Kathrin Wagner

The Role of Non‐Deletional Tolerance Mechanisms in a Murine Model of Mixed Chimerism with Costimulation Blockade

Peripheral and central clonal deletion are important tolerance mechanisms in models using bone marrow transplantation (BMT) with costimulation blockade (CB). However, since tolerance can be found before peripheral deletion is complete and since elimination of recipient CD4+ cells at the time of BMT prevents tolerance induction, we investigated the potential roles of regulation and anergy in such a murine model. We found that transient elimination of CD25+ cells or neutralization of IL2 immediately after BMT and CB prevented the induction of skin graft tolerance. Cotransfer into SCID mice of CD4+ cells taken from chimeras early after BMT, together with naïve recipient‐type CD4+ cells significantly prolonged donor skin graft survival. In contrast, cotransfer of CD4+ cells harvested from chimeras late after BMT did not prolong donor skin graft survival. Besides, depletion of CD25+ cells in established chimeras several months post‐BMT did not break tolerance. In vivo administration of recombinant IL2 inhibited chimerism and tolerance neither early nor late post‐BMT, arguing against a decisive role for classical anergy. Thus, CD4 cell‐mediated regulation contributes significantly to tolerance induction early after BMT, but appears to have no critical role in the maintenance of tolerance.

Enhancement of human IL-4 activity by soluble IL-4 receptors in vitro.

The recombinant form of the extracellular domain of the IL‐4 receptor (sIL‐4R) is a potential candidate to neutralize IL‐4; however, murine sIL‐4R displayed both antagonistic and agonistic activity in vivo.Here we show that human recombinant sIL‐4R induced the formation of complexed IL‐4 in supernatants of activated T cells in a dose‐dependent manner as measured by newly developed enzyme‐linked immunosorbent assays. These IL‐4/sIL‐4R complexes liberated free IL‐4 even after prolonged culturing. In contrast, in the absence of exogenously added sIL‐4R, free IL‐4 was rapidly consumed or proteolytically degraded in cultures of activated T cells. Thus, no IL‐4 bioactivity could be determined in supernatants of T cells activated in the presence of IL‐4 for 6 days. In contrast, the same cultures carried out in the presence of sIL‐4R showed marked IL‐4 bioactivity. While low concentrations of sIL‐4R enhanced IL‐4‐driven inhibiton of IFN‐γ production by activated T cells, higher concentrations neutralized IL‐4. Together, human sIL‐4R, besides its activity as an antagonist to IL‐4, also possesses protective and agonistic functions for IL‐4, which may be relevant for clinical studies aiming to neutralize IL‐4 in vivo.

Demonstration of the therapeutic potential of non-anaphylactogenic anti-IgE antibodies in murine models of skin reaction, lung function and inflammation

BACKGROUND Allergies and allergic asthma are believed to be mediated by allergen-specific IgE antibodies. We have investigated the therapeutic potential of inhibiting endogenous IgE by a non-anaphylactogenic anti-mouse IgE antibody 1-5 with respect to its effects on antigen-induced skin reaction, lung function changes and lung inflammation in mice. METHODS Mice were immunized with benzylpenicillinoyl-KLH or ovalbumin, and antigen-mediated skin reaction, bronchoconstriction, bronchopulmonary hyperresponsiveness (BHR) and lung eosinophilic inflammation determined in anti-IgE 1-5-treated versus untreated animals. RESULTS Application of anti-IgE 1-5 inhibited (by 90%) the serum IgE and, 3-4 days after onset of treatment, blocked the antigen-induced skin reaction. Furthermore, the antibody also inhibited (by 90%) the antigen-induced infiltration of eosinophils into the lung. This latter effect seems to be mediated by blocking the IgE-CD23 interaction and indicates that lung eosinophilic inflammation also depends on IgE. Moreover, when applied to rats passively sensitized with mouse IgE, antibody 1-5 inhibited the antigen-induced bronchoconstriction. A similar effect could be seen in actively immunized mice, where antibody 1-5 was able to inhibit (by 70%) the ovalbumin-induced bronchoconstriction as well as BHR. CONCLUSIONS In summary, non-anaphylactogenic anti-IgE antibodies can markedly inhibit IgE levels and IgE-mediated allergic reactions. Since bronchoconstriction, BHR and lung eosinophilic inflammation can be suppressed, such antibodies may be attractive principles for the treatment of allergic asthma.

New concepts of IgE regulation.

B cell switch to IgE expression is mediated by IL-4 and is regarded as a T helper cell-related phenomenon. In this overview we describe that IgE switch can also be induced by mast cell/basophil like cells (from splenic non-B, non-T cells), activated by IgE receptor cross-linking and/or IL-3 which results in IL-4 production by these cells. Furthermore, activated mast cells produce their own growth factors, IL-3 and GM-CSF. Thus, activation of mast cells can provoke an ongoing local allergic reaction as long as antigen confrontation is maintained, a process which is sustained by further IgE production as well as renewal of mast cells. It is furthermore demonstrated that in certain established immune situations the IgE response may become independent of IL-4, namely in the spontaneous in vitro IgE expression of cells from atopic individuals as well as in an in vitro antigen-induced secondary IgE response of spleen cells derived from previously immunized mice. Thus, IgE-switched B cells may persist in vivo and may represent a pool of potentially IgE-producing cells. Finally, a selective inhibition of the IgE response is described in vitro and in vivo by the use of so-called non-anaphylactic monoclonal anti-IgE antibodies. Such antibodies bind to surface IgE+ B cells, but not to IgE-sensitized mast cells, and thereby inhibit IgE responses. Non-anaphylactic antibodies blocked the binding of allergen-specific IgE to mast cells by competing with the Fc epsilon on these cells. As a consequence they do not induce but rather prevent allergen-induced mediator release by mast cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Establishment of a memory in vitro murine IgE response to benzylpenicillin and its resistance to suppression by anti-IL-4 antibody.

Regulation of a memory IgE antibody response may be different from the induction of a primary response and may, therefore, be more relevant to the study of allergic diseases and the therapeutic manipulation of IgE antibody formation. In this paper a murine hapten-specific in vitro memory IgE antibody response to benzylpenicilloyl(BPO)-KLH is described. The response was analyzed by determining the number of antibody-producing cells (APC) in an ELISA spot assay. Of the total number of BPO-specific APC (10,000 APC/10(6) cultured spleen cells), about 1% were IgE-producing cells (100/10(6) cultured cells), as detected on day 6 of culture. The level of the antibody response is antigen dose-dependent, and the detected APC are BPO specific. The memory IgE response is not inhibited by the addition of anti-IL-4 antibody (11B11), even at a high excess. In the presence of the mitogen lipopolysaccharide, it has been shown that switch of B cells to IgE is induced by IL-4, a process which can be inhibited by anti-IL-4 antibody. Because the antigen-induced IgE response cannot be inhibited by anti-IL-4 antibody, in vitro responding cells derived from BPO-KLH-preimmunized mice may, therefore, have already switched in vivo to IgE. On the other hand, B cells switching to IgE in a situation of cognate T-B cell interaction might receive IL-4 in a transsynaptical way from T cells which might not be accessible to inhibition by anti-IL-4 antibody. The identification of the two possibilities in situations of established allergic disorders will be decisive for determining whether pharmacological inhibition of IL-4 (or IL-4-induced switch)--e.g., by putative low molecular weight compounds--will ever be a meaningful approach to suppress allergic diseases.

A sensitive and efficient induction system for murine IgE single cell analysis at the clonal level

A culture system is described which permits the analysis of IgE expression by single murine cells within clones of B cells. The system is based on the use of a CB5.1 stroma cell line as a feeder which optimally supports the IL-4-induced switch to IgE of LPS-stimulated B cells in culture. In this system 100 U/ml IL-4 induces the switch to IgE, in 3-5% of B cells and the switch frequency to IgG1 was as high as 2%. Five ng IgE or 12 ng IgG1 were produced per clone containing on average 13-15 PFC. The detection of single IgE secreting B cells was possible due to two newly developed, highly specific rat anti-mouse IgE antibodies used in a sandwich-ELISA. The frequency of IgE-secreting B cells was enhanced 2.5 times when the fibroblastoid CB5.1 cells rather than thymocytes were used as feeder cells. CB5.1 cells supported the differentiation of B cells to IgM-PFC almost as well as rat thymocytes (which have, to date, been used as the standard feeder layer) whereas the amount of secreted IgG1 was about 3 times lower than in thymocyte cultures. Optimal switching to IgE occurred at concentrations of IL-4 which were 10-fold lower than that required for IgG1 expression, a situation quite opposite to that observed in the rat thymocyte-supported culture system. In confirmation of established data the switch of B cells to IgE or IgG1 occurred randomly. The advantages of CB5.1 cells as feeder cells are (1) the use of a homogeneous and defined cell line, (2) their limited release of defined lymphokines (IL-6 and GM-CSF), and (3) the low degradation and consumption of cytokine factors. The combination of the CB5.1 cell line with a highly specific IgE ELISA assay made it possible to analyse the appearance of IgE producing cells within a developing B cell clone.

An in vitro murine model of a penicillin specific IgE anamnestic response

A new murine model system was developed to study hapten specific IgE anamnestic antibody forming cell (AFC) responses induced in vitro. BALB/c mice injected intraperitoneally with BPO-KLH (10 micrograms) in aluminum hydroxide gel (alum) on day 0 and 21 were killed on day 42. Spleen cells (4 X 10(7] were cultured for 0-8 days in the presence of BPO-KLH (0.25-2500 ng/ml), after which the numbers of BPO specific IgE AFC were enumerated in an ELISPOT assay at 37 degrees C using BPO-BSA coated plates. Peak BPO specific IgE anamnestic AFC responses (approximately 40 AFC/10(7) cultured cells) occurred when cells were cultured with 100 ng/ml BPO-KLH for 5 days; peak BPO specific IgG1 and IgA responses (approximately 3000 and approximately 280 AFC/10(7) cultured cells) were detected one day earlier with the same antigen concentration. No ELISPOTs of any isotype were detected if: cells were cultured in the absence of specific antigen; cells from alum treated or unsensitized mice were used; assay wells were coated with BSA alone; isotype specific antibodies were omitted; or assays were performed in the presence of cycloheximide or at 4 degrees C. Cellular interactions in IgE anamnestic AFC responses were studied using the newly developed model system. We found that these responses were partially T cell independent, and that spleens from sensitized and unsensitized adult mice, as well as neonatal mice, contained cells which suppressed these responses.

Transfected Chinese hamster ovary cells as a model system for cytokine immunocytochemistry and in situ hybridisation.

The presence of cytokine producing cells is most easily revealed by techniques measuring the secreted cytokines in culture supernatants or body fluids. However, these techniques only measure the bulk cytokine release by a given, often mixed cell population. To demonstrate cytokine production at the single cell level, immunocytochemistry (ICC) and in situ hybridisation (ISH) are now widely used techniques. To establish these techniques, an easily accessible model system is needed which permits the evaluation of different ICC and ISH protocols. It can be used to demonstrate the specificity of the antibodies and may serve as a positive control for samples of unknown cytokine content. Here we propose the use of Chinese hamster ovary (CHO) cells transfected to express one specific cytokine as such a model system. Its usefulness is demonstrated by the characterisation of six monoclonal antibodies to human interleukin‐4 and the establishment of two in situ hybridisation protocols.