Karin Seid

Dynamics of microglial activation after human traumatic brain injury are revealed by delayed expression of macrophage-related proteins MRP8 and MRP14

Abstract Human traumatic brain injury (TBI) is ideally suited for investigation of the kinetics of human microglial cell activation as the onset of lesion formation is precisely defined. The present study provides evidence of a distinct delay in macrophage/microglia response following TBI. Eighteen brains of patients who had survived TBI for 1 h to 6 months were analysed by immunohistology. Samples of contusional and non-contusional areas were studied using antibodies directed against antigens of microglia/ macrophages [major histocompatibility complex class II, CD4, interleukin (IL)-16, macrophage-related protein (MRP) 8 and MRP14]. IL-16, a natural ligand to CD4, was expressed constitutively by numerous microglial cells in all cases throughout the brain. CD4 could be detected regularly on perivascular cells. MRP8 and MRP14, which are only expressed on activated macrophages and microglial cells, could be detected only within brains with a survival time of more than 72 h post TBI. In addition, proliferation of microglia detected by MIB-1 was not present until 72 h. This delayed expression of the activation markers MRP8 and MRP14 and the proliferation marker MIB-1 is comparable to experimental closed head injuries but strictly different from acute activation found in ischemic brains.

Allograft‐inflammatory factor‐1 in rat experimental autoimmune encephalomyelitis, neuritis, and uveitis: Expression by activated macrophages and microglial cells

Allograft inflammatory factor‐1 (AIF‐1) is a Ca2+binding peptide expressed predominantly by activated monocytes. In order to investigate the role of AIF‐1 in autoimmune lesions of the rat nervous system, we have used a synthetic gene to express AIF‐1 in E.coli and have produced monoclonal antibodies against AIF‐1.

Localization of endothelial‐monocyte‐activating polypeptide II (EMAP II), a novel proinflammatory cytokine, to lesions of experimental autoimmune encephalomyelitis, neuritis and uveitis: Expression by monocytes and activated microglial cells

Endothelial‐Monocyte‐Activating Polypeptide II (EMAP II) is a proinflammatory cytokine and chemoattractant of macrophages. In order to investigate the role of EMAP II in autoimmune lesions of the rat nervous system, we have used a synthetic gene to express EMAP II in E. coli and have produced monoclonal antibodies against EMAP II. Monoclonal antibodies are suited to demonstrate EMAP II in ELISAs, Western blots, and paraffin‐embedded tissue sections. EMAP II was localized to monocytes/macrophages with rather selective staining of a minor rat monocyte subpopulation of lymphoid tissues such as spleen, lymph nodes or follicles of the gut. In the normal brain, cells of the perivascular but not parenchymal microglia were stained. We then investigated expression of EMAP II during experimental autoimmune encephalomyelitis (EAE), neuritis (EAN), and uveitis (EAU). Within the local inflammatory lesions infiltrating macrophages are prominently stained. In the diseased brain, EMAP II‐positive microglial cells are not only found in the direct vicinity of the inflammatory infiltrate, but widespread activation is seen in the parenchyma. This is the first demonstration that EMAP II is present in autoimmune lesions. Immunostaining of microglial cells is noteworthy, as these cells are strategically placed regulatory elements of CNS immunosurveillance. EMAP II might be a factor regulating monocyte chemoattraction, endothelial cell activation and a regulator of microglial cell reactivity in autoimmune inflammation of the central nervous system. GLIA 20:365–372, 1997.

Effects of autoantigen and dexamethasone treatment on expression of endothelial-monocyte activating polypeptide II and allograft-inflammatory factor-1 by activated macrophages and microglial cells in lesions of experimental autoimmune encephalomyelitis, neuritis and uveitis.

Abstract Endothelial-monocyte activating polypeptide II (EMAP II) and allograft-inflammatory factor-1 (AIF-1) are two proteins produced by activated monocytes and microglial cells. We now report expression of these factors during experimental therapy of rat neuroautoimmune diseases. Comparative analysis of two therapeutic strategies, treatment with high doses of recombinant autoantigens or with dexamethasone, revealed unexpected differences. High doses of autoantigen were most effective in experimental autoimmune encephalomyelitis and neuritis (EAE and EAN), but less effective in experimental autoimmune uveitis (EAU). Low and high doses of dexamethasone treatment greatly reduced the severity of EAE, EAN and EAU at day 11, but a relapse was observed between days 21 and 26. Only rather limited expression of EMAP II and AIF-1 is seen in the normal central nervous system (CNS). This constitutive expression is not abolished by dexamethasone treatment. In inflammatory autoimmune lesions of the rat CNS, prominent AIF-1 and EMAP II staining was seen with macrophages and monocytes. In particular, parenchymal microglial cells were now activated to express AIF-1 and EMAP II. In accordance with prevention of neurological signs, histological observations revealed that accumulation of activated monocytes expressing EMAP II and AIF-1 in the CNS or peripheral nervous system and the massive expression of these factors by parenchymal microglial cells is inhibited by high doses of autoantigen. Dexamethasone prevented or abolished local expression of EMAP II and AIF-1 at days 10–16. However, an acute and severe relapse occurred in encephalomyelitis between days 20–26. In these cases, a smoldering expression of EMAP II and AIF-1 persisting long after cessation of neurological signs was observed. Thus, expression of EMAP II and AIF-1 by infiltrating activated macrophages is a marker of disease activity and expression of these factors could be used to demonstrate ‘silent’ lesions in the CNS and prolonged microglial cell activation. Apparently, AIF-1 and EMAP II immunoreactivity are tools to stage activation of monocytes and microglial cells in inflammatory lesions.

Leukocyte chemotactic factor, a natural ligand to CD4, is expressed by lymphocytes and microglial cells of the MS plaque

The leukocyte chemotactic factor (LCF) is a proinflammatory cytokine and natural soluble ligand to the human CD4 molecule. LCF is produced by CD4+ and CD8+ T lymphocytes and is considered essential to the influx of CD4+ T lymphocytes and macrophages into an inflammatory lesion. In order to investigate the role of LCF in the multiple sclerosis (MS) lesion, we have used a synthetic gene to express LCF in E. coli and have produced monoclonal antibodies against LCF. Monoclonal antibodies are suited to demonstrate LCF in ELISAs, Western blots and paraffin‐embedded tissue sections. In the MS lesion, immunopositive lymphocytes and microglial cells, notably, have been found. This is the first demonstration that LCF is present in MS lesions. Immunostaining of microglial cells is noteworthy, as these cells are strategically placed regulatory elements of CNS immunosurveillance and like other cells of the monocytic lineage express CD4 molecules. Thus, LCF might be a paracrine factor regulating T‐lymphocyte chemoattraction and an autocrine molecule regulating microglial cell immune reactivity.

Allograft inflammatory factor-1 defines a distinct subset of infiltrating macrophages/microglial cells in rat and human gliomas.

Abstract Allograft inflammatory factor-1 (AIF-1) is a Ca2+-binding peptide that constitutes a potential modulator of macrophage activation and function during the immune response of the brain. Peptides termed microglia response factor-1 or ionized calcium-binding adaptor molecule-1 have been reported to be identical with AIF-1. We have investigated the expression of AIF-1 in the rat C6 glioblastoma and 9L gliosarcoma tumor models and additionally assessed AIF-1 expression in a diverse range of human astrocytomas by immunohistochemistry. AIF-1 was expressed by activated microglial cells and a subset of infiltrating macrophages in areas of infiltrative tumor growth and in compact tumor areas in both rat and human gliomas. Double-labeling experiments in rats and humans characterized the nature and the functional status of AIF-1+ cells. AIF-1 expression was detected in cells expressing major histocompatibility complex class II molecules and in a subset of activated macrophages/microglial cells. All MRP-8+ cells coexpressed AIF-1. In humans, there was a strong correlation of AIF-1-expressing activated macrophages/microglial cells with tumor malignancy (P < 0.0001). These results suggest that AIF-1 defines a distinct subset of tumor-associated activated macrophages/ microglial cells.

Transient in vivo activation of rat brain macrophages/microglial cells and astrocytes by immunostimulatory multiple CpG oligonucleotides

Certain DNA sequences containing motifs of unmethylated CpG nucleotides are immunostimulatory and might contribute to the development of inflammatory lesions after infections. CpG motifs might further contribute to side effects of oligonucleotide-based therapeutic approaches. Here we have analyzed the effects of intracranial injections of synthetic CpG oligonucleotides. We observed that oligonucleotides with several unmethylated CpG motifs, but not methylated or inverted GpC motifs, stimulated microglial cells and astrocytes of the rat brain. This transient, self-limiting response is maximal at day 3 after injection and subsides until day 5. Activated microglial cells could be identified to produce two novel monocytic peptides, the allograft inflammatory factor-1 (AIF-1) and endothelial monocyte activating polypeptide II (EMAP II). Astrocytes were similarly activated as shown by expression of the enzyme heme-oxygenase-1 (HO-1). Glial cell proliferation (expression of PCNA) or aptosis was not observed. Thus immunostimulatory DNA activates the local innate immune defense system of the brain, and might contribute transiently to infectious, inflammatory and degenerative responses of the central nervous system.

Traumatic Brain Injury Induces Prolonged Accumulation of Cyclooxygenase-1 Expressing Microglia/Brain Macrophages in Rats

Inflammatory cellular responses to brain injury are promoted by proinflammatory messengers. Cyclooxygenases (prostaglandin endoperoxide H synthases [PGH]) are key enzymes in the conversion of arachidonic acid into prostanoids, which mediate immunomodulation, mitogenesis, apoptosis, blood flow, secondary injury (lipid peroxygenation), and inflammation. Here, we report COX-1 expression following brain injury. In control brains, COX-1 expression was localized rarely to brain microglia/macrophages. One to 5 days after injury, we observed a highly significant (p < 0.0001) increase in COX-1+ microglia/macrophages at perilesional areas and in the developing core with a delayed culmination of cell accumulation at day 7, correlating with phagocytic activity. There, cell numbers remained persistently elevated up to 21 days following injury. Further, COX-1+ cells were located in perivascular Virchow-Robin spaces also reaching maximal numbers at day 7. Lesion-confined COX-1+ vessels increased in numbers from day 1, reaching the maximum at days 5-7. Double-labeling experiments confirmed coexpression of COX-1 by ED-1+ and OX-42+ microglia/ macrophages. Transiently after injury, most COX-1+ microglia/macrophages coexpress the activation antigen OX-6 (MHC class II). However, the prolonged accumulation of COX-1+, ED-1+ microglia/macrophages in lesional areas enduring the acute postinjury inflammatory response points to a role of COX-1 in the pathophysiology of secondary injury. We have identified localized, accumulated COX-1 expression as a potential pharmacological target in the treatment of brain injury. Our results suggest that therapeutic approaches based on long-term blocking including COX-1, might be superior to selective COX-2 blocking to suppress the local synthesis of prostanoids.

Localization of endostatin in rat and human gliomas

Endostatin is a potent inhibitor of endothelial cell proliferation, angiogenesis, and tumor growth. Its occurrence and localization has not yet been examined in human brain tumors. The authors report the production of a monoclonal antibody and detection of endostatin in rat and human gliomas by immunohistochemistry.

Heme oxygenase-1 in lesions of rat experimental autoimmune encephalomyelitis and neuritis

The enzyme heme oxygenase-1 (HO-1) is reducing heme to the gaseous mediator carbon monoxide, to iron and the antioxidant biliverdin. The inducible expression of HO-1 is considered a protective cellular mechanism against reactive oxygen intermediates. Further, carbon monoxide (CO) is a regulator of cGMP synthesis, of NO-synthetases and cyclooxygenases, thereby indirectly modulating reactive processes. Here we report expression of HO-1 in rat experimental autoimmune encephalomyelitis (EAE) and neuritis (EAN). With both models, similar results were obtained: HO-1 was localized predominantly to infiltrating, monocytic, but only rarely to ramified microglial cells or astrocytes surrounding the inflammatory lesions. Prominent expression by monocytic cells was seen from day 11 after immunization correlating with the development of neurologic disease. Further, local expression is persistent for long after cessation of neurologic signs. Thus, HO-1 could be considered a factor in the formation and resolution of inflammatory autoimmune lesions of the nervous system.