Monocytes, but not macrophages, produce the eosinophil cationic protein.

The eosinophil cationic protein (ECP) is a cytotoxic protein with ribonuclease activity, produced and stored in bone marrow eosinophil myelocytes. Mature circulating eosinophils contain about 10 pg ECP per cell. The aim of this study was to investigate the possibility that monocytes produce and store ECP. By results from flow cytometry and specific protein measurement it is shown that human monocytes contain ECP (monocytes about 10 fg ECP per cell). RT-PCR analysis indicated the presence of mRNA coding for ECP in blood monocytes but not in alveolar macrophages. Furthermore, mRNA coding for ECP and low amounts of the protein were found in three myeloid cell lines representing different stages of monocytic differentiation. Differentiation of U-937 cells to macrophages induced lowered transcription of the ECP gene and reduced protein production. Immunohistochemical staining of lung tissue indicated that lung macrophages do not contain ECP. It is concluded that ECP is produced to a low extent by human monocytes and that the production is shut down during macrophage differentiation. This might indicate an alternative transcriptional regulation of the ECP gene in the monocytic lineage compared to the eosinophil lineage.

A molecular model of type I allergy: identification and characterization of a nonanaphylactic anti-human IgE antibody fragment that blocks the IgE-FcepsilonRI interaction and reacts with receptor-bound IgE.

BACKGROUND: The IgE-mediated activation of effector cells and antigen-presenting cells through the high-affinity receptor for IgE (FcepsilonRI) represents a key pathomechanism in type I allergy and many forms of asthma. OBJECTIVE: We sought to establish an in vitro molecular model for the interaction of human FcepsilonRI, IgE, and the corresponding allergen and to identify monoclonal anti-human IgE antibodies with a therapeutic profile different from previously established anti-IgE antibodies. METHODS: Human FcepsilonRI alpha chain, a human monoclonal allergen-specific IgE antibody (chimeric Bip 1), and the corresponding allergen, the major birch pollen allergen Bet v 1, were produced as recombinant proteins and analyzed by means of circular dichroism and native overlays, respectively. Using this molecular model, as well as negative stain immunoelectron microscopic analysis, and in vitro cultivated human basophils, we characterized mouse anti-human IgE antibodies. RESULTS: We established a molecular model for the interaction of human IgE with FcepsilonRI. Using this molecular model, we identified a nonanaphylactic anti-human IgE antibody fragment (Fab12), which blocked the IgE-FcepsilonRI interaction and reacted with effector cell-bound IgE. CONCLUSION: Fab12 represents a candidate molecule for therapy of atopy and asthma because it can be used for the depletion of circulating IgE antibodies, as well as for the depletion of IgE-bearing cells.

An in vitro model for the allergen-IgE-FcARI interaction.

BACKGROUND: The interaction of immune complexes consisting of allergens and allergen-specific IgE with the high-affinity Fcepsilon receptor represents the key event in the induction of symptoms in type I allergic individuals. Immediate-type symptoms result from the release of biological mediators due to allergen-induced cross-linking of FcepsilonRI receptors on mast cells and basophils, whereas FcepsilonRI-mediated presentation of allergen-IgE complexes may contribute to late-phase symptoms through enhanced T cell activation. The interaction of allergens/allergen-specific IgE/FcepsilonRI represents, therefore, an important target for therapeutic intervention strategies in type I allergy. METHODS AND RESULTS: A molecular model of the allergen-IgE-FcepsilonRI interaction was established. It consists of recombinant purified Bet v 1, the major birch pollen allergen, a chimeric Bet v 1 specific monoclonal IgE antibody, and the baculovirus-expressed purified human alpha chain of FcepsilonRI. The chimeric Bet v 1-specific IgE antibody consists of the light chain and the heavy chain variable region of a mouse monoclonal Bet v 1 specific antibody, Bip 1, and the constant region of human IgE. The interaction of rBet v 1, chimeric Bip 1, and human alpha chain was investigated by overlay experiments. Nitrocellulose-immobilized recombinant alpha chains was incubated with chimeric Bip 1 and, for control purposes, with mouse-derived Bip 1. Bound chimeric Bip 1 was detected with 125I-labeled rBet v 1. The specific interaction of rBetv 1, chimeric Bip 1, and recombinant human alpha chain is demonstrated. We thus establish a molecular model of the allergen/IgE/alpha chain interaction. The usefulness of the described in vitro system is exemplified by the identification of a mouse monoclonal antihuman IgE antibody which blocked the IgE-alpha chain interaction. CONCLUSIONS: The module system consisting of rBet v 1, chimeric Bip 1, and recombinant alpha chain may be used for the identification of competitors of the allergic effector reaction by means of high throughput screening of compounds or by combinatorial chemistry.

Targeted superantigens for immunotherapy of haematopoietic tumours.

With the exception of childhood common acute lymphoblastic leukaemia (cALL), treatment of other hematopoietic B cell lineage tumours such as non-Hodgkin's lymphoma (B-NHL), adult ALL and multiple myeloma (MM) is unsatisfactory. Similarly, the therapeutic outcome of acute and chronic myeloid leukaemia (AML, CML) is frequently dismal. At the same time, leukaemia/lymphoma cells represent ideal targets for immunotherapy. The present review summarizes our preclinical experience with a novel type of cytotoxic T cell based immunotherapy for B-lineage and myeloid tumours. Staphylococcal enterotoxin-derived superantigens (SAgs) are among the most potent T cell activators known, linking the T cell receptor to HLA-DR on natural target cells. SAgs were genetically engineered to reduce DR binding and were then fused to Fab parts of tumour-directed monoclonal antibodies (mAbs). Using these "targeted" SAgs, highly efficient lysis of B-lineage (B-NHL, B-CLL, ALL, MM) and myeloid (AML, CML) tumour cells by T-cells was achieved in vitro and in an animal model. We are entering an interesting era of innovative cancer therapy based on novel man-made biotherapeutic agents.

Characterization of the joining chain (J-chain) promoter.

A 300 b.p. promoter from the mouse joining chain (J-chain) gene was studied with regard to functional activity and protein/DNA interactions. The promoter only stimulated expression of a chloramphenicol-acetyl-transferase (CAT) reporter gene when an enhancer was present in the construct, regardless of whether the construct was transfected into cell lines that did or did not express an endogenous J-chain. Furthermore, deletion mutants lacking the 5' portion of the promoter were transcribed at a higher rate than the intact promoter in both J-chain positive and J-chain negative B-cell lines but not in untransformed B lymphocytes stimulated by lipopolysaccharide, indicating the presence of a negative control element in the 5' portion of the J-chain promoter active in tumour cells only. The octamer element in the J-chain promoter was found to bind Oct proteins, albeit with a low affinity. The penta-deca (p.d.) element in the J-chain promoter bound proteins in extracts from untransformed B cells but not in the tested cell lines. The protein binding to the J-chain p.d. element did not compete efficiently with a p.d. element from the SP6 kappa promoter. A protein binding to the 5' portion of the J-chain was expressed in some cell lines but not in others; neither a negative nor a positive correlation to J-chain expression could be seen. It was concluded that the J-chain promoter is equivalent to a kappa promoter and that differentiation-specific J-chain expression is governed by distal, positive control elements located outside the analysed region.

Functional modularity in the SP6 kappa promoter.

The requirements of the SP6 kappa promoter for transcriptional activation were studied in nontransformed murine B lymphocytes stimulated with lipopolysaccharide. Three different DNA motifs, besides the TATA-box, were needed for restoration of transcriptional activation to the same magnitude as seen with the native SP6 kappa promoter. The decamer motif (TNCTTTGCAT) was found to induce transcription alone and point-mutation of this element reduced transcription to negligible levels, although the other two required elements were present. The penta-decamer element (TGCAG/CCTGTGNCCAG) did not stimulate transcription alone, but activated transcription synergistically in conjunction with the decamer motif. This synergism required the presence of a third pyrimidine rich element (CCCT) in the decamer 3' flanking sequence. The pyrimidine rich element could partly be substituted for by an E-box core motif (CANNTG) 3' of, but not by the kappa Y motif (CTTCCTTA) 5' of, the decamer. Proteins interacting specifically with the penta-decamer element were detected by band-shift assay. The decamer 3' flanking sequence of the SP6 kappa promoter was found to modify the binding of endogenous Oct2 isoforms to the decamer motif i B lymphocytes, but not in CHO cells transfected with various Oct2 isoforms. Thus, complex protein/DNA interactions can be observed in the SP6 kappa promoter which correlate functionally with a synergism in transcriptional activation.

'E-boxes' as promoter elements in B cell lines and untransformed B lymphocytes.

The penta-deca (pd) promoter element from the SP6 kappa promoter and the muE2 box from the Ig heavy chain intron enhancer were analysed in an electrophoretic mobility shift assay (EMSA), utilising cell extracts from total mouse splenic B cells before and after stimulation with lipopolysaccharide. With both probes a changed EMSA pattern was observed after stimulation of the cells, where higher molecular weight DNA/protein complexes became dominant. When the pd and muE2 sequence elements were cloned in front of a TATA box and analysed for their transcriptional promoting activity in lipopolysaccharide stimulated B cells, they were inactive. On the contrary, the same constructs were transcriptionally active in some but not all B cell lines. Thus, E-boxes display functional heterogeneity as transcriptional activators depending on host cell.

Regulation of immunoglobulin transcription.

B lymphocytes express membrane-bound immunoglobulin molecules as antigen-specific, clonally distributed, cell surface receptors. Molecules with the same fine specificity can be secreted in large amounts into the body fluids by progeny of the clone, subsequent to antigenic challenge and appropriate stimulation into terminal differentiation. While this review is limited to the regulation of immunoglobulin transcription, it should be noted that immunoglobulin expression is regulated at the level of DNA, RNA and protein. A DNA recombination process determines the specificity of the antibody to be expressed, the immunoglobulin RNA level is regulated qualitatively and quantitatively, as is the mature immunoglobulin protein.

Effects of phorbol esters on B-cell gene expression.

Splenic B lymphocytes were stimulated with lipopolysaccharide alone or in combination with phorbol-12,13-dibutyrate, a protein kinase C-activating phorbol ester. The effect of the treatment was analysed at the single cell level with in situ RNA/RNA hybridization. Hybridization with a kappa light chain probe revealed that the whole population had shifted towards a low, but significant, expression of immunoglobulin mRNA. Analysis at the population level was performed by DNA/RNA and RNA/RNA hybridization experiments. It was found that the steady-state levels of mRNA for kappa light chain, IgM heavy chain and J chain were reduced by phorbol ester treatment, while the steady-state level of mRNA for IgD heavy chain was increased. Steady-state levels of mRNA for Ia antigen and alpha-actin were marginally affected.

Regulation of immunoglobulin transcription rates and mRNA processing in proliferating normal B lymphocytes by activators of protein kinase C.

Immunoglobulin gene expression in normal splenic B lymphocytes stimulated with lipopolysaccharide was selectively down-regulated by anti-IgM antibodies and a protein-kinase C-activating phorbol ester, phorbol 12,13-dibutyrate. This control was concomitant with a decreased rate of transcription of the IgM gene while "polymerase pausing" was induced in the IgD gene. The suppression was resistant to treatment with cycloheximide, indicating that it was not caused by a labile repressor protein. The down-regulation of immunoglobulin gene expression affected only the secretory form of immunoglobulin, while the mRNA levels for the membrane-bound form of immunoglobulin remained unaltered. We conclude that the mechanisms controlling immunoglobulin gene expression in untransformed B lymphocytes differ from those operating in tumors derived from the same cell lineage.