The molecular mechanism of B cell activation by toll-like receptor protein RP-105.

The B cell-specific transmembrane protein RP-105 belongs to the family of Drosophila toll-like proteins which are likely to trigger innate immune responses in mice and man. Here we demonstrate that the Src-family protein tyrosine kinase Lyn, protein kinase C beta I/II (PKCbetaI/II), and Erk2-specific mitogen-activated protein (MAP) kinase kinase (MEK) are essential and probably functionally connected elements of the RP-105-mediated signaling cascade in B cells. We also find that negative regulation of RP-105-mediated activation of MAP kinases by membrane immunoglobulin may account for the phenomenon of antigen receptor-mediated arrest of RP-105-mediated B cell proliferation.

The B-cell-specific Src-family kinase Blk is dispensable for B-cell development and activation.

The B-cell lymphocyte kinase (Blk) is a src-family protein tyrosine kinase specifically expressed in B-lineage cells of mice. The early onset of Blk expression during B-cell development in the bone marrow and the high expression levels of Blk in mature B cells suggest a possible important role of Blk in B-cell physiology. To study the in vivo function of Blk, mice homozygous for the targeted disruption of the blk gene were generated. In homozygous mutant mice, neither blk mRNA nor Blk protein is expressed. Despite the absence of Blk, the development, in vitro activation, and humoral immune responses of B cells to T-cell-dependent and -independent antigens are unaltered. These data are consistent with functional redundancy of Blk in B-cell development and immune responses.

The level of C/EBP protein is critical for cell migration during Drosophila oogenesis and is tightly controlled by regulated degradation.

The C/EBP transcription factor, Slbo, is required for migration of border cells during Drosophila oogenesis. Unexpectedly, we find that neither increase nor decrease of Slbo activity is tolerated in border cells. Correct protein level is in part ensured by cell type-specific regulated turnover of Slbo protein. Through genetic screening, we identify two genes that are involved in this regulation. The Ubp64 ubiquitin hydrolase acts as a stabilizer of Slbo protein. A novel gene, tribbles, is a negative regulator of slbo in vivo. Tribbles acts by specifically targeting Slbo for rapid degradation via ubiquitination.

Expression of human lymphocyte IgE receptor (Fc epsilon RII/CD23). Identification of the Fc epsilon RIIa promoter and its functional analysis in B lymphocytes.

Two species, Fc epsilon RIIa and Fc epsilon RIIb, of the human low-affinity receptor for IgE (Fc epsilon RII/CD23) have recently been described. They differ by only six amino acids in the cytoplasmic N-terminus and are generated by different cell-specific transcriptional start sites that lead to distinct 5'-leader sequences in the corresponding mRNA. In this study, we present the analysis of the promoter which is regulating the expression of the B cell-specific Fc epsilon RIIa. Our data show that this promoter is flanked by several long repetitive elements that are influencing transcription in the Burkitt lymphoma B cell line Jijoye. Serial deletions of the 5'-flanking region of the promoter revealed two major regulatory segments that have either inhibitory or enhancing effects on transcription. In addition, IL-4 caused a two- to four-fold up-regulation of the Fc epsilon RIIa promoter activity and the DNA element responsible was mapped within the first 250 bp of the 5'-flanking region. These results were confirmed by transferring the DNA segment containing the putative IL-4 responsive element to a heterologous thymidine kinase promoter. It was concluded that IL-4 augments the Fc epsilon RIIa expression by transcriptional regulation.

Transgene CD23 expression on lymphoid cells modulates IgE and IgG1 responses.

Transmembrane (m) and soluble (s) forms of CD23 perform activities related to various immune functions. Abnormal expression patterns of CD23 on lymphoid cells have been associated with certain pathologic conditions. To explore the effects of CD23 when it is overexpressed, on lymphoid cell development and immune function in vivo, transgenic mice were generated. These mice overexpressed either mCD23 or a 38-kDa molecular form of sCD23. Transgene expression under the control of Thy-1-regulatory sequences and the mouse Ig heavy chain enhancer (E mu) was prominent in thymus, spleen, bone marrow, and lymph nodes. Cells that expressed the transgenes included most thymocytes, peripheral CD4+ and CD8+ T cells, IgM+/highIgD-/low immature B cells, and IgMlowIgDhigh mature B cells. To resolve the expression pattern in the B cell lineage unambiguously, we used mice that carried a transgene and a disrupted endogenous CD23 gene simultaneously. Neither mCD23 nor sCD23 overexpression caused significant alterations in lymphoid cell maturation. In addition, basal serum levels of IgE and IgG1 proved to be normal. In three different experimental immune response paradigms, mCD23 transgenic, but not sCD23 and nontransgenic mice proved to be impaired in increasing serum levels of polyclonal IgE up to expected levels. In addition, mCD23 transgenic mice showed below normal increases of serum IgG1 levels in two of the three immune responses. In the presence of activated T cells and appropriate lymphokines, B cells from mCD23 mice secreted normal amounts of IgE and IgG1 in vitro, which suggests that there was no serious impairment of the T-B cell contact required for Ig production. In addition, there is no evidence for a significant role of mCD23 in IgE clearance. Therefore, we discuss alternative mechanisms by which mCD23+ B and/or T cells influence Ig production.

Immunoglobulin E-binding site in Fc epsilon receptor (Fc epsilon RII/CD23) identified by homolog-scanning mutagenesis.

The IgE-binding site of the human low-affinity receptor for IgE (Fc epsilon RII/CD23) has previously been mapped to the extracellular domain between amino acid residues 160 and 287. We now have investigated which conformational epitope within this domain specifies the receptor-ligand interaction. The analysis of homolog-scanning mutants expressed in mammalian cells demonstrates that amino acid side chains that affect IgE binding are located in two discontinuous segments, between residues 165-190 and 224-256. The overall structure of the chimeric binding domains, as probed with 11 conformation-sensitive monoclonal antibodies, is generally not distorted, except by replacement of residues 165-183. In this region, disruption of binding function appears to be caused by global conformational constraints on the binding site. Substitution and deletion mutants demonstrate that six out of eight extracellular cysteines, Cys163, Cys174, Cys191, Cys259, Cys273, and Cys282, are necessary for IgE binding and are most likely involved in intramolecular disulfide bridges. We show that the Fc epsilon RII domain delineated by Cys163 and Cys282 encodes all the structural information required to form the IgE-binding site.

Impaired cerebral cortex development and blood pressure regulation in FGF-2-deficient mice.

Fibroblast growth factor-2 (FGF-2) has been implicated in various signaling processes which control embryonic growth and differentiation, adult physiology and pathology. To analyze the in vivo functions of this signaling molecule, the FGF-2 gene was inactivated by homologous recombination in mouse embryonic stem cells. FGF-2-deficient mice are viable, but display cerebral cortex defects at birth. Bromodeoxyuridine pulse labeling of embryos showed that proliferation of neuronal progenitors is normal, whereas a fraction of them fail to colonize their target layers in the cerebral cortex. A corresponding reduction in parvalbumin-positive neurons is observed in adult cortical layers. Neuronal defects are not limited to the cerebral cortex, as ectopic parvalbumin-positive neurons are present in the hippocampal commissure and neuronal deficiencies are observed in the cervical spinal cord. Physiological studies showed that FGF-2-deficient adult mice are hypotensive. They respond normally to angiotensin II-induced hypertension, whereas neural regulation of blood pressure by the baroreceptor reflex is impaired. The present genetic study establishes that FGF-2 participates in controlling fates, migration and differentiation of neuronal cells, whereas it is not essential for their proliferation. The observed autonomic dysfunction in FGF-2-deficient adult mice uncovers more general roles in neural development and function.

Mice deficient in CD23 reveal its modulatory role in IgE production but no role in T and B cell development.

To assess roles of CD23 in lymphocyte development and immune function in vivo, CD23-deficient mice (CD23-/-) were generated. Mice heterozygous with respect to the defective allele (CD23+/-) display 50% reduced levels of CD23 expression on CD23+ cell types. This pattern is consistent with a lack of parental or tissue-specific imprinting of the CD23 gene. Neither a 50% reduced level nor a complete lack of CD23 caused profound changes in lymphocyte compartments (thymocytes, peripheral T cells, and B-1 and B-2 B cells). The lack of CD23 also did not significantly alter in vitro the proliferative response of B cells triggered via the Ag receptor in combination with CD40 ligand, IL-2, and/or IL-4. Effects on polyclonal Ig production were tested in a Th2 cell-driven immune response in vivo after infection with Nippostrongylus brasiliensis, a parasite that dramatically enhances CD23 expression on B cells. In both primary and secondary immune responses, heterozygous CD23+/- mice developed slightly higher and CD23-/- mice similar serum IgE and IgG1 levels as compared with CD23+/+ (wild-type) mice. The increase in blood eosinophil counts was similar in all three types of mice. These findings show that after N. brasiliensis infection 1) a complete lack of CD23 in vivo neither prohibits nor significantly alters quantitatively polyclonal IgE levels in serum, and 2) a 50% reduction in cell-surface CD23 expression (CD23+/- mice) correlates with slightly increased serum IgE levels.

Mice reconstituted with DNA polymerase beta-deficient fetal liver cells are able to mount a T cell-dependent immune response and mutate their Ig genes normally.

The ubiquitously expressed, error-prone DNA polymerase beta (polbeta) plays a role in base excision repair, and the involvement of this molecule in the nonhomologous end joining (NHEJ) process of DNA repair has recently been demonstrated in yeast. Polbeta-deficient mice are not viable, and studies on conditional mutants revealed a competitive disadvantage of polbeta(-/-) vs. wild-type cells. We show here that polbeta-deficient mice survive up to day 18.5 postcoitum, but die perinatally; a circumstance that allowed the investigation of a potential role of polbeta in lymphocyte development by transfer of fetal liver cells (FLC) derived from polbeta(-/-) embryos into lethally irradiated hosts. FLC transfers using mutant cells lead to an almost normal reconstitution of the lymphocyte compartment, indicating that polbeta-deficiency does not prevent V(D)J recombination, which is known to employ factors of the NHEJ pathway. Mice reconstituted with polbeta(-/-) FLC mount a normal T cell-dependent immune response against the hapten (4-hydroxy-3-nitrophenyl) acetyl (NP). Moreover, germinal center B cells from NP-immunized reconstituted mice show normal levels and patterns of somatic point mutations in their rearranged antibody genes, demonstrating that polbeta is not critically involved in somatic hypermutation.