The development of IgE+ memory B cells following primary IgE immune responses.

We studied whether long-lived IgE+ memory B cells develop following three types of primary IgE immune responses. Immunization of mice with anti-IgD antibody induced a T cell-dependent, interleukin (IL)-4-dependent primary IgE response and the formation of IgE isotype switched (IgE+) memory B cells. These IgE+ memory B cells could be stimulated in vivo by injection with goat anti-IgE antibodies to produce a profound IL-4-independent memory IgE response. By contrast, both infection of mice with Nippostrongylus brasiliensis or repeated immunization with benzylpenicilloyl-keyhole limpet hemocyanin (BPO-KLH) in alum stimulated good primary IgE responses and profound memory T cell-dependent antigen-specific IgE responses, but failed to induce the development of long lived IgE+ memory B cells because they could not be recalled with goat anti-IgE antibodies. Mice receiving double immunizations combining anti-IgD with either N. brasiliensis infection or BPO-KLH immunization mounted significant goat anti-IgE-induced secondary IgE responses, but no N. brasiliensis or BPO-KLH-specific IgE could be detected. This indicates that the N. brasiliensis and BPO-KLH induced immune responses do not suppress the development of IgE+ B cells, but rather, do not provide the necessary conditions for their formation. Taken together these data indicate that long-lived IgE+ B cells fail to develop during the primary IgE response to N. brasiliensis infection or BPO-KLH immunization. By contrast, significant numbers of IgE+ memory B cells form during the primary IgE immune response induced by anti-IgD immunization. Our observations suggest that immunization protocols involving membrane IgD cross-linking and limited duration of cognate T cell help are necessary for the formation of IgE+ memory B cells. It will be important to determine the relevance of membrane IgD interaction with allergens, as this would influence the design of new therapies for the treatment of allergy and asthma.

Efficient control of gene expression by single step integration of the tetracycline system in transgenic mice.

Tetracycline-regulated gene expression in eukaryotic cell lines, plants, and transgenic mice has become a powerful tool for the analysis of eukaryotic gene expression and function. The system consists of two plasmids, one encoding the transactivator protein under control of a viral cytomegalovirus promoter, and the second being the tet-operator minimal promoter driving the gene of interest. Here we show that these control elements, when integrated in cis on a single plasmid, allow efficient and tight control of reporter gene expression in vitro and in vivo. Dependent on the route of administration of tetracycline, gene expression can be partially or fully repressed in transgenic mice, whereas removal of the antibiotic induces the reporter gene in various tissues to levels up to 800-fold more than the two-plasmid system. In addition, crossing and analysis of animals transgenic for the individual components of the system are unnecessary, and genetic segregation of the control elements during breeding is prevented.

Tumor necrosis factor (TNF)-induced cutaneous necrosis is mediated by TNF receptor 1.

Tumor necrosis factor (TNF) is a central mediator of immune and inflammatory responses. Its activities have been shown to be mediated by two distinct receptors, TNFR1 (p55) and TNFR2 (p75). The cytoplasmic domains of both TNF receptors are unrelated, suggesting that they link to different intracellular signaling pathways. To determine their role in vivo in lipopolysaccharide (LPS)- and TNF-induced skin inflammatory necrosis, TNFR1-, TNFR2-, and TNF lymphotoxin-alpha (LT alpha)-deficient mice were used. Skin abscesses were experimentally induced with local application of TNF or LPS. Large macroscopic ulcerations were observed in TNF-injected wild-type animals and to a slightly lesser extent in TNFR2-deficient mice with tissue destruction in both cases extending deep into the dermis. Tissue destruction was accompanied by an intense immune infiltrate composed mainly of neutrophils, lymphocytes, and macrophages. TNFR1-deficient and TNFR1/TNFR2-double-deficient mice, however, did not exhibit any ulceration and showed only a very mild inflammatory infiltrate. In TNF/LT alpha-double ligand0-deficient animals, a moderate epidermal necrosis was observed with a reduced inflammatory infiltrate compared to wild-type animals. As with TNF injections, subcutaneous injection of LPS induced a comparable pattern of skin necrosis in wild-type and TNF receptor mutant mice, yet a slightly more acute inflammatory level was observed regardless of the type of animal tested. As found for TNF-induced skin necrosis, the extent of LPS-induced skin necrosis was reduced in TNF/LT alpha-deficient mice compared to wild-type animals. The present data strongly suggest that TNFR1, rather than TNFR2, is engaged in LPS- and TNF-induced skin necrosis and highlight the predominant role played by TNF in LPS-induced inflammatory diseases.

Establishment of the role of IL-6 and TNF receptor 1 using gene knockout mice.

Cytokines are known to be key players in host response to infection, immunological disorders, and tissue injury in the attempt of an organism to overcome the insult and restore homeostasis. Another important aspect of cytokines, however, is their normal physiological role during development in the unchallenged organism. The most elegant way to analyze both of these functions is to introduce targeted mutations in embryonic stem cells in order to create new mouse strains deficient for a given cytokine and identify the functions that are consequently impaired or lost. This review summarizes the mutant phenotypes of mice carrying a null mutation in the cytokine IL-6 gene or the tumor necrosis factor receptor 1 (Tnfr1) gene. Results for interferon-- and interferon-gamma receptor-deficient mice are included for comparison.

T cell receptor repertoire of gamma delta cells generated from the 14-day embryonic mouse thymus.

Whole, undisrupted 14 day mouse fetal thymus lobes cultured in the presence of 10 U/ml IL-2 generate a heterogeneous population of gamma delta-expressing T cells. Phenotypic analysis has shown that the majority of gamma delta T cells in such cultures stain with the anti-V gamma 3-specific mAb 536. To investigate the V gamma T-cell receptor diversity of cultured fetal thymocytes, cDNA was prepared and amplified using the polymerase chain reaction. The DNA fragments obtained were subsequently cloned and sequenced and compared with those obtained from fresh and organ-cultured 14 day fetal thymus lobes. Results obtained tend to support a positive selection model of gamma delta T cell differentiation.

Composition of the outer membrane of Proteus mirabilis in relation to serum sensitivity in progressive stages of cell form defectiveness.

A serum-resistant strain of Proteus mirabilis was used to determine whether changes in the composition of surface components could be detected following induction of progressive stages of cell form defectiveness by beta-lactam antibiotics. The critical stage was the conversion from filaments to the spheroplast form, which was accompanied by increased susceptibility to the bactericidal action of human serum. Inner and outer membranes of the bacterium, its filament form and its spheroplast form were separated by sucrose density-gradient centrifugation after digestion of peptidoglycan, followed by osmotic lysis of the cells. Outer membranes of the bacterial and the filament forms sedimented at the same density, whilst the outer membrane fraction of the spheroplast form sedimented in a region of lesser density. In addition, the amounts of two major outer-membrane proteins as well as the O-polysaccharide content of the lipopolysaccharide were reduced in the spheroplast form. These results indicate a general disorganization in structure and assembly of components in regard to their interactions with one another in the outer membrane of the spheroplast form.