Critical roles for interleukin-4 and interleukin-5 during respiratory syncytial virus infection in the development of airway hyperresponsiveness after airway sensitization.

In mice, respiratory syncytial virus (RSV) infection can enhance the consequences of allergic airway sensitization, resulting in lung eosinophilia and the development of airway hyperresponsiveness (AHR) to inhaled methacholine (MCh). To delineate a role for interleukin-5 (IL-5), interleukin-4 (IL-4), and interferon gamma (IFN-gamma) in mediating the effects of RSV infection on subsequent allergic sensitization, we treated BALB/c mice with anti-IL-5 during acute RSV infection but not during subsequent exposure to ovalbumin (OVA). IL-5-deficient and IL-4-deficient mice were also treated with IL-5 either during acute RSV infection or during the sensitization period. Airway responsiveness to inhaled MCh was assessed and numbers of lung eosinophils were monitored. Anti-IL-5 treatment during RSV infection reduced AHR and lung eosinophilia after subsequent exposure to allergen. In IL-5-deficient or IL-4-deficient mice lung eosinophilia and AHR after RSV infection and allergen exposure were also markedly reduced. IL-5 administration during RSV infection restored the responses to allergen in both IL-5- and IL-4-deficient mice. However, IL-5 administration only during sensitization restored these responses in IL-4-deficient but not in IL-5-deficient animals. IFN-gamma-deficient mice developed AHR and some lung eosinophilia after allergen exposure alone and when RSV infection preceded allergen, these responses were enhanced. We conclude that both IL-5, particularly during acute infection, and IL-4 are critical in mediating the effects of RSV infection on allergic airway sensitization, resulting in the development of AHR and lung eosinophilia.

B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals.

Only mature B lymphocytes can enter the lymphoid follicles of spleen and lymph nodes and thus efficiently participate in the immune response. Mature, long-lived B lymphocytes derive from short-lived precursors generated in the bone marrow. We show that selection into the mature pool is an active process and takes place in the spleen. Two populations of splenic B cells were identified as precursors for mature B cells. Transitional B cells of type 1 (T1) are recent immigrants from the bone marrow. They develop into the transitional B cells of type 2 (T2), which are cycling and found exclusively in the primary follicles of the spleen. Mature B cells can be generated from T1 or T2 B cells. Mice with genetic deletions of elements participating in the B cell receptor signaling cascade display developmental arrest at the T1 or T2 stage. The analysis of these defects showed that the development of T2 and mature B cells from T1 precursors requires defined qualitative and quantitative signals derived from the B cell receptor and that the induction of longevity and maturation requires different signals.

IL-5 and eosinophils are essential for the development of airway hyperresponsiveness following acute respiratory syncytial virus infection.

Viral respiratory infections can cause bronchial hyperresponsiveness and exacerbate asthma. In mice, respiratory syncytial virus (RSV) infection, which induces an immune response dominated by IFN-gamma, results in airway hyperresponsiveness (AHR) and eosinophil influx into the airways, both of which are prevented by pretreatment with anti-IL-5 Ab. To delineate the role of IL-5, IL-4, and IFN-gamma in the development of RSV-induced AHR and lung eosinophilia, we tested the ability of mice deficient in each of these cytokines to develop these symptoms of RSV infection. Mice deficient in either IL-5, IL-4, or IFN-gamma were administered infectious RSV intranasally, and 6 days later, airway responsiveness to inhaled methacholine was assessed by barometric body plethysmography, and numbers of lung eosinophils and production of IFN-gamma, IL-4, and IL-5 by mononuclear cells from peribronchial lymph nodes were monitored. RSV infection resulted in airway eosinophilia and AHR in both IL-4- and IFN-gamma-deficient mice, but not in IL-5-deficient mice. Reconstitution of IL-5-deficient mice with IL-5 restored these responses and enhanced the responses in IL-4-deficient mice. Anti-VLA-4 (very late Ag-4) treatment prevented lung eosinophilia and AHR following RSV infection and IL-5 reconstitution. We conclude that in response to RSV, IL-5 is essential for the influx of eosinophils into the lung and that eosinophils in turn are critical for the development of AHR. IFN-gamma and IL-4 are not essential for these responses to RSV infection.

Porin from the halophilic species Ectothiorhodospira vacuolata: cloning, structure of the gene and comparison with other porins.

The gene coding for the anion-specific porin of the halophilic eubacterium Ectothiorhodospira (Ect.) vacuolata was cloned and sequenced, the first such gene so analyzed from a purple sulfur bacterium. It encodes a precursor protein consisting of 374 amino acid (aa)-residues including a signal peptide of 22-aa residues. Comparison with aa sequences of porins from several other members of the Proteobacteria revealed little homology. Only two regions showed local homology with the previously sequenced porins of Neisseria species, Comamonas acidovorans, Bordetella pertussis, Alcaligenes eutrophus, and Burkholderia cepacia. Genomic Southern blot hybridization studies were carried out with a probe derived from the 5' end of the gene coding for the porin of Ect. vacuolata. Two related species, Ect. haloalkaliphila and Ect. shaposhnikovii, exhibited a clear signal, while the extremely halophilic bacterium Halorhodospira (Hlr.) halophila (formerly Ect. halophila) did not show any cross-hybridization even at low stringency. This result is in good accordance with a recently proposed reassignment within the family Ectothiorhodospiraceae, which included the separation of the extremely halophilic species into the new genus Halorhodospira.

In vivo analysis of the cytoplasmic domain of mIgE antibodies.

All immunoglobulin molecules exist in two forms, one integrated into the plasma membrane and displayed on the cell surface as the B cell receptor [1], the other secreted into tissue fluids as the soluble antibodies. Any given B lymphocyte has the potential of producing both forms. The membrane-bound form and the secreted form produced by a single B cell have identical light chains and identical heavy chains except for a short segment at the C terminus. This segment consists of a 'spacer' sequence, followed by a stretch of hydrophobic amino acids (transmembrane region) and the cytoplasmic region which differs in size between the Ig classes. Little is known about the function of intracellular tails of IgG, IgA and IgE. They differ from those of IgM and IgD, which have only three intracellular amino acids (Lys Val Lys). The intracellular parts of IgG, IgA and IgE are longer. Using a gene targeting technique by homologous recombination in ES cells [2, 3], combined with the prokaryotic CRE-recombinase system [4], we constructed two mice. One with the membrane exons and a mutated cytoplasmic tail in place (KVKdelta tail), and one with essentially only the sequence coding for the secreted form of IgE (delta M1M2). Measurements of the steady-state level of IgE showed that in delta M1M2 mice IgE can only be detected at a minimal level, whereas in KVKdelta tail mice serum IgE is reduced by about 50%. These data allow us to speculate about a specific function of the cytoplasmic tail of mIgE antibodies. We think that the cytoplasmic tail of IgE is involved in signal transduction which leads to the expression of high quantities of secreted IgE.

Effect of transmembrane and cytoplasmic domains of IgE on the IgE response.

B cells use immunoglobulin M (IgM) and IgD as antigen receptors, but after contact with antigen they can switch and use IgG, IgA, or IgE. In mice lacking the transmembrane and cytoplasmic domains of IgE, serum IgE is reduced by more than 95 percent and, after immunization, specific responses are negligible. In mice lacking most of the cytoplasmic tail of IgE, serum IgE levels are reduced by 50 percent and specific responses are reduced by 40 to 80 percent, without a clear secondary response. Thus, membrane expression is indispensable for IgE secretion in vivo, and the cytoplasmic tail influences the degree and quality of the response.

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.

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.

Transitional B cells are the target of negative selection in the B cell compartment.

B lymphocytes recognize antigen through membrane-bound antigen-receptors, membrane IgM and IgD (mIgM and mIgD). Binding to foreign antigens initiates a cascade of biochemical events that lead to activation and differentiation. In contrast, binding to self-antigens leads to death or to inactivation. It is commonly believed that the B cells acquire the ability to discriminate between self and nonself in the early phases of development. We report here that immature B cells, which have just emerged from the mIgMneg, B220pos pool, are not deleted upon binding of self-antigen. In vivo, developing B cells become sensitive to tolerance induction in a relatively late window of differentiation, when they are in transition from the immature (HSAbright, B220dull) to the mature (HSAdull, B220bright) stage. In the transitional B cells, early markers of differentiation such as Pgp1 (CD44) and ThB reach the highest level of expression, while the expression of CD23 and mIgD, late markers of differentiation, and expression of class II MHC, progressively increases. Most of the transitional B cells, but only few of the mature and of the immature B cells, express the fas antigen, while mature B cells, but not immature and transitional B cells, express bcl-2 protein. mIgM is present in low amounts in immature B cells, reaches the highest level of expression in transitional B cells and is down-regulated in mature resting B cells, where it is coexpressed with mIgD. The high expression of mIgM, the presence of the fas antigen and the absence of bcl-2 protein is compatible with the high sensitivity of transitional B cells to negative selection. In vitro, immature B cells die rapidly by apoptosis after cross-linking of mIgM. This result, combined with the resistance of immature B cells to elimination in vivo, suggests that early in development the stroma cell microenvironment modulates signals transduced through mIgM. The functional and phenotypic division of IgMpos bone marrow B cells in three compartments not only allows to define the target population of physiological processes like negative selection, but will also be a helpful tool for an accurate description of possible developmental blocks in mutant mice.

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.

The IgM antigen receptor of B lymphocytes is associated with prohibitin and a prohibitin-related protein.

The two major classes of antigen receptors on murine B lymphocytes, mIgM and mIgD, are both contained in a complex with two additional molecules, Ig-alpha and Ig-beta, which permit signal transduction. Accordingly, early biochemical events after antigen binding to either receptor are similar; biological effects, however, are different. Here, we describe three newly discovered intracellular proteins of 32, 37 and 41 kDa molecular mass, that are non-covalently associated with mIgM, but not with mIgD. These proteins coprecipitate with mIgM in Triton X-100 and Nonidet P-40, but not in digitonin lysates. In addition, mIgM is to some extent associated with 29 and 31 kDa proteins that are predominantly associated with mIgD (see accompanying paper). Amino acid sequencing of p32 and p37 identified p32 as mouse prohibitin; this was corroborated by Western blot analysis with antibodies specific for rat prohibitin. p37 is a newly discovered protein. cDNA clones for both proteins were isolated and sequenced. The deduced amino acid sequence of p32 is identical to that of rat prohibitin. p37 is highly homologous to p32. Since prohibitin was identified as an inhibitor of cell proliferation, its association with mIgM, but not mIgD, could explain the different biological events elicited after engagement of each receptor.

Two new proteins preferentially associated with membrane immunoglobulin D.

The IgM and IgD classes of antigen receptor can perform different functions on B cells. However, so far no class-specific components communicating with the cytoplasm have been found in the two antigen receptors. We have employed a new biotinylation protocol to search for intracellular membrane Ig-associated proteins. Here we describe two proteins of 29 and 31 kDa that are associated with membrane IgD and to some extent with membrane IgM. The membrane IgM molecule is associated specifically with three proteins of 32, 37 and 41 kDa. The purification and sequencing of the two mIgD-associated proteins revealed that they are novel proteins which are related to each other. These proteins may be the missing link between the antigen receptor and the cytoskeleton and may contribute to functional differences between membrane IgM and membrane IgD.

Negative feedback regulation of IgE synthesis by murine CD23.

Immunoglobulin E is found in nanogram amounts in normal human and mouse serum. It is increased during parasitic infestations and mediates allergy. CD23, the low-affinity receptor for IgE (Fc epsilon RII), has been proposed as an important regulator of IgE synthesis. The type-II transmembrane lectin CD23 is expressed in the mouse on B cells and follicular dendritic cells. In humans there are two forms of CD23 which differ in their intracellular amino-terminal 6/7 amino acids; expression of the A-form corresponds to that of murine CD23, whereas the B-form is also found on T and other haematopoietic cells. CD23 has been implicated in cellular adhesion, antigen presentation, as a growth and differentiation factor for human B, T and plasma cells, and as a signal transduction molecule (reviewed in refs 3, 8). Here we disrupt the gene coding for murine CD23 (ref. 9) to clarify the role of CD23 in vivo and find that B- and T-cell development is normal in these CD23-deficient mice. Immune responses to the helminth Nippostrongylus brasiliensis are unaffected. In contrast, immunization with thymus-dependent antigens leads to increased and sustained specific IgE antibody titres compared with controls. Formation of germinal centres is normal. These results suggest that murine CD23 acts as a negative feedback component of IgE regulation.

Immunoglobulin D-deficient mice can mount normal immune responses to thymus-independent and -dependent antigens.

To examine the in vivo function of IgD we generated mice deficient for IgD by gene targeting. The IgD-mice show a reduced B-cell compartment with 30-50% less B cells in the spleen and lymph nodes but show a normal pre-B-cell compartment. The surface-IgD- B cells express two to three times more surface IgM than B cells of control animals. Serum concentrations of the immunoglobulin isotypes of IgD- mice are almost normal, indicating that surface-IgD expression is not necessary for class switching of B cells. Immunization experiments showed that IgD- mice could respond well to thymus-dependent and -independent antigens. After immunization normal germinal centers developed in the IgD- mice. These data suggest that IgD is not necessary for the induction of immune responses but may be important in homeostasis of cells in the B-cell compartment.

Disruption of the murine IL-4 gene blocks Th2 cytokine responses.

Murine T-helper clones are classified into two distinct subsets (Th1 and Th2) on the basis of their patterns of lymphokine secretion. Th1 clones secrete interleukin-2 (IL-2), tumour necrosis factor-beta (TNF-beta) and interferon-gamma (IFN-gamma), whereas Th2 clones secrete IL-4, IL-5 and IL-10 (ref. 1). These subsets are reciprocally regulated by IL-4, IL-10 and IFN-gamma and differentially promote antibody or delayed-type hypersensitivity responses. To evaluate whether IL-4 is required for mounting Th2 responses, we generated IL-4-mutant mice (IL-4-/-) and assessed the cytokine secretion pattern of T cells both from naive and Nippostrongylus brasiliensis infected mice. CD4+ T cells from naive IL-4-/- mice failed to produce Th2-derived cytokines after in vitro stimulation. The levels of Th2 cytokines IL-5, IL-9 and IL-10 from CD4+ T cells obtained after nematode infection were significantly reduced. The reduced IL-5 production in IL-4-/- mice correlated with reduced helminth-induced eosinophilia, which has been shown to be dependent on IL-5 in vivo. We conclude that IL-4 is required for the generation of the Th2-derived cytokines and that immune responses dependent on these cytokines are impaired.