discuss the clinicopathologic correlation.

Leukocyte traffic in the central nervous system: the participants and their roles.

While the central nervous system has long been considered immunologically privileged, over the past decade it has become evident that a wide variety of leukocyte types traffic through the nervous system. It is also apparent that the rules governing the trafficking of these disparate cell types are different for each. Some arrive, and probably depart, continuously as part of normal physiology. Others only appear to seek a specific antigen or in response to tissue damage. In this review the nature and function of individual cell types are discussed and our current knowledge regarding the parameters governing their entry into the CNS is examined.