Widespread immunoreactivity for neuronal nuclei in cultured human and rodent astrocytes

The monoclonal antibody (mAb) neuronal nuclei (NeuN) labels the nuclei of mature neurons in vivo in vertebrates. NeuN has also been used to define post-mitotic neurons or differentiating neuronal precursors in vitro . In this study, we demonstrate that the NeuN mAb labels the nuclei of astrocytes cultured from fetal and adult human, newborn rat, and embryonic mouse brain tissue. A non-neuronal fibroblast cell line (3T3) also displayed NeuN immunoreactivity. We confirmed that NeuN labels neurons but not astrocytes in sections of P10 rat brain. Western blot analysis of NeuN immunoreactive species revealed a distribution of bands in nucleus-enriched fractions derived from the different cell lines that was similar, but not identical to adult rat brain homogenates. We then examined the hypothesis that the glial fibrillary acidic protein/NeuN-double positive population of cells might correspond to neuronal precursors. Although the NeuN-positive astrocytes were proliferating, no evidence of neurogenesis was detected. Furthermore, expression of additional neuronal precursor markers was not detected. Our results indicate that primary astrocytes derived from mouse, rat, and human brain express NeuN. Our findings are consistent with NeuN being a selective marker of neurons in vivo , but indicate that studies utilizing NeuN-immunoreactivity as a definitive marker of post-mitotic neurons in vitro should be interpreted with caution.     

A Monoclonal Antibody Recognizing K- but Not γ- and δ-Opioid Receptors

A monoclonal antibody (mAb), KA8 that interacts with the kappa-opioid receptor binding site was generated. BALB/c female mice were immunized with a partially purified kappa-opioid receptor preparation from frog brain. Spleen cells were hybridized with SP2/0AG8 myeloma cells. The antibody-producing hybridomas were screened for competition with opioid ligands in a modified enzyme-linked immunosorbent assay. The cell line KA8 secretes an IgG1 (kappa-light chain) immunoglobulin. The mAb KA8 purified by affinity chromatography on protein A-Sepharose CL4B was able to precipitate the antigen from a solubilized and affinity-purified frog brain kappa-opioid receptor preparation. In competition studies, the mAb KA8 decreased specific [3H]ethylketocyclazocine ([3H]EKC) binding to the frog brain membrane fraction in a concentration-dependent manner to a maximum to 72%. The degree of the inhibition was increased to 86% when mu- and delta-opioid binding was suppressed by 100 nM [D-Ala2,NMe-Phe4,Gly-ol]-enkephalin (DAGO) and 100 nM [D-Ala2,L-Leu5]-enkephalin (DADLE), respectively, and to 100% when mu-, delta-, and kappa 2-sites were blocked by 5 microM DADLE. However, the mu-specific [3H]DAGO and the delta-preferring [3H]DADLE binding to frog brain membranes cannot be inhibited by mAb KA8. These data suggest that this mAb is recognizing the kappa- but not the mu- and delta-subtype of opioid receptors. The mAb KA8 also inhibits specific [3H]naloxone and [3H]EKC binding to chick brain cultured neurons and rat brain membranes, whereas it has only a slight effect on [3H]EKC binding to guinea pig cerebellar membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Susceptibility of astrocytes to class I MHC antigen-specific cytotoxicity

Cell-mediated immune mechanisms contribute to tissue injury within the central nervous system (CNS) in a number of experimental diseases, including experimental allergic encephalomyelitis and some viral infections, and may mediate lesion formation in multiple sclerosis. We investigated the conditions under which murine astrocytes can become susceptible targets of cytotoxic T cells. We demonstrate that mouse astrocytes in vitro can be susceptible targets of class I major histocompatibility complex (MHC)-specific cytotoxicity mediated by L3 cytotoxic T lymphocytes (CTL). Expression of appropriate class I MHC antigen on the astrocytes is a requirement, because only cells bearing the H-2d phenotype are susceptible to lysis by L3 cells. BALB/c-H-2dm2 astrocytes lacking the specific determinant recognized by L3 cells are not susceptible to lysis. Astrocyte lysis can, however, occur under culture conditions in which MHC antigen expression is immunocytochemically low or undetectable. Cytolysis can be inhibited by pretreatment of the effector L3 cells with either anti-Lyt-2 monoclonal antibody (mAb) or anti-clonotypic mAb and by preincubation of the glial target cells with an appropriate anti-H-2 antibody (anti-H-2Ld). mAb to lymphocyte function-associated antigen does not inhibit cytotoxicity of the L3 clone against glial cells. Knowledge regarding the role of CTL within the CNS, including the surface molecules involved in glial cell lysis, could further the development of immunotherapies designed to effect immune reactivity within the CNS.     

Immunohistochemical localization of P-glycoprotein in rat brain and detection of its increased expression by seizures are sensitive to fixation and staining variables.

The MDR1 gene product, P-glycoprotein (P-gp), was shown to confer multidrug resistance to cancer cells, but its overexpression is also suggested to be involved in pharmacoresistance of epilepsy by acting as an energy-dependent drug-efflux pump in the blood-brain barrier (BBB). In normal brain tissue, P-gp is almost exclusively expressed by capillary endothelial cells (EC) of the BBB, whereas little or no expression is detected in other cell types. Increased P-gp expression was observed after seizures, but localization of this increase, i.e., within brain capillary EC or within parenchymal or perivascular astrocytes, which contribute to the BBB function, is controversial. To test whether these antithetic data arise from unusual properties of the antigen itself, we compared different immunohistochemical techniques and monoclonal or polyclonal antibodies to P-gp in normal rat brain and rat brain after kainate-induced seizures. Using acetone-fixed cryostat sections of snap-frozen tissue, strong P-gp labeling was detected in EC and, after seizures, in hippocampal neurons, but not in astrocytes. In contrast, EC and neuronal P-gp immunolabeling were not seen in paraformaldehyde-fixed sections, whereas both perivascular and parenchymal astrocytes exhibited strong P-gp labeling after seizures. The lack of P-gp labeling in EC by paraformaldehyde fixation, was reversed by treatment of the sections with acetate/ethanol. These experiments demonstrate that various fixation conditions have a striking effect on the immunohistochemical localization of P-gp in rat brain and detection of its increased expression by seizures. When data obtained from different immunohistochemical techniques are taken together, seizures seem to induce overexpression of P-gp in four different cell types, i.e., EC, perivascular astrocytes, parenchymal astrocytes, and neurons.     

Oxygen tension regulates the maturation of the blood-brain barrier.

The oxygen tension during the development of vascular systems influences vascular vessel formation through regulating angiogenesis. We studied the effect of hypoxia/reoxygenation (H/R) to explain its role in concert with astrocytes involvement in the development of the blood-brain barrier (BBB). On the basis of the fact that the disappearance of hypoxic regions and the decreased expression of vascular endothelial growth factor (VEGF) were observed by immunohistochemistry in a development-dependent manner in rat cerebral cortex, we examined the effects of astrocytes on the BBB-like properties of ECV304 cells by exposing astrocytes to H/R. Conditioned medium of reoxygenated astrocytes inhibited [(3)H]thymidine incorporation and tube formation of ECV 304 cells. When astrocytes were exposed to reoxygenation, the expression of VEGF was reduced, whereas the expression of angiopoietin-1 and thrombospondin-1 was enhanced. Moreover, [(3)H]sucrose permeability assay revealed that astrocytes enhance the barrier function of ECV 304 cells in coculture model within 5 h of reoxygenation. Correspondingly, the occludin expression of ECV 304 cells was slightly increased by the conditioned medium of reoxygenated astrocytes. In conclusion, our study suggests that reoxygenation of astrocytes may act as a significant driving force for the maturation of the BBB during brain development through oxygen-regulated gene(s).     

Identification and molecular cloning of a novel brain-specific receptor protein that binds to brain injury-derived neurotrophic peptide. Possible role for neuronal survival

Brain injury-derived neurotrophic peptide (BINP) is a synthetic 13-mer peptide that supports neuronal survival and protects hippocampal neurons in primary cultures from cell death caused by glutamate. We have developed a monoclonal antibody named mAb 6A22 against the 40-kDa BINP-binding protein, p40BBP. mAb 6A22 inhibits binding between BINP and rat brain synaptosomes and abolishes the protective effect of BINP. The antigen of mAb 6A22 should be the BINP-binding protein that mediates the neuroprotective action of BINP. Using an expression cloning approach with mAb 6A22, we isolated a cDNA encoding a novel receptor protein that shows binding activity of BINP. COS7 cells transfected with the cloned cDNA show binding of BINP and cell surfaces that are stained by 6A22. The mRNA for p40BBP is specific for the rat brain and is increased after birth. From immunohistochemical studies using mAb 6A22, p40BBP increased after kainic acid treatment in rat hippocampal neurons.     

Fibronectin is expressed by astrocytes cultured from embryonic and early postnatal rat brain

In early primary cultures from newborn rat brain, few glial fibrillary acidic protein (GFAP)-positive glial cells expressed intracytoplasmic immunoreactivity for fibronectin. After the second week in culture, however, fibronectin was expressed by a distinct population of GFAP-positive flat astrocytes, irrespective of which brain region was studied. In cerebellar cultures, these cells were more abundant than in cortical or neostriatal cultures and often formed a major population of the GFAP-positive cells. The difference in fibronectin expression between cerebellum and the other areas studied was statistically significant. When cultures were started from 9-day-old postnatal rat brain, fibronectin-positive astrocytes appeared earlier than in those from newborn animals, in all areas studied. Further, especially in the case of cerebellum, the number of fibronectin-positive astrocytes increased as a function of time in culture. In cultures started from whole brains of 12-day-old rat embryos, fibronectin was expressed within 24 h in culture by all the cells with morphology of flat astrocytes, positive for vimentin but negative for GFAP. These results indicate that astrocytes cultured from newborn and early postnatal rat brain are a heterogeneous population of cells: depending on the brain region studied and also depending on the age of brain tissue or the time in culture, less than 1-60% of the GFAP-positive flat astrocytes expressed fibronectin. This, together with the fact that fibronectin was present in early embryonic brain cells in culture, suggests that fibronectin may be a prerequisite for the development or interactions of brain cells.     

Schwann Cell Marker Defined by a Monoclonal Antibody (224–58) with Species Cross-Reactivity. I. Cellular Localization

We have demonstrated by indirect immunofluorescence the cellular localization of a monoclonal antibody (mAb 224-58), produced after immunization of a mouse with human central nervous system (CNS) myelin. Serologically, mAb 224-58 was found to be specific for 3'-sulfomonogalactosylglycolipids, namely 3'-sulfogalactosylceramide (SGC) and 3'-sulfogalactosyl 1-O-alkyl ether 2-O-acylglycerol (seminolipid). This mAb did not bind to SGC-containing tissues such as kidney, liver, spleen, or brain, nor to muscle. However mAb 224-58 did stain positively mouse, rat, and human peripheral nerve sections. In these latter sections, mAb 224-58 was bound to Schwann cell bodies and processes. The specificity of mAb 224-58 for Schwann cells was ascertained on teased rat sciatic nerves and rat Schwann cell cultures. Cells positive for mAb 224-58 were also positive for laminin, and negative for Thy 1-1 antigens both in teased fibers and Schwann cell cultures. In addition, in teased nerve preparations, mAb 224-58-positive cells were also galactosylceramide (GalC)- and SGC-positive. Isolated Schwann cells also expressed 224-58 antigen, even after prolonged time in culture. On testis sections, which contain both SGC and seminolipid, the SGC-positive cells, i.e., the spermatogonia, were always 224-58-negative. But the other germinal cells were 224-58-positive. This suggests that although 224-58 does not discriminate between SGC and seminolipid in serological tests, these lipids in their naturally occurring membrane acquire a spatial configuration that renders them distinguishable to their respective antibody.     

Cloning, expression, and functional characterization of the substrate binding subunit of rat type II iodothyronine 5'-deiodinase

Type II iodothyronine 5'-deiodinase catalyzes the bioactivation of thyroid hormone in the brain. In astrocytes, this approximately 200-kDa, membrane-bound enzyme is composed of at least one p29 subunit, an approximately 60-kDa, cAMP-induced activation protein, and one or more unidentified catalytic subunit(s). Recently, an artificial type II-like selenodeiodinase was engineered by fusing two independent cDNAs together; however, no native type II selenodeiodinase polypeptide is translated in the brain or brown adipose tissue of rats. These data suggest that the native type II 5'-deiodinase in rat brain is unrelated to this artificial selenoprotein. In this report, we describe the cloning of the 29-kDa subunit (p29) of type II 5'-deiodinase from a lambdazapII cDNA library prepared from cAMP-induced astrocytes. The 3.3-kilobase (kb) cDNA encodes an approximately 30-kDa, 277-amino acid long, hydrophobic protein lacking selenocysteine. Northern blot analysis showed that a 3.5-kb p29 mRNA was present in tissues showing type II 5'-deiodinase activity such as brain and cAMP-stimulated astrocytes. Domain-specific, anti-p29 antibodies specifically immunoprecipitated enzyme activity. Overexpression of exogenous p29 or a green fluorescence protein (GFP)-tagged p29 fusion protein led to a >100-fold increase in deiodinating activity in cAMP-stimulated astrocytes, and the increased activity was specifically immunoprecipitated by anti-GFP antibodies. Steady-state reaction kinetics of the enzyme in GFP-tagged p29-expressing astrocytes are identical to those of the native enzyme in brain. Direct injection of replication-deficient Ad5-p29(GFP) virus particles into the cerebral cortex of neonatal rats leads to a approximately 2-fold increase in brain type II 5'-deiodinating activity. These data show 1) that the 3.3-kb p29 cDNA encodes an essential subunit of rat type II iodothyronine 5'-deiodinase and 2) identify the first non-selenocysteine containing subunit of the deiodinase family of enzymes.     

A rat anti-mouse T4 monoclonal antibody (H129.19) inhibits the proliferation of Ia-reactive T cell clones and delineates two phenotypically distinct (T4+, Lyt-2,3-, and T4-, Lyt-2,3+) subsets among anti-Ia cytolytic T cell clones

Hybridoma H129 .19 was derived by fusion between spleen cells of a Lou / Ws1 rat immunized with an Lyt-1+,2- anti-I-Ak cytolytic T lymphocyte (CTL) clone and the nonsecreting myeloma X63-Ag8.653. The monoclonal antibody (mAb) H129 .19 (IgG2a, kappa) was selected for its capacity to inhibit the lytic potential of the immunizing clone. H129 .19 identified a monomorphic determinant on a 55 m.w. murine T cell differentiation antigen, which appeared to be homologous to the human T4 molecule in that: 1) H129 .19 reacted with 80% adult thymocytes, with a subset of splenic T cells, and with the interleukin 2 (IL 2)-producing EL4 thymoma; 2) The mAb bound to and inhibited the IL 2 production and the proliferation of various allo- or soluble antigen-reactive T cell clones that recognized restriction or activating determinants on the I-A or I-E molecules, respectively; 3) H129 .19 did not inhibit the proliferation and/or cytolysis of Lyt-2,3+ T cells specific for class I MHC antigen; and 4) Among six anti-Iak CTL clones examined in this study, the mAb H129 .19 reacted with two I-Ak-specific, Lyt-2,3- clones on which it exerted strong cytolysis inhibiting effect at the effector cell level. By contrast, two other anti-I-Ak and two anti-I-Ek CTL clones were found to express the Lyt-2,3+,T4- cell surface phenotype. The cytolytic potential of the latter clones was not inhibited by anti-Lyt-2,3 mAb. These studies strongly suggest that the mouse T4 molecule facilitates the recognition of class II MHC antigen by most but not all T cells.     

Identification and characterization of the ligand binding subunit of a kainic acid receptor using monoclonal antibodies and peptide mapping

Monoclonal antibodies (mAb) and a polyclonal antiserum were produced against a kainic acid receptor (KAR) purified from frog brain. Several of the mAb and the antiserum immunoprecipitated [3H]kainic acid binding activity from solubilized preparations of frog brain and labeled a group of proteins on immunoblots that migrated at Mr = 48,000. These results confirm that the ligand binding subunit of the frog brain KAR is contained in the Mr = 48,000 proteins. Immunoblots from different frog tissues demonstrated that the antibody reactivity was highly concentrated in the frog nervous system with no detectable immunoreactivity observed in non-neuronal tissues. The purified KAR was radioiodinated and subjected to two-dimensional gel electrophoresis and autoradiography. A series of proteins was detected at Mr = 48,000 with isoelectric points from 5.5 to 6.3. The anti-KAR mAb and the antiserum reacted with the same group of proteins from frog whole brain after separation by two-dimensional gel electrophoresis. Peptide maps of the 125I-labeled KAR separated by two-dimensional gel electrophoresis demonstrated that the group of proteins clustered at Mr = 48,000 is homologous. mAb KAR-B1 reacted on immunoblots with a protein in rat brain with a Mr = 99,000. This protein comigrated with an unreduced form of the KAR in frog brain. It was present in rat cerebral cortex, hippocampus, and cerebellum but was not detected in thalamus, globus pallidus, or brain stem, nor was it detected in rat non-neuronal tissues. The presence of the Mr = 99,000 immunoreactive polypeptide in discrete areas of rat brain suggests that this protein may be part of a mammalian KAR or a related receptor.     

Characterization of the MRC OX40 antigen of activated CD4 positive T lymphocytes--a molecule related to nerve growth factor receptor.

The antigen recognized by the monoclonal antibody (mAb) MRC OX40 is present on activated rat CD4 positive T lymphocytes but not other cells. cDNA clones were isolated from an expression library using the MRC OX40 mAb and the protein sequence for the OX40 antigen deduced. It contains a typical signal sequence and a single putative transmembrane sequence of 25 predominantly hydrophobic amino acids giving an extracellular domain of 191 amino acids and a cytoplasmic domain of 36 amino acids. The sequence of the extracellular domain includes a cysteine-rich region with sequence similarities with the low affinity nerve growth factor receptor (NGFR) of neurons and the CD40 antigen present on human B cells. Within this region three cysteine-rich motifs can be recognized in OX40 compared with four similar motifs in both NGFR and CD40. OX40, CD40 and NGFR constitute a new superfamily of molecules with expression including lymphoid cells (OX40, CD40) and neuronal cells (NGFR). This is reminiscent of the immunoglobulin superfamily whose molecules are variously found at the surface of lymphoid or brain cells or both.     

Tubulin is a neuronal target of autoantibodies in Sydenham's chorea

Sydenham's chorea is a CNS disorder and sequela of group A streptococcal infection where deposition of Abs in brain may result in movement and neuropsychiatric abnormalities. We studied human mAbs 24.3.1, 31.1.1, and 37.2.1 derived from chorea and selected for cross-reactivity with group A streptococci and brain Ags. Our novel findings reveal that Sydenham's chorea mAbs target a 55-kDa brain protein with an N-terminal amino acid sequence of MREIVHLQ corresponding to beta-tubulin. Chorea mAb specificity for purified brain tubulin was confirmed in ELISA and Western immunoblot, and significant levels of anti-tubulin IgG were found in acute chorea sera and cerebrospinal fluid. Lysoganglioside G(M1) inhibited binding of chorea mAbs to tubulin and mAb reactivity with human caudate and putamen brain sections was blocked by anti-tubulin mAb. The chorea mAbs labeled both intra- and extracellular Ags of a neuronal cell line providing evidence suggesting mimicry between intracellular brain protein tubulin and extracellular lysoganglioside. In addition, chorea mAb 24.3.1 and acute chorea sera induced calcium/calmodulin-dependent protein kinase II activity in human neuronal cells. Nucleotide sequence analysis of the chorea mAb V(H) genes revealed that mAb 24.3.1 V(H) gene was encoded by the V(H)1 germline gene family which encodes other anti-ganglioside V(H) genes associated with motor neuropathies. mAb recognition of tubulin and the neuronal cell surface with initiation of cell signaling and dopamine release supports an emerging theme in autoimmunity whereby cross-reactive or polyreactive autoantibodies against intracellular Ags recognize cell surface epitopes potentially leading to disease.     

Astrocytes in injury states rapidly produce anti-inflammatory factors and attenuate microglial inflammatory responses

Microglia are known to be a primary inflammatory cell type in the brain. However, microglial inflammatory responses are attenuated in the injured brain compared to those in cultured pure microglia. In the present study, we found that astrocytes challenged by oxygen-glucose deprivation (OGD) or H(2) O(2) released soluble factor(s) and attenuated microglial inflammatory responses. Conditioned medium prepared from astrocytes treated with OGD (OGD-ACM) or H(2) O(2) (H(2) O(2) -ACM) significantly reduced the levels of interferon-γ (IFN-γ)-induced microglial inflammatory mediators, including inducible nitric oxide synthase, at both the mRNA and protein levels. The anti-inflammatory effect of astrocytes appeared very rapidly (within 5min), but was not closely correlated with the extent of astrocyte damage. Both OGD-ACM and H(2) O(2) -ACM inhibited STAT nuclear signaling, as evidenced by a reduction in both STAT-1/3 binding to the IFN-γ-activated site and IFN-γ-activated site promoter activity. However, both phosphorylation and nuclear translocation of STAT-1/3 was unchanged in IFN-γ-treated microglia. The active component(s) in OGD-ACM were smaller than 3kDa, and displayed anti-inflammatory effects independent of protein synthesis. These results suggest that, in the injured brain, astrocytes may act as a controller to rapidly suppress microglial activation.     

Interaction of monoclonal antibodies with alpha-bungarotoxin and (-)-nicotine binding sites in goldfish brain. Identification of putative nicotinic acetylcholine receptor subtypes

Monoclonal antibodies raised against the nicotinic acetylcholine receptor of Electrophorus electricus electroplaque have been used as probes to characterize putative nicotinic acetylcholine receptors in goldfish brain. One monoclonal antibody (mAb), mAb 47, recognized a protein which binds both (-)-[3H]nicotine and 125I-alpha-bungarotoxin with high affinity. Another monoclonal antibody (mAb 172) recognized a protein which binds (-)-[3H]nicotine but not 125I-alpha-bungarotoxin. Both antibodies precipitated a protein(s) (biosynthetically labeled with [35S]methionine) in the absence, but not in the presence, of excess purified nicotinic acetylcholine receptor from Torpedo nobiliana. The dilution of mAb 47 that precipitated half of the maximum amount of 125I-alpha-bungarotoxin binding protein was the same as that which precipitated half of the maximum amount of (-)-[3H]nicotine binding activity. When used in combination, the two antibodies precipitated more (-)-[3H]nicotine radioactivity than either antibody alone. The (-)-[3H]nicotine and 125I-alpha-bungarotoxin binding component-mAb complexes were characterized by sucrose density centrifugation. In the presence of either mAb 172 or 47, the (-)-[3H] nicotine binding component migrated further into the gradient, but only mAb 47 shifted the 125I-alpha-bungarotoxin peak. Incubation of solubilized brain extract with alpha-bungarotoxin-coupled Sepharose reduced the amount of (-)-[3H]nicotine radioactivity precipitated by mAb 47 but not by mAb 172. These data suggest that the antibodies may recognize distinct subtypes of (-)-nicotine binding sites in goldfish brain, one subtype which binds both 125I-alpha-bungarotoxin and (-)-[3H]nicotine and a second subtype which binds only (-)-[3H] nicotine.     

Characterization of a monoclonal antibody reactive with a glycolipid antigen expressed by tumorigenic and certain immortalized,non-tumorigenic rat esophageal epithelial cell lines

A monoclonal antibody (mAb 5G) was produced against a tumorigenic rat esophageal epithelial cell line, designated B2T. Using an enzyme-linked immunosorbent assay, immunofluorescence assay (IFA), thin-layer chromatography (TLC) and immunoperoxidase staining, it was found that mAb 5G reacted specifically with a glycolipid antigen expressed by three tumorigenic rat esophageal epithelial cell lines, and two out of the three nontumorigenic, immortalized rat esophageal epithelial cell lines tested; but did not react with primary cultures of normal rat esophageal epithelial cells or fibroblasts. mAb 5G did not bind to rat respiratory tract carcinoma cell lines, to immortalized rat tracheal epithelial cell lines, or to primary cultures of normal rat tracheal epithelial cells. In addition, mAb 5G did not react with any of the human or mouse cell lines tested. In IFA experiments, mAb 5G stained imprints prepared from in vivo propagated B2T tumor tissues, but did not react with normal rat esophageal, tracheal, lung, liver, and kidney tissues. The antigen was identified by TLC as a neutral glycolipid, consisting of two bands, withR _F = 0.45 and 0.41, which migrated in proximity to the ceramide trihexoside standard on TLC plates. Densitometric scanning of the antigen bands indicated that the tumorigenic rat esophageal cell lines possessed 50%–90% more mAb-5G-reactive antigen than the nontumorigenic esophageal cell lines. The results show that mAb 5G reacts specifically with a glycolipid antigen expressed by tumorigenic and certain non-tumorigenic, immortalized rat esophageal epithelial cell lines that might be at the late stages of transformation and early malignancy.     

A Thy-1.1-specific monoclonal alloantibody activates both mouse and rat T cells

In an attempt to further evaluate the role of Thy-1 in the antigen-independent triggering of mouse T cells, we have examined the activating properties of two Thy-1.1-specific mouse monoclonal antibodies (mAb). These reagents were established from an (A.TH X A.TL)F1 hybrid mouse (Thy-1b) immunized with IL-2 producing (BALB/c (Thy-1b) X BW5147 (Thy-1a)) T hybridoma cells. Although both mAb recognized the same Thy-1.1 determinant, one mAb of the gamma 3,kappa class (H171-146) was found to induce several T hybridoma cells to produce IL-2, and AKR thymocytes or cloned helper T cells to proliferate, whereas another mAb of the gamma 1,kappa class (H171-112) failed to do so even in the presence of phorbol myristic acetate (PMA). Increased IL-2 responses of T hybridoma cells were observed when the cell bound Thy-1.1-specific mAb were crosslinked by goat anti-mouse Ig (GaMIg) antibodies. Both a T-cell activating rat anti-Thy-1.2 mAb and the anti-Thy-1.1 mAb H171-146, although directed at distinct cell surface molecules, synergistically stimulated IL-2 production by T hybridoma cells. In addition, the mouse mAb H171-146 was found to stimulate LOU/M rat thymocytes to proliferate in the presence of exogenous IL-2. These data demonstrate that T cells can use Thy-1 as a signal-transducing molecule in both mouse and rat species, and support the notion that the activating properties of Thy-1.1-specific mAb are influenced by their heavy chain isotypes.     

Pathways of dehydroepiandrosterone formation in rat brain glia

In peripheral steroidogenic tissues, dehydroepiandrosterone (D) is formed from pregnenolone (P) by the microsomal cytochrome P450c17 enzyme. Although some steroidogenic P450s have been found in brain tissue, no enzyme has been shown to possess P450c17 activity. We recently demonstrated the presence of an alternative, Fe(2+)-dependent pathway responsible for D formation from alternative precursors in rat glioma cells. We and others could not find P450c17 mRNA and protein in rat brain, but demonstrate herein the presence of Fe(2+)-dependent alternative pathway for D formation in rat brain cortex microsomes. Using primary cultures of differentiating rat glial cells, we observed that P450c17 mRNA and protein were present in O-2A oligodendrocyte precursors and mature oligodendrocytes. In the presence of P, O-2A and mature oligodendrocytes formed D. Addition of Fe(2+) together with submaximal concentrations of P increased D formation by these cells. Treatment of oligodendrocytes with the P450c17 inhibitor SU 10603 in the presence or absence of P failed to inhibit D production. These data suggest that D formation in oligodendrocytes occurs independently of the P450c17 protein present in the cells. In isolated type I astrocytes we did not find neither P450c17 mRNA nor protein. These cells responded to Fe(2+) by producing D and addition of P together with Fe(2+) further increased D synthesis. SU 10603 failed to inhibit D formation by astrocytes. Taken together these results suggest that in differentiating rat brain oligodendrocytes and astrocytes D is formed via a P450c17-independent and oxidative stress-dependent alternative pathway.     

Cellular and subcellular localization of peripheral benzodiazepine receptors after trimethyltin neurotoxicity

The peripheral benzodiazepine receptor (PBR) is currently used as a marker of inflammation and gliosis following brain injury. Previous reports suggest that elevated PBR levels in injured brain tissue are specific to activated microglia and infiltrating macrophages. We have produced hippocampal lesions using the neurotoxicant trimethyltin (TMT) to examine the cellular and subcellular nature of the PBR response. Degenerating, argyrophilic pyramidal neurons were observed in the hippocampus at 2 and 14 days after TMT exposure. Reactive microglia were also evident at both times with a maximal response observed at 14 days, subsiding by 6 weeks. Astrocytosis was observed at 14 days and 6 weeks, but not 2 days, after TMT administration, suggesting that the onset of the astroglia response is delayed, but more persistent, compared with microgliosis. Morphological evidence from [3H]PK11195 microautoradiography and PBR immunohistochemistry indicates that both astrocytes and microglia are capable of expressing high levels of PBR after injury. This was confirmed by double labeling of either Griffonia simplicifolia isolectin B4, a microglial-specific marker, or glial fibrillary acidic protein, an astrocyte-specific protein with PBR fluorescence immunohistochemistry. These results demonstrate that PBR expression is increased after brain injury in both activated microglia and astrocytes. Our findings also provide the first evidence for in situ nuclear localization of PBR in glial cells.