A specific histochemical marker (lectinRicinus communis agglutinin-1) for normal human microglia, and application to routine histopathology

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234

Nitric oxide (NO) has been implicated as a pathogenic mediator in a variety of central nervous system (CNS) We have reported (1) that Borna disease virus, rabies virus, and herpes simplex virus induce the increased expression of inducible nitric oxide synthase (iNOS) mRNA in the brains of intrathecally infected mice and rats. We and others (2) also found that the induction of experimental allergic encephalomyelitis resulted in a similar increase in iNOS mRNA expression, suggesting that a similar phenomenon may occur in the brains of patients with multiple sclerosis (MS). We investigated this hypothesis by detecting iNOS mRNA in extracts of brains from patients who died with MS and from extracts of brain tissues from patients who died from nonneurological diseases. We have also colocalized the iNOS expressing cells by using immunocytochemical techniques combined with reverse transcriptase (RT) driven-in situ PCR and demonstrated the presence of iNOS protein by the same double labeling procedures. Finally, as a surrogate marker for nitric oxide (NO) presence, we have immunocytochemically localized nitroty-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.rosine adducts in histological sections of brain from MS patients. MATERIALS AND METHODSSources and Selection of Specimens. The samples used in these studies were collected from patients with an antemortem diagnosis of MS based on clinical presentation and reconfirmed by histopathology after death. All MS samples were found to contain pathognomonic MS plaques; control brains were examined and found to be pathologically unremarkable. In most cases, autopsy samples were of frontal lobes, which provided mixed white and gray matter.In two of the MS cases, special efforts were made to preserve the tissue morphology. Small portions of the brain specimens were frozen in liquid nitrogen immediately after sectioning and then stored at -70°C. This procedure preserved the architecture of the brain significantly better than direct freezing of fresh brain specimens at -80°C, which results in ice crystal formation in the tissue and distorts its architecture.Cell Culture and Cytokine Activation. A549 human pulmonary epithelial cells were purchased from the American Type Culture Collection. Cells were grown in Ham's F-12 medium supplemented with 10% (vol/vol) fetal calf serum, penicillin (50 units/ml), streptomycin (50 ,ug/ml), and 5 ,tM L-glutamine. Cells were seeded into 6-well tissue culture clusters (Falcon) for Northern blot analysis or seeded into 8-well Lab-Tek culture slides (Nunc) for RT-in situ PCR. For those cultures stimulated for the expression of iNOS, interleukin 1(3 (IL-1,B at 100 units/ml), tumor necrosis factor a (TNF-a at 10 ng/ml), and interferon y (IFN-,y at 500 units/ml) were added for 8 h.For use in Northern blot analysis, the cells were scraped from the culture substrate, washed three ti...

Section: Discussionmentioning

confidence: 99%

“…Our experiments employed RCA-1, a selective marker of endothelial cells and microglia in human nervous tissue (11,12), to identify cells expressing iNOS mRNA. Microglia and endothelial cells are easily differentiable on morphological criteria.…”

Section: Discussionmentioning

confidence: 99%

Activation of the inducible form of nitric oxide synthase in the brains of patients with multiple sclerosis.

Bagasra

Michaels²,

Zheng³

et al. 1995

441

234

“…Cellular localization. In both LPS-treated and untreated primary cultures, >95% of the cells could be identified as astrocytes as defined by GFAP staining (Fig 3 a and c), and 3-5% of the cells were identified as microglia by staining with rhodamine-or biotinlabeled lectin RCA-1 (25). Microglial content was not increased by LPS treatment.…”

mentioning

confidence: 84%

“…To detect astrocytes, rabbit anti-bovine GFAP poly- clonal serum was used (23), and for NOS, a rabbit antimacrophage NOS serum (a gift of Q.-W. Xie, H. J. Cho, and C. Nathan) that specifically detects the inducible form of NOS (24). Microglia were visualized by incubating fixed cells with rhodamine-or biotin-labeled RCA-1 (10 gg/ml) for 1 hr at room temperature (25). After removal of the primary antibody or biotin-labeled lectin, cells were visualized by the peroxidase-antiperoxidase method using diaminobenzidine as chromogen (Vector Laboratories).…”

mentioning

confidence: 99%

Induction of calcium-independent nitric oxide synthase activity in primary rat glial cultures.

Galea

Feinstein

Reis

1992

440

302

Exposure of primary cultures of neonatal rat cortical astrocytes to bacterial lipopolysaccharide (LPS) results in the appearance of nitric oxide synthase (NOS) activity. The induction of NOS, which is blocked by actinomycin D, is directly related to the duration of exposure and dose of LPS, and a 2-hr pulse can induce enzyme activity. Cytosol from LPS-treated astrocyte cultures, but not from control cultures, produces a Ca2+-independent conversion of L-arinine to L-Citrulline that can be completely blocked by the specific NOS inhibitor NG-monomethyl-L-arginine. The induced NOS activity exhibits an apparent K. of 16.5 FM for L-arginine and is dependent on NADPH, FAD, and tetrahydrobiopterin. LPS also induces NOS in C6 glioma cells and microglial cultures but not in cultured cortical neurons. The expression of NOS in astrocytes and microglial cells has been confirmed by immunocytochemical staining using an antibody to the inducible NOS of mouse macrophages and by histochemical staining for NADPH diaphorase activity. We conclude that glial cells of the central nervous system can express an inducible form of NOS similar to the inducible NOS of macrophages. Inducible NOS in glia may, by generating nitric oxide, contribute to the neuronal damage associated with cerebral ischemia and/or demyelinating diseases.

“…RCA-1 lectin binds to ␤-Dgalactose residues and labels microglial cells in adult human brain (21); or rabbit anti-ionized calcium binding adapter molecule 1 (Iba1) antibody (dilution 1:400, Imai, Japan (22). Iba1 is specifically expressed in cells of the monocyte͞macrophage lineage, including microglia.…”

Section: Methodsmentioning

confidence: 99%

Human CD34⁺cells differentiate into microglia and express recombinant therapeutic protein

Asheuer

Pflumio

Benhamida

et al. 2004

156

103

In rodents, bone marrow-derived cells enter the brain during adult life. Allogeneic bone marrow transplantation is used to treat genetic CNS diseases, but the fate of human bone marrow and CD34 ؉ cells within the brain remains to be elucidated. The present study demonstrates that cells derived from human CD34 ؉ cells, isolated from either cord blood or peripheral blood, migrate into the brain after infusion into nonobese diabetic͞severe combined immunodeficient mice. Both types of CD34 ؉ -derived cells differentiate into perivascular and ramified microglia. The lentiviral transfer of genes into CD34 ؉ cells before infusion does not modify the differentiation of human CD34 ؉ cells into microglia, allowing new transgenic proteins to be expressed in these cells. The transplantation of CD34 ؉ cells could thus be used for the treatment of CNS diseases. U p to 20% of the total nonneuronal cell population is made of microglia (1). Microglia are ubiquitously distributed in the CNS and play a major role in the response to infectious, traumatic, inflammatory, and ischemic processes, as well as in degenerative CNS diseases, such as Alzheimer's disease, multiple sclerosis, or Parkinson's disease. The adult brain contains two subsets of microglia: the resting microglia, which ramify throughout the brain parenchyma, and the perivascular microglia, which resemble peripheral macrophages (1, 2).Following a long debate about their origin, microglia are now believed to be derived from bone marrow as liver, spleen, or lung macrophages (2). Murine bone marrow-derived cells enter the CNS and differentiate into microglia (3-5). In mice, the turnover of perivascular microglia reaches 30% 1 year after engraftment, whereas the turnover of ramified microglia is much slower (6, 7). However, the subset of bone marrow-derived cells that are the progenitors of microglia has not been characterized. In mice, transplantation of transduced bone marrow cells allows the expression of glucocerebrosidase or GFP in microglia (8, 9). In humans, there are very little data documenting the fate of bone marrow-derived cells in the CNS after bone marrow transplantation (BMT) (10, 11). However, that BMT is used to treat genetic CNS diseases like Hurler disease or X-linked adrenoleukodystrophy (ALD) (12, 13) suggests that bone marrow cells may serve as vehicles for the delivery of genes into the human CNS.Transplantation of CD34 ϩ hematopoietic cells is replacing that of whole bone marrow cells for many applications, including autotransplantation in cancer, non-HLA genoidentical BMT, and gene therapy (14-16). Human CD34 ϩ cells can be easily collected from cord blood at birth or from peripheral blood after cytokine mobilization. We studied the migration, differentiation, and distribution of human CD34 ϩ cells purified either from umbilical cord blood (UCB) or from mobilized peripheral blood (MPB) in the brain of nonobese diabetic͞severe combined immunodeficient (NOD͞SCID) mouse. Our results demonstrate that a fraction of these cells, when infused into NOD͞SCI...