Ontogeny and distribution of the murine B cell Fc gamma RII.

The distribution and expression of the IgG FcRII (Fc gamma RII) on normal murine B cells was examined. Using multicolor flow cytometry, spleens from neonatal mice of increasing age and adult bone marrow were analyzed for expression of the Fc gamma RII. In addition, B cells from peripheral lymphoid organs, as well as panel of B cell tumors, were tested. The results demonstrate that the Fc gamma RII is expressed on all pre-B cells and immature B cells in the neonatal spleen and adult bone marrow, on all mature B cells in peripheral lymphoid organs, and on switched B cells in Peyer's patches. Furthermore, the Fc gamma RII was found to be present on B cell tumors representative of all stages of B cell maturation and differentiation. Taken together, the results indicate that Fc gamma RII is expressed during the entire lifetime of the B cell. In addition, examination of spleen cells from neonatal mice revealed a large number of pre-B cells, phenotypically defined as B220+, IgM-. These pre-B cells were present at birth, peaked in number between 2 and 3 wk of age, and became a minor population by day 30. Further phenotypic analysis of these cells demonstrated the expression of the BLA-1 and BP-1 Ag, and the lack of T cell and NK cell markers, thus confirming their assignment to the B cell lineage. Finally, the Fc gamma RII present on these pre-B cells was shown to be functional, by virtue of its ability to bind aggregated IgG.     

Immunoglobulin heavy chain switch region recombination within a retroviral vector in murine pre-B cells.

We have employed a retroviral vector, ZN(Smu/S gamma 2b)tk1, as a substrate for detecting the presence of immunoglobulin heavy chain constant region (CH) gene switch (S) recombination activity in murine pre-B cells. ZN(Smu/S gamma 2b)tk1 contains a neomycin (neo) resistance gene in addition to the herpes simplex virus thymidine kinase (Htk) gene which is positioned between murine Smu and S gamma 2b sequences. Stable acquisition of the ZN(Smu/S gamma 2b)tk1 vector was selected in G-418 and switch region recombination within these proviruses was selected by resistance to the drug bromodeoxyuridine (BUdR). Fluctuation analyses of ZN(Smu/S gamma 2b)tk1 infected 18-8tk- and 38B9tk- pre-B lines revealed Htk gene inactivations with apparent frequencies of 5 X 10(-5) and 1 X 10(-5) events/cell/generation, respectively, while G-418 resistant Ltk- fibroblasts lost the HTK phenotype at an apparent rate of 4 X 10(-8). Southern blot analysis demonstrated that switch recombination caused the deletion of the Htk gene in all pre-B clones examined while the loss of Htk in Ltk- clones was not mediated by S region recombination. In 21 out of 24 pre-B clones, the recombinations involved the tandemly repetitive portions of the Smu and S gamma 2b sequences. These results demonstrate that the CH gene S region segments inserted into ZN(Smu/S gamma 2b)tk1 are sufficient for B-cell-specific recombination/deletion within the S region tandem repeats.     

Secondary genomic rearrangement events in pre-B cells: VHDJH replacement by a LINE-1 sequence and directed class switching.

We describe rearrangement events which alter expression from a productive VHDJH rearrangement in an Abelson murine leukemia virus-transformed pre-B cell line. One such rearrangement results in replacement of the initially expressed variable region gene by a site-specific join between the open reading frame of a LINE-1 repetitive element and a remaining JH segment. We discuss this event in the context of the 'accessibility' model of recombinase control, and with respect to similar rearrangements involved in oncogene activation. In another subclone of the same pre-B cell line, altered heavy chain expression resulted from a mu to gamma 2b class switch recombination which occurred by a recombination-deletion mechanism but involved a complex inversion. We provide evidence that the germline gamma 2b region is specifically expressed in pre-B cell lines and early in normal development. We propose that the predisposition of pre-B cell lines to switch to gamma 2b production may reflect a normal physiological phenomenon in which the switch event is directed by an increased 'accessibility' of the germline gamma 2b locus to switch-recombination enzymatic machinery. Our findings support the hypothesis that the apparently distinct recombination systems involved in variable region gene assembly and heavy chain class switching are both directed by the accessibility of their substrate gene segments.     

Isotype switching in murine pre-B cell lines

Isotype switching at the pre-B cell stage was studied by employing A-MuLV-transformed cell lines. Two gamma 2b-producing cell lines that did not have other cytoplasmic heavy chains or light chains were established from A-MuLV-transformed cell lines. One clone (SL2-1-52) arose spontaneously from a non-Ig-producing cell line (SL2-1) during in vitro culture. Another clone (AT11-2-24-6-99) underwent isotype switching from a mu-producing cell line (AT11-2-24-6), which had been derived from another non-Ig-producing line (AT11-2). Southern blot analysis of two gamma 2b-producing clones was performed in comparison to that of their respective parent clones. The results showed that isotype switching can operate at the stage of pre-B cells by a CH gene deletion mechanism without utilizing the switch region. In addition, the possibility is presented that deletion of the intervening CH genes could occur prior to the formation of the functional V region-coding DNA segment. This indicates that the prior expression of mu chains is not obligatory for the expression of other isotypes and that isotype commitment could occur in pre-B cells.     

Role of associated gamma-chain in tyrosine kinase activation via murine Fc gamma RIII.

Type III receptors for the Fc portion of IgG (Fc gamma RIII), initially characterized on macrophages and NK cells, are also expressed on several pre-B cell lines. Surface expression of Fc gamma RIII requires the association of the ligand binding alpha-chain with homodimeric gamma-chains. Type II Fc gamma R is homologous to Fc gamma RIII alpha-chain in the extracellular portion and differs in the transmembrane and cytoplasmic domains. The role of Fc gamma R in cell activation was investigated by expressing Fc gamma RIII and the lymphocyte-specific b1 isoform of Fc gamma RII (Fc gamma RIIb1) in an Fc gamma R-negative, sIgG-positive B-cell line. We found that, in contrast to Fc gamma RIIb1, Fc gamma RIII triggers the same events of cell activation as sIG i.e. Ca2+ mobilization, tyrosine phosphorylation and IL-2 secretion. By expressing cytoplasmic domain-lacking Fc gamma RIII alpha-chain in the absence or in the presence of gamma-chains, we demonstrated that cell activation via Fc gamma RIII requires the co-expression of gamma-chains, and is independent of the cytoplasmic portion of the alpha-chain. Furthermore, the cytoplasmic portion of the gamma-chain, fused to the extracellular and transmembrane domains of Fc gamma RII confers on the chimeric receptor the ability to trigger cell activation. Mutation of one tyrosine residue in the cytoplasmic domain of the gamma-chain prevented triggering of cytoplasmic signals. We therefore demonstrate that a tyrosine-containing motif, present in the cytoplasmic domain of the associated gamma-chain, is necessary and sufficient to trigger cell activation via Fc gamma RIII.     

Developmental differences in B cell receptor-induced signal transduction

We have compared early signaling events at various stages of B cell differentiation using established mouse cell lines. Clustering of pre-B cell antigen receptor (BCR) or BCR induced the tyrosine phosphorylation of various proteins in all cells, although the phosphorylation pattern differed. In spite of the pre-BCR-induced tyrosine phosphorylation, we could not detect an intracellular Ca(2+) signal in pre-B cells. However, co-clustering of the pre-BCR with CD19 did induce Ca(2+) mobilization. In contrast to the immature and mature B cells, where the B cell linker protein (BLNK) went through inducible tyrosine phosphorylation upon BCR clustering, we observed a constitutive tyrosine phosphorylation of BLNK in pre-B cell lines. Both BLNK and phospholipase C (PLC)gamma were raft associated in unstimulated pre-B cells, and this could not be enhanced by pre-BCR engagement, suggesting a ligand-independent PLC gamma-mediated signaling. Further results indicate that the cell lines representing the immature stage are more sensitive to BCR-, CD19- and type II receptors binding the Fc part of IgG (Fc gamma RIIb)-mediated signals than mature B cells.     

The pre-B cell receptor signaling for apoptosis is negatively regulated by Fc gamma RIIB.

Many studies have shown that FcgammaRIIB is a negative regulator of B cell receptor signaling, and even though FcgammaRIIB is expressed through all developmental stages of the B cell lineage, its involvement in pre-B cell receptor (pre-BCR) signaling has not been examined. To investigate FcgammaRIIB function at the pre-B cell stage, we have established pre-BCR positive pre-B cell lines from normal mice and FcgammaRIIB-deficient mice, named PreBR and Fcgamma(-/-)PreBR, respectively. These cell lines are able to differentiate into immature B cells in vitro by removal of IL-7. In PreBR, apoptosis was moderately induced by F(ab')(2) anti-mu Ab, but not by intact anti-mu Ab. Phosphorylation of SH2-containing inositol 5-phosphatase (SHIP) and Dok, which are involved in FcgammaRIIB signaling, was induced by anti-mu cross-linking in PreBR. In contrast, apoptosis was strongly induced by both the F(ab')(2) and intact anti-mu Abs in Fcgamma(-/-)PreBR, and the level of phosphorylation of SHIP or Dok was much lower in Fcgamma(-/-)PreBR than those observed in PreBR. Restoration of FcgammaRIIB to Fcgamma(-/-)PreBR followed by anti-mu cross-linking blocked severe apoptosis, and up-regulated SHIP and Dok phosphorylation. The results demonstrate that FcgammaRIIB negatively regulates pre-BCR-mediated signaling for apoptosis.     

A matched set of rat/mouse chimeric antibodies. Identification and biological properties of rat H chain constant regions mu, gamma 1, gamma 2a, gamma 2b, gamma 2c, epsilon, and alpha

Rat C regions mu, gamma 1, gamma 2a, gamma 2b, gamma 2c, epsilon, and alpha have been characterized by means of chimeric antibody technology. A set of rat/mouse Ag-specific (anti-4-hydroxy-3-nitrophenacetyl) antibodies was constructed that differ only in the H chain constant region but carry identical V region and L chain, both of which are of mouse origin. All rat constant regions could be expressed and m.w. were as expected from the protein sequence. A slight variation in mobility within the IgG subclasses allowed us to establish a hierarchy for the sizes of the four gamma H chains; gamma 2b greater than gamma 1 greater than gamma 2c greater than gamma 2a. Rat IgG2c and IgG2b could be purified on both protein A and protein G while rat IgG2a could only be purified on protein G. Rat IgM and IgG2b were the most potent in C-mediated hemolysis. This was not simply a consequence of the amount of C1q bound because IgG2c bound C1q efficiently but was relatively poor in cell lysis. In ADCC using human effector and target cells, IgG2b and IgG1 were the most effective.     

Functional role and immunocytochemical localization of the gamma a and gamma b forms of the Na,K-ATPase gamma subunit

The gamma subunit of the Na,K-ATPase is a member of the FXYD family of type 2 transmembrane proteins that probably function as regulators of ion transport. Rat gamma is present primarily in the kidney as two main splice variants, gamma(a) and gamma(b), which differ only at their extracellular N termini (TELSANH and MDRWYL, respectively; Kuster, B., Shainskaya, A., Pu, H. X., Goldshleger, R., Blostein, R., Mann, M., and Karlish, S. J. D. (2000) J. Biol. Chem. 275, 18441-18446). Expression in cultured cells indicates that both variants affect catalytic properties, without a detectable difference between gamma(a) and gamma(b). At least two singular effects are seen, irrespective of whether the variants are expressed in HeLa or rat alpha1-transfected HeLa cells, i.e. (i) an increase in apparent affinity for ATP, probably secondary to a left shift in E(1) <--> E(2) conformational equilibrium and (ii) an increase in K(+) antagonism of cytoplasmic Na(+) activation. Antibodies against the C terminus common to both variants (anti-gamma) abrogate the first effect but not the second. In contrast, gamma(a) and gamma(b) show differences in their localization along the kidney tubule. Using anti-gamma (C-terminal) and antibodies to the rat alpha subunit as well as antibodies to identify cell types, double immunofluorescence showed gamma in the basolateral membrane of several tubular segments. Highest expression is in the medullary portion of the thick ascending limb (TAL), which contains both gamma(a) and gamma(b). In fact, TAL is the only positive tubular segment in the medulla. In the cortex, most tubules express gamma but at lower levels. Antibodies specific for gamma(a) and gamma(b) showed differences in their cortical location; gamma(a) is specific for cells in the macula densa and principal cells of the cortical collecting duct but not cortical TAL. In contrast, gamma(b) but not gamma(a) is present in the cortical TAL only. Thus, the importance of gamma(a) and gamma(b) may be related to their partially overlapping but distinct expression patterns and tissue-specific functions of the pump that these serve.     

Circulating cellfree Fc gamma 2b/gamma 1 receptor in normal mouse serum: its detection and specificity

By using intact or Fab fragments of rat monoclonal antibodies against murine Fc gamma 2b/gamma 1 receptor, in a solid phase radioimmunoassay, we demonstrated the occurrence of circulating cellfree Fc gamma 2b/gamma 1 receptors (Cf-Fc gamma 2b/gamma 1R) in normal mouse serum. These Cf-Fc gamma 2b/gamma 1R were removed from serum by affinity chromatography by using Sepharose columns coupled with IgG but not by Sepharose coupled with F(ab')2 fragments. Furthermore, the material retained by and eluted from the Sepharose IgG column reacted with the monoclonal antibody; these results support a direct relationship between the antigenic and the functional Cf-Fc gamma 2b/gamma 1R that were detected in serum. This Cf-Fc gamma 2b/gamma 1R was found in all the 39 normal mouse sera that were tested. The results seemed to indicate that aging may be associated with increased levels of Cf-Fc gamma 2b/gamma 1R and that levels of circulating Fc gamma R may be under genetic regulation. By forming complexes with circulating IgG within the blood stream, such Cf-Fc gamma 2b/gamma 1R may modulate some of the functions in which the Fc portion of Ig is involved.     

Allelic exclusion at the TCR delta locus and commitment to gamma delta lineage: different modalities apply to distinct human gamma delta subsets

Expression of a beta-chain, as a pre-TCR, in T cell precursors prevents further rearrangements on the alternate beta allele through a strict allelic exclusion process and enables precursors to undergo differentiation. However, whether allelic exclusion applies to the TCR delta locus is unknown and the role of the gamma delta TCR in gamma delta lineage commitment is still unclear. Through the analysis of the rearrangement status of the TCR gamma, delta, and beta loci in human gamma delta T cell clones, expressing either the TCR V delta 1 or V delta 2 variable regions, we show that the rate of partial rearrangements at the delta locus is consistent with an allelic exclusion process. The overrepresentation of clones with two functional TCR gamma chains indicates that a gamma delta TCR selection process is required for the commitment of T cell precursors to the gamma delta lineage. Finally, while complete TCR beta rearrangements were observed in several V delta 2 T cell clones, these were seldom found in V delta 1 cells. This suggests a competitive alpha beta/gamma delta lineage commitment in the former subset and a precommitment to the gamma delta lineage in the latter. We propose that these distinct behaviors are related to the developmental stage at which rearrangements occur, as suggested by the patterns of accessibility to recombination sites that characterize the V delta 1 and V delta 2 subsets.     

Structure and expression of Fc gamma receptors on mouse suppressor T cell hybridomas

Antigen-specific and idiotype-specific mouse suppressor T cell hybridomas were analyzed for the presence and specificity of Fc gamma receptors (Fc gamma R) by EA rosetting and by flow microfluorometry with the use of monoclonal antibodies. We found that four hybridomas expressed Fc gamma R specific for IgG1 and IgG2b, one of which became Fc gamma R- during prolonged culture. Four other hybridomas and the fusion parent, BW5147, consistently lacked Fc gamma R. The 125I-labeled Fc gamma R were isolated from surface radioiodinated hybridoma cells solubilized with 1% Nonidet P-40, were purified by using single or repetitive chromatography on mouse IgG-Sepharose columns, and were analyzed by SDS-PAGE. An 125I-labeled 56,000 to 61,000 Mr macromolecule was isolated from each of the Fc gamma R+ hybridomas, but from none of the Fc gamma R- hybridomas nor from BW5147 cells. This macromolecule rebound to insolubilized mouse IgG1, IgG2b, and human Fc fragments, but not to insolubilized mouse IgG2a, IgG3, or IgA or human F(ab')2 fragments, consistent with the specificity observed for Fc gamma R on intact hybridoma cells. The mouse suppressor T cell Fc gamma R differs in size and specificity from mouse B cell Fc gamma R. A 70,000 Mr protein expressed on all hybridomas and on BW5147 cells was radiolabeled and, despite preclearing with ovalbumin-Sepharose, bound to the mouse IgG-Sepharose columns, presumably due to mouse antibodies to gp-70. This macromolecule was completely and specifically removed by using goat antiserum to gp-70.     

miR-582 negatively regulates pre-B cell proliferation and survival through targeting Hif1α and Rictor

B cell development in bone marrow (BM) is a multi-staged process involving pro-B, pre-B, immature B, and mature B cells, among which pre-B cells undergo vigorous proliferation, differentiation, apoptosis, and gene rearrangement. While several signaling pathways participate in pre-B cell development have been clarified, detailed intrinsic mechanisms regulating pre-B cell proliferation and survival have not been fully understood. In the current study, we report that miR-582 regulates pre-B cell proliferation and survival. miR-582 is enriched in pre-B cells. Deletion of miR-582 in mice expanded the BM pre-B cell population in a cell-autonomous manner as shown by competitive BM transplantation. We show that forced miR-582 overexpression inhibited pre-B cell proliferation and survival, whereas downregulation of miR-582 by siRNA significantly promoted pre-B cell proliferation and survival in vitro. We identified that Hif1α and Rictor are authentic targets of miR-582 in pre-B cells as shown by reporter assays. Moreover, miR-582 overexpression reduced the expression of Hif1α and its downstream molecule Glut1, as well as Rictor and mTORC2 activity as shown by attenuated AKT and FoxO1 phosphorylation, while miR-582 knockdown showed opposite effects. miR-582 knockdown-induced increases in pre-B proliferation and survival was abrogated by Hif1α and Rictor inhibitors. Together, miR-582 functions as a negative regulator of pre-B cell proliferation and survival by simultaneously targeting Hif1α and mTORC2 signaling that regulates metabolism in early B cell development.Subject terms: Developmental biology, Cell proliferation     

Gene conversion and polymorphism: generation of mouse immunoglobulin gamma 2a chain alleles by differential gene conversion by gamma 2b chain gene

We have determined the complete nucleotide sequence of the C57BL/6 allele of the mouse immunoglobulin gamma 2a chain gene. A comparison with the BALB/c gamma 2a gene for 1912 nucleotides reveals that the two alleles exhibit extensive divergence, since there are 138 single-base-pair differences and 8 insertions or deletions. We have compared the two gamma 2a alleles with the two corresponding gamma 2b alleles, which differ in only 12 positions. It appears that among the 134 differences between the two gamma 2a alleles, 70 are at positions where gamma 2a and gamma 2b are identical in the BALB/c haplotype and 54 are at positions where gamma 2a and gamma 2b are identical in the C57BL/6 haplotype. All these results suggest that nonreciprocal gene conversion between nonallelic genes can introduce sequence homogeneity in linked genes and can generate extensive divergence and polymorphism in allelic genes. We suggest that the gamma 2a and gamma 2b gene ancestors freely diverged after duplication, and that the conversion events were promoted by a deletion shortening the distance between the two loci.     

Properties of the gamma subunit of phosphorylase kinase

Enzymatic properties of the isolated, active gamma subunit of phosphorylase kinase were characterized. Kinetic parameters indicated that the gamma subunit binds the substrates MgATP and phosphorylase b as well as the holoenzyme with a Km (MgATP) of 98 microM and a Km (phosphorylase b) of 80 microM at pH 8.2, but maximal velocities are significantly lower than the holoenzyme's. Unlike the gamma-calmodulin complex, the gamma subunit activity is dependent on pH in the range of pH 6.2-9.0, with a ratio of activity at pH 6.8 to activity at pH 8.2 of 0.5-0.6. Calmodulin activates the gamma subunit more at low pH than at high pH. ADP inhibits the gamma subunit in a competitive manner with a Ki of 60 microM. Free Mg2+ stimulates gamma subunit activity 3.5-fold at both pH 6.8 and 8.2. MnATP is equivalent to MgATP as a substrate for the enzyme, but free Mn2+ inhibits gamma subunit activity. Several protein substrates of holophosphorylase kinase were found also to be phosphorylated by the gamma subunit. These included kappa-casein, myelin basic protein, the troponin complex, and troponin T alone. In the troponin complex, the proportion of 32P incorporated by the gamma subunit into troponin I compared with troponin T was not Ca2+ dependent, but with the holoenzyme, this proportion was changed greatly by Ca2+ concentration.     

Evolution of the rat immunoglobulin gamma heavy-chain gene family

The sequences of the four immunoglobulin gamma heavy chains of the rat (gamma 1, gamma 2a, gamma 2b, gamma 2c) have been determined. These sequences reveal that the rat genes have evolved differently from the closely related mouse gamma genes (gamma 1, gamma 2a, gamma 2b, gamma 3): in rat two of the four genes (gamma 2a and gamma 1) are 94% homologous to each other and best resemble the single mouse gamma 1 gene. Rat gamma 2b is equivalent to the mouse gamma 2a/gamma 2b pair as regards both nucleotide sequence and antibody effector functions whilst rat gamma 2c resembles mouse gamma 3. In evolutionary terms this suggests the existence of a set of three common C gamma genes before separation of rat and mouse as individual species. In addition, two independent duplication events must have occurred after species separation affecting different constant regions; this yielded rat gamma 2a and gamma 1 as a recently evolved pair and mouse gamma 2a and gamma 2b as a different pair. Furthermore, the sequence comparisons reveal several other features of interest; rat IgG2b lacks two amino acids in CH1 which are conserved in all other sequenced gamma chains. Residues believed to be essential for monocyte interaction (FcRI) are retained only in rat gamma 2b and not in the other rat gamma genes whilst a particular motif involved in C1q interaction shows a variation in both rat IgG1 and rat IgG2a which has not been observed previously.