Marinus C. Lamers

CD22 is a negative regulator of B-cell receptor signalling

BACKGROUND . Antibody responses are triggered by binding of antigen to the B-cell antigen receptor (BCR). The strength of the resulting signal determines the outcome of the response, which may vary from the induction of tolerance to the antigen, to the production of specific high-affinity antibodies. Additional cell-surface proteins assist the BCR in its function, and can facilitate or inhibit an antibody response. CD22 is a BCR-associated transmembrane protein, the cytoplasmic tail of which contains three immunoreceptor tyrosine-based inhibitory motifs. These motifs are phosphorylated upon BCR-crosslinking, and can bind the tyrosine phosphatase SHP-1, a putative negative regulator of signalling from the BCR. In order to assess the role of CD22 in vivo, we have generated CD22(-/-) mice by targeted gene inactivation. RESULTS . In CD22(-/-) mice, B-cell development is normal. There are normal numbers of peripheral B cells, but these have a more mature phenotype. In addition, recirculating B cells are absent from the bone marrow. However, the distribution of the two B-cell subtypes, B-1 and B-2, is normal. After BCR-crosslinking in vitro, splenic CD22(-/-) B cells show an increased Ca2+ influx and a lower survival due to an increased induction of apoptosis. In contrast, there is an increased proliferative response to the B-cell mitogen lipopolysaccharide (LPS). A shorter average lifespan in the B-cell compartment is also found in vivo. Furthermore, T-cell independent immune responses are impaired, whereas T-cell dependent responses are normal. CONCLUSIONS . The absence of CD22 expression lowers the signalling threshold for BCR-crosslinking and can thus influence the fate of the B cell. We propose that the low threshold leads to hyperresponsiveness of the B cells and a chronic basal activation. In this model, engagement of the receptor without T-cell help leads to an increased induction of apoptosis, thus explaining the shorter lifespan of CD22(-/-) B cells and the low response to T-cell independent antigens. The alteration in B-cell phenotype and the higher levels of LPS-reactivity are attributable to the chronic basal stimulation.

Direct Toll-like receptor 2 mediated co-stimulation of T cells in the mouse system as a basis for chronic inflammatory joint disease

The pathogenesis of chronic inflammatory joint diseases such as adult and juvenile rheumatoid arthritis and Lyme arthritis is still poorly understood. Central to the various hypotheses in this respect is the notable involvement of T and B cells. Here we develop the premise that the nominal antigen-independent, polyclonal activation of preactivated T cells via Toll-like receptor (TLR)-2 has a pivotal role in the initiation and perpetuation of pathogen-induced chronic inflammatory joint disease. We support this with the following evidence. Both naive and effector T cells express TLR-2. A prototypic lipoprotein, Lip-OspA, from the etiological agent of Lyme disease, namely Borrelia burgdorferi, but not its delipidated form or lipopolysaccharide, was able to provide direct antigen-nonspecific co-stimulatory signals to both antigen-sensitized naive T cells and cytotoxic T lymphocyte (CTL) lines via TLR-2. Lip-OspA induced the proliferation and interferon (IFN)-γ secretion of purified, anti-CD3-sensitized, naive T cells from C57BL/6 mice but not from TLR-2-deficient mice. Induction of proliferation and IFN-γ secretion of CTL lines by Lip-OspA was independent of T cell receptor (TCR) engagement but was considerably enhanced after suboptimal TCR activation and was inhibitable by monoclonal antibodies against TLR-2.

The riddle of the dual expression of IgM and IgD.

Signalling through the B cell antigen receptor (BCR) is required for peripheral B lymphocyte maturation, maintenance, activation and silencing. In mature B cells, the antigen receptor normally consists of two isotypes, membrane IgM and IgD (mIgM, mIgD). Although the signals initiated from both isotypes differ in kinetics and intensity, in vivo, the BCR of either isotype seems to be able to compensate for the loss of the other, reflected by the mild phenotypes of mice deficient for mIgM or mIgD. Thus, it is still unclear why mature B cells need expression of mIgD in addition to mIgM. In the current review we suggest that the view that IgD has a simpIy definable function centred around the basic signalling function should be replaced by the assumption that IgD fine tunes humoral responses, modulates B cell selection and homeostasis and thus shapes the B cell repertoire, defining IgD to be a key modulator of the humoral immune response.

Inefficient processing of mRNA for the membraneform of IgE is a genetic mechanism to limit recruitment of IgE-secreting cells

Immunoglobulin E (IgE) is the key effector element in allergic diseases ranging from innocuous hay fever to life‐threatening anaphylactic shock. Compared to other Ig classes, IgE serum levels are very low. In its membrane‐bound form (mIgE), IgE behaves as a classical antigen receptor on B lymphocytes. Expression of mIgE is essential for subsequent recruitment of IgE‐secreting cells. We show that in activated, mIgE‐bearing B cells, mRNA for the membrane forms of both murine and human epsilon (ϵ) heavy chains (HC) are poorly expressed compared to mRNA for the secreted forms. In contrast, in mIgG‐bearing B cells, mRNA for the membrane forms of murine gamma‐1 (γ1) and the corresponding human γ4 HC are expressed at a much higher level than mRNA for the respective secreted forms. We show that these findings correlate with the presence of deviant polyadenylation signal hexamers in the 3′ untranslated region (UTR) of both murine and human ϵ genes, causing inefficient processing of primary transcripts and thus poor expression of the proteins and poor recruitment of IgE‐producing cells in the immune response. Thus, we have identified a genetic steering mechanism in the regulation of IgE synthesis that represents a further means to restrain potentially dangerous, high serum IgE levels.

Targeting the extracellular membrane-proximal domain of membrane-bound IgE by passive immunization blocks IgE synthesis in vivo.

The classical allergic reaction starts seconds or minutes after Ag contact and is committed by Abs produced by a special subset of B lymphocytes. These Abs belong to the IgE subclass and are responsible for Type I hyperreactivity reactions. Treatment of allergic diseases with humanized anti-IgE Abs leads primarily to a decrease of serum IgE levels. As a consequence, the number of high-affinity IgE receptors on mast cells and basophils decreases, leading to a lower excitability of the effector cells. The biological mechanism behind anti-IgE therapy remains partly speculative; however, it is likely that these Abs also interact with membrane IgE (mIgE) on B cells and possibly interfere with IgE production. In the present work, we raised a mouse mAb directed exclusively against the extracellular membrane-proximal domain of mIgE. The interaction between the monoclonal anti-mIgE Ab and mIgE induces receptor-mediated apoptosis in vitro. Passive immunization experiments lead to a block of newly synthesized specific IgEs during a parallel application of recombinant Bet v1a, the major birch pollen allergen. The decrease of allergen-specific serum IgE might be related to tolerance-inducing mechanisms stopping mIgE-displaying B cells in their proliferation and differentiation.

Migration of antibody secreting cells towards CXCL12 depends on the isotype that forms the BCR

Truncation of the cytoplasmic tail of membrane‐bound IgE in vivo results in lower serum IgE levels, decreased numbers of IgE‐secreting plasma cells and the abrogation of specific secondary immune responses. Here we present mouse strain KN1 that expresses a chimeric ε‐γ1 BCR, consisting of the extracellular domains of the ε gene and the transmembrane and cytoplasmic domains of the γ1 gene. Thus, differences in the IgE immune response of KN1 mice reflect the influence of the “γ1‐mediated signalling” of mIgE bearing B cells. KN1 mice show an increased serum IgE level, resulting from an elevated number of IgE‐secreting cells. Although the primary IgE immune response in KN1 mice is inconspicuous, the secondary response is far more robust. Most strikingly, IgE‐antibody secreting cells with “γ1‐signalling history” migrate more efficiently towards the chemokine CXCL12, which guides plasmablasts to plasma cell niches, than IgE‐antibody secreting cells with WT “ε‐signalling history”. We conclude that IgE plasmablasts have an intrinsic, lower chance to contribute to the long‐lived plasma cell pool than IgG1 plasmablasts.

The Upstream Regulatory Region of the Carbamoyl-phosphate Synthetase I Gene Controls Its Tissue-specific, Developmental, and Hormonal Regulation in Vivo

The carbamoyl-phosphate synthetase I gene is expressed in the periportal region of the liver, where it is activated by glucocorticosteroids and glucagon (via cyclic AMP), and in the crypts of the intestinal mucosa. The enhancer of the gene is located 6.3 kilobase pairs upstream of the transcription start site and has been shown to direct the hormone-dependent hepatocyte-specific expression in vitro. To analyze the function of the upstream region in vivo, three groups of transgenic mice were generated. In the first group the promoter drives expression of the reporter gene, whereas the promoter and upstream region including the far upstream enhancer drive expression of the reporter gene in the second group. In the third group the far upstream enhancer was directly coupled to a minimized promoter fragment. Reporter-gene expression was virtually undetectable in the first group. In the second group spatial, temporal, and hormonal regulation of expression of the reporter gene and the endogenous carbamoyl-phosphate synthetase gene were identical. The third group showed liver-specific periportal reporter gene expression, but failed to activate expression in the intestine. These results show that the upstream region of the carbamoyl-phosphate synthetase gene controls four characteristics of its expression: tissue specificity, spatial pattern of expression within the liver and intestine, hormone sensitivity, and developmental regulation. Within the upstream region, the far upstream enhancer at −6.3 kilobase pairs is the determinant of the characteristic hepatocyte-specific periportal expression pattern of carbamoyl-phosphate synthetase.

Regulation of IgE Synthesis. Lessons from the Study of IgE Transgenic and CD23‐deficient Mice

Allergy could be defined as an inappropriate response of the immune system to an in principle hamiless challenge from environmental antigens: the hair of a cat, the excrement ol a house mite or the pollen of grasses and trees. The inappropriateness lies in the class of antibodies that dominates the response, namely IgE antibodies. In normal, healthy individuals, the frequency of IgE-producing cells is low. and IgE is present in very low quantities in .serum. In mice, the serum levels of laboratory strains range from 20()-3()() nanograms to a few micrograms per ml. depending on strain and age. Serum IgE has a very short half-life (Haba et al. 1985). IgE binds to IgE receptors, of which two classes are known-the high-and the low-affinity receptor and to other structures, e.g. FcYReceptors (Takizawa et al. 1992) and Mac-2 (Drickamer 1988). Bound to the IgE receptor, in particular to Ihe high-affinity receptor, the life span of an IgE molecule is much longer. An allergic reaction results normally from the cross-linking of IgE antibodies bound to the high-affinity receptor on mast cells or on basophilic granulocytes by the specific allergen. Following this event, which can take place years after the initial contact with the allergen, vasoactive suhstances are released causing the local or generalized inflammatory symptoms that are characteristic of the allergic reaction. Estimates of the number of people suffering from allergic disorders in the Western worid range from 5-15%, and are tending to increase. The reasons for

Prohibitin and prohibitone are contained in high-molecular weight complexes and interact with α-actinin and annexin A2

The closely related proteins prohibitin (p32) and prohibitone (p37) are evolutionarily conserved with homologues found from cyanobacteria to man. They are thought to be exclusively mitochondrial and have been assigned many-rather different-functions, ranging from a role in lifespan, in mitochondrial inheritance and as chaperones of mitochondrial proteases in yeast. Evidence for a localisation outside of mitochondria has been brought forward in mammalian cells, where they influence cell-cycle progression and are found in association with cell surface receptors. We have employed a yeast two-hybrid screen to identify other interacting proteins and have identified alpha-actinin and annexin A2 as binding partners for prohibitin and prohibitone. Coprecipitation experiments supported the putative binding between prohibitin and prohibitone on the one hand and annexin A2 or alpha-actinin on the other hand in intact cells. Surface plasmon resonance analysis was used to determine relative affinities between prohibitin and alpha-actinin and between prohibitone and annexin A2 and alpha-actinin, respectively. We further show that prohibitin and prohibitone can also form homomeric (preferentially tetrameric) and heteromultimeric complexes, with significant affinities.

B-cell maturation in chimaeric mice deficient for the heat stable antigen (HSA/mouse CD24)

The murine differentiation marker heat stable antigen (HSA) is a GPI-anchored surface glycoprotein showing strong expression on immature B- and T-lymphocytes and gradually reduced expression during maturation. Although HSA has been suggested to be involved in adhesion and/or signalling, its function has not been clearly demonstrated so far. In order to elucidate the function of HSA, we analysed chimaeric mice that were generated by targeted disruption of both HSA alleles in ES cells. These mice contain normal numbers of peripheral B-cells and normal serum IgM and IgG titres of ES cell-derived allotype, demonstrating that HSA expression on B-cells is not an absolute requirement for their maturation. However, a reduction in immature B-cells in the bone marrow and an altered degree of bone marrow and blood chimaerism suggest that HSA expression influences the maturation of B-cells.