Ontogeny of Ia and IgD on IgM-bearing B lymphocytes in mice.

We used immunofluorescence to examine the developmental relationship of Ia and IgD on B cells. Pre-B cells in fetal liver did not express Ia. Only very few surface IgM-positive (sIgM+) B cells in fetal spleen were found to be Ia+ and were weakly stained for Ia. After birth there was a linear increase in the proportion of sIgM+ spleen cells which expressed Ia, reaching 95% by 9 days. Adult bone marrow also contains a sizeable proportion of sIgM+ Ia- cells. Unstimulated cells from fetal or newborn liver and spleen expressed Ia at the same rate in culture. Anti-Ia antisera suppressed the LPS-induced differentiation of IgM and IgG plasma cells in cultures of neonatal lymphocytes. Ia was also detected on IgM and IgG plasma cells in vitro suggesting that lipopolysaccharide (LPS)-stimulated B cells by may express Ia antigens, induced by LPS, or appearing as part of normal differentiation. IgD did not appear on sIgM+ cells until 3 days of age and then rose slowly to reach adult levels later than Ia antigens.

Myelin-specific proteins and glycolipids in rat Schwann cells and oligodendrocytes in culture.

We have used antibodies to identify Schwann cells and oligodendrocytes and to study the expression of myelin-specific glycolipids and proteins in these cells isolated from perinatal rats. Our findings suggest that only Schwann cells which have been induced to myelinate make detectable amounts of galactocerebroside (GC), sulfatide, myelin basic protein (BP), or the major peripheral myelin glycoprotein (P0). When rat Schwann cells were cultured, they stopped making detectable amounts of these myelin molecules, even when the cells were associated with neurites in short-term explant cultures of dorsal root ganglion. In contrast, oligodendrocytes in dissociated cell cultures of neonatal optic nerve, corpus callosum, or cerebellum continued to make GC, sulfatide and BP for many weeks, even in the absence of neurons. These findings suggest that while rat Schwann cells require a continuing signal from appropriate axons to make detectable amounts of myelin-specific glycolipids and proteins, oligodendrocytes do not. Schwann cells and oligodendrocytes also displayed very different morphologies in vitro which appeared to reflect their known differences in myelinating properties in vivo. Since these characteristic morphologies are maintained when Schwann cells and oligodendrocytes were grown together in mixed cultures and in the absence of neurons, we concluded that they are intrinsic properties of these two different myelin-forming cells.

Heterogeneity of surface immunoglobulin on murine B lymphocytes.

Surface IgM on murine splenic lymphocytes was capped using a rhodamine-coupled anti-mu chain system. The cells were then reacted with a polyspecific, fluorescein-coupled anti-mouse immunoglobulin (Ig) under non-capping conditions. A large number (31-41% of total Ig-bearing cells) which gave red caps with anti-mu subsequently stained green peripherally with anti-Fab under the non-capping conditions. The remaining stained cells were divided between those showing only red caps (17-29% of Ig-bearing cells) and those showing only green rings (37-45% of Ig-bearing cells). These data are interpreted by assuming that most of the staining with the second reagent (fluorescein-coupled anti-mouse Ig) is due to the presence of surface associated IgD.

Location of nucleotide pyrophosphatase and alkaline phosphodiesterase activities on the lymphocyte surface membrane.

1. Isolated mouse spleen lymphocytes hydrolysed UDP-galactose added to the medium. Nucleotide pyrophosphatase activity that accounted for this hydrolysis was enriched to a similar extent as alkaline phosphodiesterase and 5'-nucleotidase in a lymphocyte plasma-membrane fraction. 2. The cell surfaces of mouse spleen and thymus lymphocytes were iodinated with 125I by using the lactoperoxidase-catalysis method. Detergent extracts of the cells were mixed with a purified anti-(mouse liver plasma-membrane nucleotide pyrophosphatase) antiserum and the immunoprecipitates analysed by polyacrylamide-gel electrophoresis. Only one major radioactive component, similar in size (apparent mol.wt 110000-130000) to the liver enzyme, was observed. 3. Electrophoresis of an iodinated spleen plasma-membrane fraction indicated peaks of radioactivity, including one of apparent mol.wt 110000-130000. 4. When detergent extracts of spleen lymphocytes were passed through a Sepharose-bead column containing covalently attached anti-(nucleotide pyrophosphatase) antiserum, the nucleotide pyrophosphatase activity was retained by the beads, whereas protein and leucine naphthylamidase activity were eluted. 5. The results indicate that nucleotide pyrophosphatase and alkaline phosphodiesterase activities are due to the location of the same or similar enzymes at the outer aspect of the lymphocyte plasma membrane. Some possible functions of enzymes at this location are discussed.

Two glial cell lineages diverge prenatally in rat optic nerve.

Three types of glial cells have been previously described in cultures of neonatal rat optic nerve--oligodendrocytes, type 1 astrocytes, and type 2 astrocytes--which can be distinguished using three different antibodies: antigalactocerebroside antibodies recognize oligodendrocytes; antibodies against glial fibrillary acidic protein recognize both types of astrocytes, while the A2B5 monoclonal antibody distinguishes between the two, binding to type 2 but not type 1 astrocytes. It was subsequently shown that oligodendrocytes and type 2 astrocytes, but not type 1 astrocytes, develop in cultures of 7 day optic nerve from a common, A2B5+ progenitor cell. In the present study, the distribution of rat neural antigen-2 (Ran-2), a cell-surface antigen defined by a monoclonal antibody, has been examined on optic nerve cells. It is demonstrated that, in contrast to A2B5, Ran-2 is present on type 1 but not type 2 astrocytes in optic nerve cultures. More importantly, it is shown that Ran-2 and A2B5 antibodies react with largely nonoverlapping populations of cells in cell suspensions of embryonic Day 17 (E17) and postnatal Day 1 (P1) optic nerve, and that the Ran-2+, A2B5- population contains type 1 astrocytes and their precursors while the A2B5+,Ran-2- population contains the progenitor cells for oligodendrocytes and type 2 astrocytes. These findings provide strong evidence that the glial cells of the rat optic nerve develop as two distinct lineages--one giving rise to type 1 astrocytes and the other to oligodendrocytes and type 2 astrocytes--and that the two lineages diverge as early as E17.

Tracing the development of oligodendrocytes from precursor cells using monoclonal antibodies, fluorescence-activated cell sorting, and cell culture.

We have used antibody and complement-mediated cell killing, fluorescence-activated cell sorting and tissue culture to study the development of rat oligodendrocytes. We show that (1) three ligands that bind to the majority of CNS neurons (the monoclonal antibodies A4 and A2B5 and tetanus toxin) also bind to immature oligodendrocytes and to precursor cells in 14-day embryonic rat brain that develop into oligodendrocytes in vitro; and (2) precursor cells in 17- to 18-day embryonic rat optic nerve can develop into oligodendrocytes in vitro in the absence of living neurons.

Structure of mouse Fc receptor.

A variety of mouse cell types were externally labeled with radioactive iodine and solubilized in detergent. A single chain radioactive molecule of mol. wt. 120 000 was precipitated from lysates of surface-labeled lymphocytes, macrophages and fibroblasts by complexes between pneumococcal type 3 polysaccharide and its homologous rabbit antibody in the IgG form, but not as F(ab')2 or Facb. The same results were obtained using an alternative precipitating system, namely ovalbumin and IgG and F(ab')2 forms of rabbit anti-ovalbumin. The 120 000 mol. wt. compound could not be detected on a thymoma cell line (5178) previously known to lack the Fc receptor. On the basis of these criteria the material was therefore identified as mouse Fc receptor. Although very susceptible to proteolysis, the fragments resulting from digestion remain associated by disulfide bonds in such a way as to still bind to the antibody-antigen precipitate. The proteolytic fragmentation products (mol. wts. 75 000, 45 000, 20 000 and 10 000) only become apparent upon chemical reduction, but the ease with which the molecule is degraded explains the wide variation in mol. wts. reported for the Fc receptor, and is perhaps a clue to explain the biological role of the molecule.

Mouse immunoglobulin receptors on lymphocytes: identification of IgM and IgD molecules by tryptic cleavage and a postulated role for cell surface IgD.

The two mouse immunoglobulin receptors on lymphocytes (IgM and IgD-like) were individually digested by trypsin. The tryptic susceptibility, and the products released, were similar to those of their human counterparts. Evidence for a structural homology between human IgD and its presumed mouse conterpart has been provided by the ramarkably similar profile of fragments resulting from digestion. More definitive homology awaits sequence determination. The extreme susceptibility of surface IgD to proteolysis contrasted with the resistance of surface IgM. We therefore propose that the major role of IgD is to release a fragment (Fabdelata) following exposure to antigen and then elicit a regulatory anti-idiotype response which acts through recognition of the protease-resistant IgM idiotype remaining on the cell surface.

Macroglial cell development in embryonic rat brain: studies using monoclonal antibodies, fluorescence activated cell sorting, and cell culture.

Astrocytes, ependymal cells, and oligodendrocytes have been shown to develop on the same schedule in dissociated cell cultures of early embryonic rat brain as in vivo. Subsequent studies showed that there are two major types of astrocyte (type-1 and type-2), which, in cultures of perinatal optic nerve, develop as two distinct lineages. In such cultures, type-2 astrocytes and oligodendrocytes develop from the same, bipotential, (O-2A) progenitor cells, which differentiate into type-2 astrocytes in 10% fetal calf serum (FCS) and into oligodendrocytes in less than or equal to 0.5% FCS. In light of these findings, we now have extended our studies on macroglial cell development in rat brain and show the following: (i) The first astrocytes to develop have a type-1 phenotype, while astrocytes with a type-2 phenotype do not develop until almost 2 weeks later, just as in the optic nerve. (ii) Most importantly, type-2 astrocytes, like the other macroglial cells, develop on the same schedule in cultures of early embryonic (less than or equal to E15) brain as they do in vivo. (iii) By contrast, both oligodendrocytes and type-2 astrocytes develop prematurely in cultures of E17 brain, and FCS influences this development in the same way it does in perinatal optic nerve cultures. (iv) Type-2 astrocyte precursors are labeled by the A2B5 monoclonal antibody, as shown previously for oligodendrocyte precursors in brain and for O-2A progenitor cells in optic nerve. Taken together with our previous findings, these results suggest that oligodendrocytes and type-2 astrocytes in brain develop from bipotential O-2A progenitor cells, whose choice of developmental pathway and timing of differentiation depend on mechanisms that operate independently of brain morphogenesis.

Reconstitution of a developmental clock in vitro: a critical role for astrocytes in the timing of oligodendrocyte differentiation.

The rat optic nerve contains three types of macroglial cells: type 1 astrocytes first appear at embryonic day 16 (E16), oligodendrocytes at birth (E21), and type 2 astrocytes between postnatal days 7 and 10. The oligodendrocytes and type 2 astrocytes develop from a common, bipotential O-2A progenitor cell. We show here that although O-2A progenitor cells in E17 optic nerve prematurely stop dividing and differentiate into oligodendrocytes within 2 days in culture, when cultured on a monolayer of type 1 astrocytes, they continue to proliferate; moreover, the first cells differentiate into oligodendrocytes after 4 days in vitro, which is equivalent to the time that oligodendrocytes first appear in vivo. Our findings suggest that the timing of oligodendrocyte differentiation depends on an intrinsic clock in the O-2A progenitor cell that counts cell divisions that are driven by a growth factor (or factors) produced by type 1 astrocytes.

Two types of astrocytes in cultures of developing rat white matter: differences in morphology, surface gangliosides, and growth characteristics.

Two types of glial fibrillary acidic protein-positive (GFAP+) astrocytes were found in cultures of developing rat optic nerve. Type 1 astrocytes had a fibroblast-like morphology, did not bind tetanus toxin or the monoclonal antibody A2B5 (both of which bind to specific polysialogangliosides), and were stimulated to divide by an extract of bovine pituitary and by epidermal growth factor (EGF). Type 2 astrocytes had a neuron-like morphology, bound tetanus toxin and A2B5 antibody, and were not stimulated to divide by bovine pituitary extract or by EGF. Although both types of astrocytes were present in cultures of white matter, only type 1 astrocytes were found in cultures of gray matter. Astrocytes did not convert from one type to the other in culture: while many type 1 astrocytes adopted a neuron-like morphology when exposed to dibutyryl cyclic adenosine 3':5'-monophosphate, or pituitary or brain extracts, especially in serum-free medium, such morphologically altered cells did not bind tetanus toxin or A2B5 antibody. Although small numbers of tetanus toxin-binding, A2B5+, GFAP+ cells were present in suspensions of freshly dissected, neonatal optic nerves, most of the type 2 astrocytes in cultures of such optic nerves developed from tetanus toxin-binding, A2B5+, GFAP- cells, which were induced to express GFAP by the culture conditions. Since type 2 astrocytes have a neuron-like morphology and bind tetanus toxin and A2B5 antibody, these ligands cannot be used on their own as neuron-specific markers in central nervous system cultures.

Identification of a high molecular weight protein on the surface of murine thymus and thymus-dependent cells.

Rabbit anti-mouse Ig reacted with mouse thymocytes resulting in the formation of caps which were shed into the medium and subsequently injected into rabbits. The antiserum from these animals (AMTP) reacted strongly with thymocytes and peripheral T cells and weakly with B cells. The antiserum did not react via the Thy-1 antigen and could be made specific for T lymphocytes by absorption with B lymphocytes. By surface labeling of lymphocytes with 125I, it could be shown that the major T lymphocyte antigen recognized by AMTP was one, or possibly two, large, single chain molecules with a molecular weight of approximately 200000. This molecule was not Ig and, furthermore, the AMTP did not react with cell surface Ig of B lymphocytes. The implications of this finding for previous reports on the existence of immunoglobulin on T lymphocytes are discussed.

Immunoglobulin M receptors on memory cells of immunoglobulin G antibody-forming cell clones.

The memory cells of two antibody-forming cell clones had receptors of the IgM class, even though the clones had been producing IgG1 or IgG2a anti-2,4-dinitrophenyl antibodies for 9-15 months previously (on exposure to antigen). Thus a phenotypic switch in heavy chain constant region evidently occurred after re-exposure of these memory cells to antigen. To show that, we first removed the clonal cells' surface immunoglobins by "capping" and "stripping", with class- or subclass-specific antisera. Then, to assay their remaining receptor activity, the cells were incubated with antigen in vitro, washed and transferred (together with carrier primed cells) to irradiated recipients, and their antibody responses to this in vitro boost were assayed by iselectric focusing. Pretreatment with anti-mu serum, as well as with anti-Fab(kappa), prevented the responses of the IgG1 and IgG2a clones to an in vitro boost, while anti-gamma1 and anti-gamma2a antisera had no effect. An antiserum to the putative mouse IgD also had no effect. The anti-mu serum failed to react with the IgG1 and IgG2A clonal serum antibodies in the test tube. Some other contaminating clones were suppressed completely only by the anti-Fab serum. This result strongly suggests that switching in class commitment may occur during the differentiation of memory cells to antibody producers, and may therefore be antigen-dependent. It also implies that some apparently naive cells with surface IgM may, in reality, be B memory cells.

A quantitative immunohistochemical study of macroglial cell development in the rat optic nerve: in vivo evidence for two distinct astrocyte lineages.

We have shown previously that three antibodies--anti-galactocerebroside (GC), anti-glial fibrillary acidic protein (GFAP), and the A2B5 monoclonal antibody--can be used to help distinguish three classes of glial cells in the rat optic nerve: oligodendrocytes are GC+, GFAP-, almost all type-1 astrocytes are A2B5-, GFAP+, and almost all type-2 astrocytes are A2B5+, GFAP+. In the present study we have used these antibodies to examine the timing and sequence of the development of the three types of glial cells in vivo. We show that type-1 astrocytes first appear at embryonic Day 16 (E16), oligodendrocytes at birth (E21), and type-2 astrocytes between postnatal Days 7 and 10 (P7-10). Moreover, we demonstrate quantitatively that astrocytes in the optic nerve develop in two waves, with more than 95% of type-1 astrocytes developing before P15 and more than 95% of type-2 astrocytes developing after P15. Finally, we provide indirect evidence that type-2 astrocytes do not develop from type-1 astrocytes in vivo, supporting previous direct evidence that the two types of astrocytes develop from two serologically distinct precursor cells in vitro.

Sequential expression of immunoglobulin on developing mouse B lymphocytes: a systematic survey that suggests a model for the generation of immunoglobulin isotype diversity.

Paired immunofluorescent staining with antibodies specific for the major isotypes of mouse immunoglobulin was used to study the ontogenetic expression of diversity of cell surface immunoglobulin. The first B lymphocytes to emerge, derived from cytoplasmic IgM+ precursors, express sIgM exclusively. Between birth and 3 days of age separate populations of sIgM+ B lymphocyte acquire a second isotype: sIgD, one of the subclasses of sIgG, or sIgA. At 3 days, all splenic B lymphocytes that bear sIg or sIgA also express sIgM, but virtually none stain for sIgD. By 7 days, a substantial porportion of sIgG+ or sIgA+ lymphocytes in spleen and most of those in lymph node express both sIgM ans sIgD. Anti-mu antibody treatment from birth prevented development of B lymphocytes expressing any isotype. These observations suggest that the immature sIgM+ B lymphocyte is the pivotal cell in the generation of the different sublines of B cells and that sIgD ig or IgA. The frequency of lymphocytes bearing only sIgG or sIgA is higher in old than in young mice, suggesting that sIgD and sIgM may be lost after stimulation by antigens. The occurrence of a nearly identical distribution of sIg isotypes on B lymphocytes from athymic, pathogen-free mice suggests that primary expression of isotype diversity does not require T cells.

Comparative surface ultrastructure of adult Onchocerca volvulus recovered from human nodules by dissection or collagenase digestion.

The surface ultrastructure of male and female adult worms of Onchocerca volvulus obtained from human nodules by the technique of collagenase digestion has been compared with that of worms excised manually without the aid of enzyme treatment. No topographical differences have been identified.

Expression of intercellular adhesion molecule-1 and lymphocyte function-associated antigen-3 on human thyroid epithelial cells in Graves' and Hashimoto's diseases.

Human endocrine thyroid epithelial cells (TEC) from autoimmune thyroiditis which express HLA Class II antigens have been shown to present autoantigens to T cells for a TEC-specific immune response. Since the initiation of a specific immune response also involves antigen-receptor independent interactions between accessory molecules, such as lymphocyte function-associated antigen-1 (LFA-1) with intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-3 (LFA-3) with CD2, it was of interest to determine whether TEC can express the adhesion molecules (ICAM-1 and LFA-3) which augment the efficiency of antigen presentation. Cultured TEC were studied for their expression of ICAM-1 and LFA-3 by immunofluorescence. Those derived from Graves' disease expressed these molecules after stimulation with recombinant human interferon-gamma (IFN gamma) or with recombinant human tumour necrosis factor-alpha (TNF alpha). However, using the same stimuli, TEC from non-toxic goitre were induced to express ICAM-1, but not LFA-3. To establish whether ICAM-1 and LFA-3 on TEC were expressed in vivo during the disease process, antibodies against these molecules were incubated with frozen sections of autoimmune thyroiditis, including Graves' and Hashimoto's diseases, and non-toxic goitre. Both ICAM-1 and LFA-3 were highly expressed in the autoimmune diseases, but not in non-toxic goitre. These findings establish that TEC are able to express adhesion molecules and suggest the possible involvement of these adhesion molecules in the TEC-specific immune response in autoimmune thyroiditis.

Is reactive gliosis a property of a distinct subpopulation of astrocytes?

We have shown previously that the A2B5 monoclonal antibody distinguishes two types of glial fibrillary acidic protein-containing astrocytes in semithin frozen sections of adult rat optic nerve: A2B5- (type-1) astrocytes are found mainly at the periphery of the nerve, where they form the glial limiting membrane, while A2B5+ (type-2) astrocytes are found mainly in the interior of the nerve and constitute more than 65% of the astrocytes in the adult optic nerve. In the present study we show that although most astrocytes in semithin frozen sections of adult rat corpus callosum and optic nerve are A2B5+, the great majority of reactive astrocytes in similar sections of corpus callosum examined 20 weeks after a stab lesion, and in optic nerve examined 20 weeks after adult transection, are A2B5-. Although both A2B5+ and A2B5- astrocytes are stimulated to synthesize DNA in the first week after transection, adult optic nerves examined 20 weeks after transection contain only half as many astrocytes as do normal optic nerves: While A2B5+ astrocytes are reduced almost 10-fold, A2B5- astrocytes are increased by about 25%. We consider the simplest interpretation of these findings to be that type-1 astrocytes are largely responsible for forming glial scars in adult white matter following either a stab lesion or Wallerian degeneration and that in transected optic nerves, most type-2 astrocytes eventually die, possibly because they depend on axons for their long-term survival.

Detection of in vivo production of tumour necrosis factor-alpha by human thyroid epithelial cells.

We have established previously that human thyroid epithelial cells (TEC) from patients with autoimmune thyroiditis are able to synthesize cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6). This paper examines TEC in sections from autoimmune thyroiditis for the in vivo production of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) using the combined techniques of in situ hybridization and immunohistochemistry. Thyroid tissue from patients with Graves' disease, Hashimoto's disease and non-toxic goitre was examined and both mRNA and the protein of TNF-alpha were detected in TEC on frozen sections. Representative figures of only Graves' samples are illustrated in this paper. In contrast, using the same methods, IFN-gamma was detected only in the infiltrating cells and not in TEC of thyroid tissue from the patients.