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).

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.

Exogenously-induced, natural killer cell-mediated neuronal killing: a novel pathogenetic mechanism.

Many human neurodegenerative diseases are characterized by the idiopathic death of cells narrowly restricted to a subset of neurons in a specific functional neuroanatomic system. Few in vivo models exist for the analysis of these types of degeneration. This report documents the death of sympathetic neurons resident in the superior cervical ganglia of rats after exposure to an exogenous chemical agent, the drug guanethidine, as being mediated by natural killer (NK) cells. This is the first in vivo model of a disorder of the nervous system in which NK cells appear to be the principal effector cell, and thus could serve a central role in dissecting the normal and pathological function of NK cells. In addition, this pathogenetic mechanism appears to represent a novel type of autoimmune reaction that could have a direct bearing on a number of human illnesses.

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.

Endosomes and Golgi vesicles in adsorptive and fluid phase endocytosis.

We studied with morphometric methods the endocytosis by pheochromocytoma cells of a conjugate of wheat germ agglutinin with ferritin (WGA-Ft) and of horseradish peroxidase (HRP). Quantitative studies indicated that WGA-Ft was cleared slowly from cell surfaces and that it was not recycled to the surface. Cells labeled with WGA-Ft for 15 min at room temperature were washed and incubated in medium containing HRP for 15 or 30 min at 37 degrees C. The greatest proportion of labeled vesicles and tubules contained only WGA-Ft (83.4% at 15 min and 85.3% at 30 min). A very small fraction of labeled vesicles and tubules contained only HRP (0.2% at 15 min and 0.9% at 30 min). Vesicles and tubules at the Golgi apparatus were labeled almost exclusively with WGA-Ft (97% at 15 min and 30 min); the rest had both labels. Most labeled lysosomes contained both labels (80.1% at 15 min and 80.8% at 30 min). Of the remainder more contained WGA-Ft alone (20% at 15 min and 10.9% at 30 min), then HRP alone (none at 15 min and 8.2% at 30 min). In contrast to the various and varying patterns of labeling with WGA-Ft and HRP of the other organelles studied, the vast majority of endosomes contained both markers (94.1% at 15 min and 100% at 30 min); the rest contained WGA-Ft only. These results demonstrate that endosomes are recipients of both fluid phase and adsorptive endocytosis markers; these findings are consistent with the hypothesis that endosomes mediate the sorting out and subsequent intracellular traffic of membrane bound and fluid phase markers. Cisterns of the Golgi apparatus did not contain WGA-Ft; in sharp contrast, when WGA-HRP was used, the cisterns of the Golgi apparatus consistently contained HRP.

Perivascular microglial cells of the CNS are bone marrow-derived and present antigen in vivo.

A crucial question in the study of immunological reactions in the central nervous system (CNS) concerns the identity of the parenchymal cells that function as the antigen-presenting cells in that organ. Rat bone marrow chimeras and encephalitogenic, major histocompatability--restricted T-helper lymphocytes were used to show that a subset of endogenous CNS cells, commonly termed "perivascular microglial cells," is bone marrow-derived. In addition, these perivascular cells are fully competent to present antigen to lymphocytes in an appropriately restricted manner. These findings are important for bone marrow transplantation and for neuroimmunological diseases such as multiple sclerosis.

A neuroendocrine marker in tissues of the immune system.

Antibodies to chromogranin, a secretory protein marker for the diffuse neuroendocrine system, were used to analyze rat lymphoreticular tissues by means of immunochemistry and immunohistochemistry. Chromogranin-positive cells were present in spleen, lymph node, thymus, and fetal liver. When these organs were gently dispersed and separated on a Ficoll gradient, the chromogranin-immunoreactive cells became enriched in the dense red-cell pellets. The unexpected distribution of these neuroendocrine cells in all immunologically relevant structures suggests that they may link the nervous and immunological systems.

Active immunization with lipopolysaccharide Pseudomonas antigen for chronic Pseudomonas bronchopneumonia in guinea pigs.

Chronic respiratory infection with Pseudomonas aeruginosa is a leading clinical problem among patients with cystic fibrosis. Because antimicrobial agents are usually ineffective in eradicating these infections, additional therapeutic or prophylactic measures should be considered. In this study, an experimental guinea pig model of chronic Pseudomonas aeruginosa bronchopneumonia was utilized to determine whether active immunization with lipopolysaccharide (LPS) P. aeruginosa antigen may favorably influence the course of this infection. Experimental pneumonia was established by tracheobronchial instillation of suspensions of microscopic agar beads, which were impregnated with viable P. aeruginosa. After 4 wk of infection, the geometric mean (reciprocal) passive hemagglutinating Pseudomonas antibody titer was 185+/-1.3, and lungs contained 16.8+/-4 x 10(3) colony-forming units Pseudomonas/ml of lung homogenate. Pseudomonas immunization, given prior to a 4-wk infection, resulted in significantly higher passive hemagglutinating titers (474+/-1.4; P < 0.05), lower numbers of viable Pseudomonas in lung tissues (2.4+/-0.6 x 10(3); P < 0.01), and reduced histopathology in lungs. In contrast, providing Pseudomonas immunization to animals 2 wk after pulmonary infection was established, offered no apparent benefit. Likewise, no protection was afforded by prophylactic immunization with a non-Pseudomonas LPS antigen (Escherichia coli J5 vaccine). Using a Raji cell assay, modified to detect circulating immune complexes in vaccinated and infected guinea pig sera, there was no evidence that active immunization increased the frequency of circulating immune complexes in infected guinea pigs. It is concluded that prophylactic immunization with Pseudomonas LPS antigen may confer protection from subsequent Pseudomonas bronchopneumonia, but that immunization during established infection is not beneficial.

Aminoguanidine, an inhibitor of inducible nitric oxide synthase, ameliorates experimental autoimmune encephalomyelitis in SJL mice.

Previous work from our laboratory localized nitric oxide to the affected spinal cords of mice with experimental autoimmune encephalomyelitis, a prime model for the human disease multiple sclerosis. The present study shows that activated lymphocytes sensitized to the central nervous system encephalitogen, myelin basic protein, can induce nitric oxide production by a murine macrophage cell line. Induction was inhibited by amino-guanidine, a preferential inhibitor of the inducible nitric oxide synthase isoform, and by NG-monomethyl-L-arginine. Aminoguanidine, when administered to mice sensitized to develop experimental autoimmune encephalomyelitis, inhibited disease expression in a dose-related manner. At 400 mg aminoguanidine/kg per day, disease onset was delayed and the mean maximum clinical score was 0.9 +/- 1.2 in aminoguanidine versus 3.9 +/- 0.9 in placebo-treated mice. Histologic scoring of the spinal cords for inflammation, demyelination, and axonal necrosis revealed significantly less pathology in the aminoguanidine-treated group. The present study implicates excessive nitric oxide production in the pathogenesis of murine inflammatory central nervous system demyelination, and perhaps in the human disease multiple sclerosis.

A novel synthetic oleanane triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid, with potent differentiating, antiproliferative, and anti-inflammatory activity.

The new synthetic oleanane triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) is a potent, multifunctional molecule. It induces monocytic differentiation of human myeloid leukemia cells and adipogenic differentiation of mouse 3T3-L1 fibroblasts and enhances the neuronal differentiation of rat PC12 pheochromocytoma cells caused by nerve growth factor. CDDO inhibits proliferation of many human tumor cell lines, including those derived from estrogen receptor-positive and -negative breast carcinomas, myeloid leukemias, and several carcinomas bearing a Smad4 mutation. Furthermore, it suppresses the abilities of various inflammatory cytokines, such as IFN-gamma, interleukin-1, and tumor necrosis factor-alpha, to induce de novo formation of the enzymes inducible nitric oxide synthase (iNos) and inducible cyclooxygenase (COX-2) in mouse peritoneal macrophages, rat brain microglia, and human colon fibroblasts. CDDO will also protect rat brain hippocampal neurons from cell death induced by beta-amyloid. The above activities have been found at concentrations ranging from 10(-6) to 10(-9) M in cell culture, and these results suggest that CDDO needs further study in vivo, for either chemoprevention or chemotherapy of malignancy as well as for neuroprotection.

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.

Bone marrow-derived elements in the central nervous system: an immunohistochemical and ultrastructural survey of rat chimeras.

This report defines the bone marrow-derived elements found in the central nervous system of adult rat radiation chimeras. Four cell types were identified which bore the major histocompatibility (MHC) class I molecules of the donor rat strain thereby indicating a marrow origin. They were: meningeal macrophages, perivascular "microglial" cells, lymphocytes and rare cells with parenchymal microglial morphology. These cells were examined by immunohistochemical methods at the light microscopic and ultrastructural levels. Extended descriptions of the perivascular marrow-derived elements and the parenchymal microglial cells are presented. These latter two cell types, which exist in humans, have a significant role in neuroimmune processes and most probably function as the antigen-presenting cells in the central nervous system of mammals.

Ultrastructural and immunohistologic characterization of guanethidine-induced destruction of peripheral sympathetic neurons.

Repeated daily injections of the adrenergic blocking agent guanethidine in high doses (50 mg/kg) to either newborn or adult rats produces a permanent sympathectomy which is thought to involve an immune-mediated mechanism. Ultrastructural examination of the acute phase of guanethidine-induced neuronal degeneration in the superior cervical ganglion demonstrated a prominent lymphocytic infiltrate in the ganglionic parenchyma and perivascular spaces. Within the satelliting cell sheath there were lymphocytes with delicate processes in intimate contact with abnormal or degenerating principal sympathetic neurons. The lymphocytic infiltrate and early evidence of neuronal degeneration developed within three days of onset of treatment and reached a maximum on the 7th day. Immunocytochemical studies of the ganglion demonstrated that the mononuclear infiltrate was composed primarily of CD-8 positive, OX-19 negative lymphocytes, which may represent a natural killer (NK) or NK-like effector cell, as well as scattered monocytes and macrophages. Guanethidine showed little effect on the prevertebral (superior mesenteric and celiac) sympathetic ganglia compared to the paravertebral (superior cervical) chain ganglia of the same animals. Considerable rat strain selectivity was noted with Lewis and Sprague-Dawley strains showing a greater degree of neuronal destruction than Fischer 344 rats. Neonatal thymectomy failed to prevent neuronal degeneration and the lymphocytic infiltrate. These features of immune neuronal killing are not typical of a characterized antibody or T-lymphocyte mediated effector mechanism, but may represent a selective NK cell or NK-like cell mediated pathogenetic pathway. Guanethidine-induced neuronal destruction appears to be a unique type of drug-induced, cell-mediated, autoimmune attack on sympathetic neurons and may provide insight into autoimmune disorders and possibly into diseases characterized by selective neuronal degeneration.

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.

Migration of hematogenous cells through the blood-brain barrier and the initiation of CNS inflammation.

The central nervous system has long been considered an immunologically privileged site. Nevertheless, cells derived from the bone marrow can and do enter the CNS in a number of circumstances. Derivatives of the monocyte/macrophage lineage appear to enter and take up residence in various structures of the CNS as part of normal ontogeny and physiology. Immunocompetent cells, such as T-lymphocytes of both CD4 and CD8 positive groups, enter the nervous system in what appears to be a random fashion when they are activated by antigenic stimulation. These lymphocytes perform the required immunological surveillance of the CNS, and initiate inflammation therein during infectious and autoimmune reactions. In this review, the evidence supporting the above observations is examined, and a hypothesis for the pathogenesis of CNS inflammatory reactions is presented.

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.

Neuropathology of immunosuppression.

Traditionally, the brain has been considered an "immunologically privileged" organ. Under normal conditions, the blood-brain barrier (BBB) is highly effective in preventing both cellular and humoral constituents of the blood from entering the brain parenchyma. In certain pathological conditions, such as viral infections and demyelinating disorders, the BBB may become altered, activated T cells and monocytes may gain access to the brain parenchyma, and microglia may assume the functions of antigen-presenting cells and macrophages. Naturally-occurring or clinically-induced immunosuppression may dramatically alter various cellular and/or humoral aspects of the immune system. Consequently, the brain may become susceptible to disorders that would otherwise be excluded or may develop more severe manifestations of diseases, such as certain infections. This review considers the neuropathologic aspects of various conditions that may be encountered in the setting of both acquired and inherited immunosuppression. The major categories include infectious, neoplastic, vascular, and metabolic disorders. The review also briefly addresses the neuropathology of complications of chemotherapeutic agents, radiotherapy, and organ transplantation inasmuch as they often occur in the clinical setting of acquired immunosuppression.

Local cerebral glucose utilization in chronic middle cerebral artery occlusion in the cat.

This study examines the correlation between local CMRglc (LCMRglc) alterations and clinicopathological changes in a chronic middle cerebral artery (MCA) occlusion model in the cat. The left MCA was occluded for a period of 2 h. The animals were grouped into mild, moderate, and severe ischemia based on the depression of the EEG 30 min after the MCA occlusion. Following release of the clip, the animals were allowed to recover for a week during which time daily neurological examinations were performed. On the seventh day [14C]2-deoxyglucose was injected for the determination of LCMRglc. Alternative blocks were processed for histological evaluation in which both neuronal and phagocytic changes were graded into four categories (0 = normal to 3 = severe). LCMRglc (mumol/100 g/min) in the ischemic hemisphere (all histological grades) was significantly lower than the metabolic rate in comparable regions of the sham MCA occlusion group. Regions with significant phagocytosis (grade 2 and 3) invariably exhibited activated glucose metabolism (57.4 +/- 8.4 and 105.9 +/- 6.8 mumol/100 g/min, respectively), which was significantly higher than in regions without phagocytosis (30.4 +/- 0.8 mumol/100 g/min). There was a significant gradient of metabolism in the central, peripheral, and boundary zone and the non-MCA territory in the animals with severe ischemic lesions. LCMRglc in the central MCA territory was well correlated with the EEG amplitude changes (r = 0.82, p less than 0.05) and the morphological score (r = -0.89, p less than 0.05). The metabolic rate was significantly depressed in both the ipsilateral and the contralateral central MCA territories in comparison with the sham occlusion animals. The depression in LCMRglc in the contralateral hemisphere correlated well with the concomitant depression in the contralateral EEG amplitude. These studies demonstrate that local heterogeneous metabolic alterations and contralateral cortical diaschisis exist chronically following temporary MCA occlusion and that the increases in local cerebral glucose metabolism seen in chronic stroke may be due to phagocytotic activity.

Intravenous lipopolysaccharide induces cyclooxygenase 2-like immunoreactivity in rat brain perivascular microglia and meningeal macrophages.

Production of prostaglandins is a critical step in transducing immune stimuli into central nervous system (CNS) responses, but the cellular source of prostaglandins responsible for CNS signalling is unknown. Cyclooxygenase catalyzes the rate-limiting step in the synthesis of prostaglandins and exists in two isoforms. Regulation of the inducible isoform, cyclooxygenase 2, is thought to play a key role in the brain's response to acute inflammatory stimuli. In this paper, we report that intravenous lipopolysaccharide (LPS or endotoxin) induces cyclooxygenase 2-like immunoreactivity in cells closely associated with brain blood vessels and in cells in the meninges. Neuronal staining was not noticeably altered or induced in any brain region by endotoxin challenge. Furthermore, many of the cells also were stained with a perivascular microglial/macrophage-specific antibody, indicating that intravenous LPS induces cyclooxygenase in perivascular microglia along blood vessels and in meningeal macrophages at the edge of the brain. These findings suggest that perivascular microglia and meningeal macrophages throughout the brain may be the cellular source of prostaglandins following systemic immune challenge. We hypothesize that distinct components of the CNS response to immune system activation may be mediated by prostaglandins produced at specific intracranial sites such as the preoptic area (altered sleep and thermoregulation), medulla (adrenal corticosteroid response), and cerebral cortex (headache and encephalopathy).