Stephanie Bunkowski

Tumour necrosis factor alpha mRNA expression in early multiple sclerosis lesions: Correlation with demyelinating activity and oligodendrocyte pathology

The precise role of tumour necrosis factor alpha (TNFα) in multiple sclerosis (MS) is still controversial. Most findings from the animal model experimental allergic encephalomyelitis have yet to be confirmed in multiple sclerosis. The aim of this study was to define the significance of TNFα with respect to the hallmark of MS, that is demyelination. Therefore, 78 lesion areas from diagnostic brain biopsies of 32 patients were analysed. Lesion demyelinating activity was classified by the presence of myelin degradation products in macrophages and macrophage activation markers. Non‐radioactive in situ hybridisation was carried out to detect TNFα mRNA expressing cells. DNA fragmentation was visualised by TdT‐mediated X‐dUTP nick end labeling. A significantly higher number of cells expressed TNFα mRNA in active demyelinating lesions than in inactive or remyelinating lesions irrespective of the extent of the inflammatory infiltrate. TNFα mRNA expression correlated with the appearance of DNA fragmentation in T lymphocytes and oligodendrocytes within the lesions. In the periplaque white matter, expression of TNFα mRNA negatively correlated with oligodendrocyte numbers. These data support previous findings from animal models and in vitro experiments. Although not proving, the current study strongly suggests a pathogenic role of TNFα in demyelination in human multiple sclerosis and gives further support for TNFα‐directed therapeutic strategies. GLIA 29:366–375, 2000.

Toll-Like Receptor Stimulation Enhances Phagocytosis and Intracellular Killing of Nonencapsulated and Encapsulated Streptococcus pneumoniae by Murine Microglia

ABSTRACT Toll-like receptors (TLRs) are crucial pattern recognition receptors in innate immunity that are expressed in microglia, the resident macrophages of the brain. TLR2, -4, and -9 are important in the responses against Streptococcus pneumoniae, the most common agent causing bacterial meningitis beyond the neonatal period. Murine microglial cultures were stimulated with agonists for TLR1/2 (Pam3CSK4), TLR4 (lipopolysaccharide), and TLR9 (CpG oligodeoxynucleotide) for 24 h and then exposed to either the encapsulated D39 (serotype 2) or the nonencapsulated R6 strain of S. pneumoniae. After stimulation, the levels of interleukin-6 and CCL5 (RANTES [regulated upon activation normal T-cell expressed and secreted]) were increased, confirming microglial activation. The TLR1/2, -4, and -9 agonist-stimulated microglia ingested significantly more bacteria than unstimulated cells (P < 0.05). The presence of cytochalasin D, an inhibitor of actin polymerizaton, blocked >90% of phagocytosis. Along with an increased phagocytic activity, the intracellular bacterial killing was also increased in TLR-stimulated cells compared to unstimulated cells. Together, our data suggest that microglial stimulation by these TLRs may increase the resistance of the brain against pneumococcal infections.

Neuronal Apoptosis in the Dentate Gyrus in Humans with Subarachnoid Hemorrhage and Cerebral Hypoxia

Apoptosis of dentate granule cells is a typical feature of several animal models of disease. In 20 autopsy cases of subarachnoid hemorrhage (SAH) and global cerebral hypoxia caused by protracted shock or respiratory failure, we evaluated by light microscopy and in situ tailing whether this pattern of neuronal damage also occurs in humans. In subarachnoid hemorrhage, 4.0/mm2 (0–13.0/mm2) a poptotic neurons were observed in the dentate gyrus, in cerebral hypoxia 3.6/mm2 (0–19.9/mm2) (p>0.05), and in 10 aged‐matched control cases dying rapidly from non‐neurological diseases 0/mm2 (0–0/mm2) (median [range]) (p<0.001 versus SAH and hypoxia). Neuronal apoptosis in the dentate gyrus was most frequent, when death occurred later than 24 hours and less than 11 days after disease onset. Neuronal damage in the hippocampus was always necrotic. It was more severe in hypoxia than in SAH (median neuronal damage score 3 [range: 0–3] versus 0 [0–3], p<0.001).

Lesion development in Marburg's type of acute multiple sclerosis: from inflammation to demyelination

We report a patient who suffered from acute inflammatory CNS demyelination and underwent two consecutive diagnostic stereotactic brain biopsies during the early disease course. The first lesion was drawn 33 days after the onset of disseminated neurological symptoms. Macrophages and T lymphocytes diffusely infiltrated small vessel walls and the white matter. mRNA for tumor necrosis factor alpha (TNFa) and inducible nitric oxide synthase (iNOS) was abundantly expressed. Myelin sheaths were entirely preserved. The second biopsy 76 days later showed confluent demyelinating lesions with a diffuse infiltration of macrophages that were positive for myelin debris, activation markers and TNFa and iNOS mRNA. IgG and C9neo deposits were found along myelin sheaths. The patient had received intravenous immunoglobulins (IVIG) prior to biopsy. Findings from this single patient affirm that demyelination follows the migration of inflammatory cells from the circulation into the white matter with subsequent inflammation and demyelination. Inflammation alone may be sufficient to cause significant clinical deficits without demyelination. Inflammatory mediators such as TNFa and NO are involved at very early stages in the pathogenetic process. IVIG treatment may lead to the deposition of immunoglobulins and to the activation of the complement cascade, but the clinical relevance of this particular finding remains uncertain.

Increased activin levels in cerebrospinal fluid of rabbits with bacterial meningitis are associated with activation of microglia

Activin, a member of the transforming growth factor superfamily, is upregulated in a number of inflammatory episodes such as septicemia and rheumatoid arthritis. In the CNS, activin has been predominantly assessed in terms of a neuroprotective role. In this report we characterized the activin response in the CNS in a rabbit model of meningitis. In normal animals, cerebrospinal fluid (CSF) activin levels were higher than those in serum, indicating an intracranial secretion of this cytokine. Following intracisternal inoculation with Streptococcus pneumoniae, activin in CSF was unchanged for the first 12 h and then rose progressively; levels were increased approximately 15‐fold within 24 h. Activin levels were correlated positively with CSF protein content and with the number of apoptotic neurons in the dentate gyrus. No apparent correlation was observed between CSF activin concentrations and bacterial titer, lactate concentrations or leukocyte density. Using immunohistochemistry, activin staining was localized to epithelial cells of the choroid plexus, cortical neurons and the CA3 region of the hippocampus, with similar staining intensities in both normal and meningitic brains. However, in meningitic brains there was also strong staining in activated microglia and infiltrating macrophages. Taken together, these results demonstrate that activin forms part of the CNS response to immune challenge and may be an important mediator to modulate inflammatory processes in the brain.

Rifampin Followed by Ceftriaxone for Experimental Meningitis Decreases Lipoteichoic Acid Concentrations in Cerebrospinal Fluid and Reduces Neuronal Damage in Comparison to Ceftriaxone Alone

ABSTRACT Rifampin (RIF) releases smaller quantities of lipoteichoic acids (LTAs) from Streptococcus pneumoniae than ceftriaxone (CRO). Due to the rapid development of resistance, RIF cannot be used as a single agent for therapy of bacterial meningitis. For this reason, we compared the effect of treatment with RIF followed by treatment with CRO (RIF-CRO) or the effect of treatment with clindamycin (CLI) followed by treatment with CRO (CLI-CRO) to that of CRO alone on the concentrations of LTAs and teichoic acids in vitro. The effects of RIF-CRO on LTA concentrations in cerebrospinal fluid (CSF) and on neuronal injury were investigated in a rabbit model of S. pneumoniae meningitis. In vitro, bacterial titers were effectively reduced by CRO, RIF-CRO, and CLI-CRO when each drug was used at 10 μg/ml. The levels of release of LTAs after the initiation of therapy were lower in RIF-CRO- and CLI-CRO-treated cultures than in cultures treated with CRO alone (P < 0.05 from 3 to 12 h after initiation of treatment). Similarly, in rabbits, the increase in the amount of LTAs in CSF was lower in RIF-CRO-treated animals than in CRO-treated animals (P = 0.02). The density of dentate apoptotic granular cells was lower after RIF-CRO therapy than after CRO therapy (medians, 58.4 and 145.6/mm2, respectively; 25th quartiles, 36.3 and 81.7/mm2, respectively; 75th quartiles, 100.7 and 152.3/mm2, respectively; P = 0.03). Therefore, initiation of therapy with a protein synthesis-inhibiting antibacterial and continuation of therapy with a combination that includes a β-lactam may be a strategy to decrease neuronal injury in bacterial meningitis.

Axonal injury, a neglected cause of CNS damage in bacterial meningitis

The contribution of axonal injury to CNS damage in bacterial meningitis was studied by histology and immunohistochemistry for amyloid-β precursor protein in humans and experimental rabbits. Axonal injury in the white matter caused predominantly but not exclusively by ischemia was detected in all autopsy cases (n = 5) and in 11 of 15 brains of rabbits 18 to 24 hours after intracisternal infection with Streptococcus pneumoniae. This suggests a substantial contribution of axonal pathology to neurologic sequelae after bacterial meningitis.

Clindamycin is neuroprotective in experimental Streptococcus pneumoniae meningitis compared with ceftriaxone

In animal models of Streptococcus pneumoniae meningitis, rifampin is neuroprotective in comparison to ceftriaxone. So far it is not clear whether this can be generalized for other protein synthesis‐inhibiting antimicrobial agents. We examined the effects of the bactericidal protein synthesis‐inhibiting clindamycin (n = 12) on the release of proinflammatory bacterial components, the formation of neurotoxic compounds and neuronal injury compared with the standard therapy with ceftriaxone (n = 12) in a rabbit model of pneumococcal meningitis. Analysis of the CSF and histological evaluation were combined with microdialysis from the hippocampal formation and the neocortex. Compared with ceftriaxone, clindamycin reduced the release of lipoteichoic acids from the bacteria (p = 0.004) into the CSF and the CSF leucocyte count (p = 0.011). This led to lower extracellular concentrations of hydroxyl radicals (p = 0.034) and glutamate (p = 0.016) in the hippocampal formation and a subsequent reduction of extracellular glycerol levels (p = 0.018) and neuronal apoptosis in the dentate gyrus (p = 0.008). The present data document beneficial effects of clindamycin compared with ceftriaxone on various parameters linked with the pathophysiology of pneumococcal meningitis and development of neuronal injury. This study suggests neuroprotection to be a group effect of bactericidal protein synthesis‐inhibiting antimicrobial agents compared with the standard therapy with β‐lactam antibiotics in meningitis.

Increased neurogenesis after experimental Streptococcus pneumoniae meningitis

Neuronal damage in the hippocampal formation is a common feature in animal models of bacterial meningitis and human disease. In mouse and rabbit models of Streptococcus pneumoniae meningitis, proliferation of neural progenitor cells quantified by bromodeoxyuridine (BrdU) incorporation was enhanced in the subgranular layer of the dentate gyrus. In mice, the density of BrdU‐labeled cells was maximal on Day 2 after infection. Approximately 60% of the cells labeled by BrdU between Days 7 and 10 after infection that remained present 28 days later had migrated into deeper layers of the dentate gyrus and differentiated into neurons, as evidenced by immunohistochemical staining for TUC‐4, MAP‐2 and beta‐tubulin. This suggests that endogenous repair mechanisms may limit consequences of neuronal destruction after meningitis.

Identification of macrophage populations expressing tumor necrosis factor-α mRNA in acute multiple sclerosis

Abstract Macrophages are involved in central nervous system (CNS) demyelination in multiple sclerosis (MS) and other demyelinating diseases. Macrophages seen in MS lesions form a heterogeneous population with respect to their stage of activation and differentiation. We have analyzed macrophages from active demyelinating lesions of a patient who died from fulminant MS of Marburg’s type to define the functional heterogeneity of different macrophage populations in acute demyelination. We examined tumor necrosis factor-α (TNF-α) mRNA expression in macrophages defined by different activation markers. The majority of TNF-α mRNA-positive cells were macrophages positive for the pan-macrophage marker KiM1P. A subgroup of TNF-α mRNA-positive macrophages was stained by the early activation marker MRP14. In contrast, macrophages positive for the acute activation marker 27E10 were entirely negative for TNF-α mRNA. In conclusion, macrophages in acute demyelinating CNS lesions are heterogeneous as shown by staining for different activation markers. This heterogeneity is also of functional relevance as certain subpopulations are involved in TNF-α mRNA expression, while others are not. This may be important for directing therapeutic strategies against well-defined pathogenic macrophage populations.