Alternative intronic polyadenylation generates the interleukin-6 trans-signaling inhibitor sgp130-E10.

Interleukin (IL)-6 signals via a receptor complex composed of the signal-transducing ß-receptor gp130 and the non-signaling membrane-bound or soluble IL-6 receptor α (IL-6R, sIL-6R), which is referred to as classic and trans-signaling, respectively. IL-6 trans-signaling is functionally associated with the development of chronic inflammatory diseases and cancer. Soluble gp130 (sgp130) variants are natural inhibitors of trans-signaling. Differential splicing yields sgp130 isoforms. Here, we describe that alternative intronic polyadenylation in intron 10 of the gp130 transcript results in a novel mRNA coding for an sgp130 protein isoform (sgp130-E10) of 70-80 kDa. The sgp130-E10 protein was expressed in vivo in human peripheral blood mononuclear cells. To assess the biological activity of sgp130-E10, we expressed this variant as Fc-tagged fusion protein (sgp130-E10Fc). Recombinant sgp130-E10Fc binds to a complex of IL-6 and sIL-6R, but not to IL-6 alone, and specifically inhibits IL-6 trans-signaling. Thus, it might play an important role in the regulation of trans-signaling in vivo.

Soluble T cell immunoglobulin and mucin domain (TIM)-1 and -4 generated by A Disintegrin And Metalloprotease (ADAM)-10 and -17 bind to phosphatidylserine.

T cell immunoglobulin and mucin domain 1 and 4 (TIM-1 and -4) proteins serve as phosphatidylserine receptors to engulf apoptotic cells. Here we show that human TIM-1 and TIM-4 proteins are targets of A Disintegrin And Metalloprotease (ADAM)-mediated ectodomain shedding resulting in soluble forms of TIM-1 and TIM-4. We identified ADAM10 and ADAM17 as major sheddases of TIM-1 and TIM-4 as shown by protease-specific inhibitors, the ADAM10 prodomain, siRNA and ADAM10/ADAM17 deficient murine embryonic fibroblasts (MEFs). TIM-1 and TIM-4 lacking the intracellular domain were efficiently cleaved after ionomycin- and PMA-treatment, indicating that the intracellular domain was not necessary for ectodomain shedding. Soluble TIM-1 and -4 were able to bind to phosphatidylserine, suggesting that soluble TIM-1 and -4 might act as negative regulators of cellular TIM-1 and -4. In summary, we describe TIM-1 and TIM-4 as novel targets for ADAM10- and ADAM17-mediated ectodomain shedding.

A disintegrin and metalloprotease (ADAM) 10 and ADAM17 are major sheddases of T cell immunoglobulin and mucin domain 3 (Tim-3).

T cell immunoglobulin and mucin domain 3 (Tim-3) dampens the response of CD4(+) and CD8(+) effector T cells via induction of cell death and/or T cell exhaustion and enhances the ability of macrophages to clear pathogens via binding to galectin 9. Here we provide evidence that human Tim-3 is a target of A disintegrin and metalloprotease (ADAM)-mediated ectodomain shedding resulting in a soluble form of Tim-3. We identified ADAM10 and ADAM17 as major sheddases of Tim-3 as shown by ADAM-specific inhibitors and the ADAM10 pro-domain in HEK293 cells and ADAM10/ADAM17-deficient murine embryonic fibroblasts. PMA-induced shedding of Tim-3 was abrogated by deletion of amino acids Glu(181)-Asp(190) of the stalk region and Tim-3 lacking the intracellular domain was not efficiently cleaved after PMA stimulation. Surprisingly, a single lysine residue within the intracellular domain rescues shedding of Tim-3. Shedding of endogenous Tim-3 was found in primary human CD14(+) monocytes after PMA and ionomycin stimulation. Importantly, the recently described down-regulation of Tim-3 from Toll-like receptor-activated CD14(+) monocytes was caused by ADAM10- and ADAM17-mediated shedding. Inhibition of Tim-3 shedding from lipopolysaccharide-induced monocytes did not influence lipopolysaccharide-induced TNFα and IL-6 but increases IL-12 expression. In summary, we describe Tim-3 as novel target for ADAM-mediated ectodomain shedding and suggest a role of Tim-3 shedding in TLR-mediated immune responses of CD14(+) monocytes.

Immunoglobulin class switching appears to be regulated by B-cell antigen receptor-specific T-cell action.

Antigen affinity is commonly viewed as the driving force behind the selection for dominant clonotypes that can occur during the T-cell-dependent processes of class switch recombination (CSR) and immune maturation. To test this view, we analyzed the variable gene repertoires of natural monoclonal antibodies to the hapten 2-phenyloxazolone (phOx) as well as those generated after phOx protein carrier-induced thymus-dependent or Ficoll-induced thymus-independent antigen stimulation. In contrast to expectations, the extent of IgM heterogeneity proved similar and many IgM from these three populations exhibited similar or even greater affinities than the classic Ox1 clonotype that dominates only after CSR among primary and memory IgG. The population of clones that were selected during CSR exhibited a reduced VH/VL repertoire that was enriched for variable domains with shorter and more uniform CDR-H3 lengths and almost completely stripped of variable domains encoded by the large VH1 family. Thus, contrary to the current paradigm, T-cell-dependent clonal selection during CSR appeared to select for VH family and CDR-H3 loop content even when the affinity provided by alternative clones exhibited similar to increased affinity for antigen.

Upd3--an ancestor of the four-helix bundle cytokines.

The unpaired-like protein 3 (Upd3) is one of the three cytokines of Drosophila melanogaster supposed to activate the JAK/STAT signaling pathway (Janus tyrosine kinases/signal transducer and activator of transcription). This activation occurs via the type-I cytokine receptor domeless, an orthologue of gp130, the common signal transducer of all four-helix bundle interleukin-6 (IL-6) type cytokines. Both receptors are known to exist as preformed dimers in the plasma membrane and initiate the acute-phase response. These facts indicate an evolutionary relation between vertebrate IL-6 and the Drosophila protein Upd3. Here we presented data which strengthen this notion. Upd3 was recombinantly expressed, a renaturation and purification protocol was established which allows to obtain high amounts of biological active protein. This protein is, like human IL-6, a monomeric-α helical cytokine, implicating that Upd3 is an "ancestor" of the four-helix bundle cytokines.

ADAM17-overexpressing breast cancer cells selectively targeted by antibody-toxin conjugates.

A disintegrin and metalloproteinase 17 (ADAM17) is significantly upregulated not only in malignant cells but also in the pro-inflammatory microenvironment of breast cancer. There, ADAM17 is critically involved in the processing of tumor-promoting proteins. Therefore, ADAM17 appears to be an attractive therapeutic target to address not only tumor cells but also the tumor-promoting environment. In a previous study, we generated a monoclonal anti-ADAM17 antibody (A300E). Although showing no complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity, the antibody was rapidly internalized by ADAM17-expressing cells and was able to transport a conjugated toxin into target cells. As a result, doxorubicin-coupled A300E or Pseudomonas exotoxin A-loaded A300E was able to kill ADAM17-expressing cells. This effect was strictly dependent on the presence of ADAM17 on the surface of target cells. As a proof of principle, both immunotoxins killed MDA-MB-231 breast cancer cells in an ADAM17-dependent manner. These data suggest that the use of anti-ADAM17 monoclonal antibodies as a carrier might be a promising new strategy for selective anti-cancer drug delivery.

The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis.

BACKGROUND: In apoptosis, proteolysis by caspases is the primary mechanism for both initiation and execution of programmed cell death (PCD). In contrast, the impact of proteolysis on the regulation and execution of caspase-independent forms of PCD (programmed necrosis, necroptosis) is only marginally understood. Likewise, the identity of the involved proteases has remained largely obscure. Here, we have investigated the impact of proteases in TNF-induced necroptosis. RESULTS: The serine protease inhibitor TPKC protected from TNF-induced necroptosis in multiple murine and human cells systems whereas inhibitors of metalloproteinases or calpain/cysteine and cathepsin proteases had no effect. A screen for proteins labeled by a fluorescent TPCK derivative in necroptotic cells identified HtrA2/Omi (a serine protease previously implicated in PCD) as a promising candidate. Demonstrating its functional impact, pharmacological inhibition or genetic deletion of HtrA2/Omi protected from TNF-induced necroptosis. Unlike in apoptosis, HtrA2/Omi did not cleave another protease, ubiquitin C-terminal hydrolase (UCH-L1) during TNF-induced necroptosis, but rather induced monoubiquitination indicative for UCH-L1 activation. Correspondingly, pharmacologic or RNA interference-mediated inhibition of UCH-L1 protected from TNF-induced necroptosis. We found that UCH-L1 is a mediator of caspase-independent, non-apoptotic cell death also in diseased kidney podocytes by measuring cleavage of the protein PARP-1, caspase activity, cell death and cell morphology. Indicating a role of TNF in this process, podocytes with stably downregulated UCH-L1 proved resistant to TNF-induced necroptosis. CONCLUSIONS: The proteases HtrA2/Omi and UCH-L1 represent two key components of TNF-induced necroptosis, validating the relevance of proteolysis not only for apoptosis, but also for caspase-independent PCD. Since UCH-L1 clearly contributes to the non-apoptotic death of podocytes, interference with the necroptotic properties of HtrA2/Omi and UCH-L1 may prove beneficial for the treatment of patients, e.g. in kidney failure.

A novel bispecific single-chain antibody for ADAM17 and CD3 induces T-cell-mediated lysis of prostate cancer cells.

ADAM17 (A disintegrin and metalloproteinase 17) is a membrane-bound protease that cleaves various cell surface proteins, including cytokines and cytokine receptors. Recently it was shown that ADAM17 is highly expressed on the surface of many cancer cells, whereas normal cells express low levels of ADAM17, implying that ADAM17 is a potential immunotherapeutic target. We have generated a monoclonal antibody against human ADAM17, which recognized the membrane proximal cysteine-rich extension of the ADAM17 protein. Unlike normal cells, tumour cell lines, such as a prostate cancer cell line, pancreatic cancer cell lines, a breast cancer cell line and a non-small lung cancer cell line, expressed ADAM17 on the cell surface. Using the sequence of the antibody we generated an ADAM17-specific scFv (single-chain variable fragment) and fused this to a CD3-specific scFv to generate a bispecific T-cell engager antibody [A300E-BiTE (bispecific T-cell engager antibody)]. Specificity was demonstrated on cells in which ADAM17 was knocked down with a specific shRNA (short hairpin RNA). A300E-BiTE recognized ADAM17 and CD3 on the cell surface of tumour cells and T-cells respectively. In the presence of primary human peripheral blood mononuclear cells or human T-cells the addition of A300E-BiTE led to ADAM17-specific killing of prostate tumour cells indicating a novel strategy for the treatment of cancer.

Multimerisation of A disintegrin and metalloprotease protein-17 (ADAM17) is mediated by its EGF-like domain.

A disintegrin and metalloprotease protein 17 (ADAM17) is a transmembrane zinc dependent metalloprotease. The catalytic activity of the enzyme results in the shedding of a broad range of membrane proteins. The release of the corresponding ectodomains induces a switch in various physiological and pathophysiological processes. So far there is not much information about the molecular mechanism of ADAM17 activation available. As for other transmembrane proteases, multimerisation may play a critical role in the activation and function of ADAM17. The present work demonstrates that ADAM17 indeed exists as a multimer in the cell membrane and that this multimerisation is mediated by its EGF-like domain.

Thymus-independent type 2 antigen induces a long-term IgG-related network memory.

Thymus-independent type 2 (TI-2) antigens occasionally induce long-lasting IgM memory, but do not prime for typical secondary IgG responses. However, contrary to current understanding, we detected several TI-2-induced long-term memory effects in subsequent thymus-dependent (TD) responses to the hapten 2-phenyloxazolone coupled to a protein carrier. The early primary TD response, even 3 months after TI-2 immunization, included non-mutated IgM as well as IgG antibodies exhibiting higher affinities than the Ox1 idiotype which dominates and has highest affinity in sole TD responses. The secondary exclusive IgG response 8 weeks later contained major hitherto non-observed clones. Somatic hypermutation on the normally dominant V(H)Ox1 gene was largely silenced while the associated VkappaOx1 exhibited the classical affinity-enhancing mutations, thus suggesting a separate regulation of this process for V(H) and V(L) genes. Mutations accumulated in genes which normally are rarely or non-expressed or non-mutating. First evidence is presented that receptor revision by V(H) replacement may occur during immune maturation in genetically non-engineered wildtype mice. We conclude that the TI-2 antigen-induced altered selection of TD Ag-inducible clones and its severe gene-specific influence on further somatic mutations and affinity maturation represents a network memory, which we hypothesize to be mediated by anti-idiotypic regulatory T cells.

Molecular basis of antibody binding to mucin glycopeptides in lung cancer.

Glycopeptides bearing Tn epitopes are emerging targets for cancer diagnosis and immunotherapy. In this study, we analyzed membrane proteins containing O-glycosylated tandem repeat (TR) sequences in lung cancer patients of different types and stages, using gene microarray data in public domain. The expression of Tn and glycopeptide epitopes on the surface of lung cancer cell lines were studied by monoclonal IgG antibodies 14A, 16A, and B72.3. The binding of mAbs to synthetic glycopeptides were studied by surface plasmon resonance. Nine mucin mRNAs were found to be expressed in lung cancer patients but at similar level to healthy individuals. At protein level, a glycopeptide epitope on cancer cell surface is preferably recognized by mAb 16A, as compared to peptide-alone (14A) or sugar-alone epitopes (B72.3). 14A and 16A favor clustered TR containing more than three TR sequences, with 10-fold lower Kd than two consecutive TR. B72.3 preferrably recognized clustered sialyl-Tn displayed on MUC1 but not other O-glycoproteins, with 100-fold stronger binding when MUC1 is transfected as a sugar carrier, while the total sugar epitopes remain unchanged. These findings indicate that clusters of both TR backbones and sugars are essential for mAb binding to mucin glycopeptides. Three rules of antibody binding to mucin glycopeptides at molecular level are presented here: first, the peptide backbone of a glycopeptide is preferentially recognized by B cells through mutations in complementarity determining regions (CDRs) of B cell receptor, and the sugar-binding specificity is acquired through mutations in frame work of heavy chain; secondly, consecutive tandem repeats (TR) of peptides and glycopeptides are preferentially recognized by B cells, which favor clustered TR containing more than three TR sequences; thirdly, certain sugar-specific B cells recognize and accommodate clustered Tn and sialyl-Tn displayed on the surface of a mucin but not other membrane proteins.

CD30 on extracellular vesicles from malignant Hodgkin cells supports damaging of CD30 ligand-expressing bystander cells with Brentuximab-Vedotin, in vitro.

The goal of targeted immunotherapy in cancer is to damage both malignant and tumor-supporting cells of the microenvironment but spare unaffected tissue. The malignant cells in classical Hodgkin lymphoma (cHL) selectively express CD30. They release this receptor on extracellular vesicles (EVs) for the tumor-supporting communication with CD30 ligand (CD30L)-positive bystander cells. Here, we investigated how CD30-positive EVs influence the efficacy of the CD30 antibody drug conjugate (ADC) Brentuximab Vedotin (SGN-35). The malignant cells and the EVs expressed the active sheddase ADAM10. ADAM10 cleaved and released the CD30 ectodomain (sCD30), causing a gradual depletion of SGN-35 binding sites on EVs and creating a soluble competitor of the ADC therapy. In a 3D semi-solid tumor microenvironment model, the EVs were retained in the matrix whereas sCD30 penetrated readily into the surrounding culture medium. This resulted in a lowered ratio of EV-associated CD30 (CD30EV) to sCD30 in the surrounding medium in comparison to non-embedded cultures. A low percentage of CD30EV was also detected in the plasma of cHL patients, supporting the clinical relevance of the model. The adherence of CD30EV but not sCD30 to CD30-/CD30L+ mast cells and eosinophils allowed the indirect binding of SGN-35. Moreover, SGN-35 damaged CD30-negative cells, provided they were loaded with CD30+ EVs.

Clonal Progression during the T Cell-Dependent B Cell Antibody Response Depends on the Immunoglobulin DH Gene Segment Repertoire.

The diversity of the third complementarity determining region of the IgH chain is constrained by natural selection of immunoglobulin diversity (DH) sequence. To test the functional significance of this constraint in the context of thymus-dependent (TD) immune responses, we immunized BALB/c mice with WT or altered DH sequence with 2-phenyloxazolone-coupled chicken serum albumin (phOx-CSA). We chose this antigen because studies of the humoral immune response to the hapten phOx were instrumental in the development of the current theoretical framework on which our understanding of the forces driving TD responses is based. To allow direct comparison, we used the classic approach of generating monoclonal Ab (mAb) from various stages of the immune response to phOx to assess the effect of changing the sequence of the DH on clonal expansion, class switching, and affinity maturation, which are hallmarks of TD responses. Compared to WT, TD-induced humoral IgM as well as IgG antibody production in the D-altered ΔD-DµFS and ΔD-iD strains were significantly reduced. An increased prevalence of IgM-producing hybridomas from late primary, secondary, and tertiary memory responses suggested either impaired class switch recombination (CSR) or impaired clonal expansion of class switched B cells with phOx reactivity. Neither of the D-altered strains demonstrated the restriction in the VH/VL repertoire, the elimination of VH1 family-encoded antibodies, the focusing of the distribution of CDR-H3 lengths, or the selection for the normally dominant Ox1 clonotype, which all are hallmarks of the anti-phOx response in WT mice. These changes in clonal selection and expansion, as well as CSR indicate that the genetic constitution of the DH locus, which has been selected by evolution, can strongly influence the functional outcome of a TD humoral response.

The disintegrin domain of ADAM17 antagonises fibroblast­carcinoma cell interactions.

The malignant phenotype of carcinoma cells depends on their ability to invade into their microenvironment promoting metastasis. Therefore, carcinoma cells overexpress many proteins, including A disintegrin and metalloproteases (ADAMs). ADAM17 is expressed by different cancer cell lines and possesses adhesive as well as enzymatic activities. To address the adhesive properties in tumour progression the recombinantly expressed soluble disintegrin domain of ADAM17 was employed. Fibroblasts and carcinoma cells adhere to the immobilized disintegrin domain. Additionally, the soluble disintegrin domain impaired fibroblast-carcinoma cell interactions and increased the shedding activity of ADAM17. Silencing of ADAM17 in fibroblasts or in carcinoma cells decreases cell-cell interaction between these cells. In summary, our results show that the adhesive properties of ADAM17 are mediated by its disintegrin domain and enables carcinoma cells to interact with their microenvironment.

The membrane-proximal domain of A Disintegrin and Metalloprotease 17 (ADAM17) is responsible for recognition of the interleukin-6 receptor and interleukin-1 receptor II.

A great number of physiological processes are regulated by the release of ectodomains of membrane proteins. A Disintegrin And Metalloprotease 17 (ADAM17) is one of the important enzymes, which mediate this process called shedding. Today, more than 70 substrates of this transmembrane metalloprotease are known. This broad spectrum raises the question how ADAM17 recognizes its substrates specifically. Differently tagged ADAM17 deletion variants were used to demonstrate that exclusively the extracellular domains of ADAM17 are needed for interaction with two of its substrates, the IL-6R and the IL-1RII; whereas the transmembrane- and cytoplasmic-region are dispensable for this process. In the extracellular part solely the membrane-proximal domain of ADAM17 is mandatory for recognition of the two type-I transmembrane proteins, but not for the interaction with the type-II transmembrane molecule TNF-α.

Toll-like receptors on the fork roads between innate and adaptive immunity.

There is a permanent interaction amid the innate and adaptive immune systems that leads to a defensive immune response against pathogens and contributes substantially to self-nonself discrimination. Toll-like receptors (TLRs) are essential molecules of the innate immune system that stimulate numerous inflammatory pathways and harmonize systemic defense against a wide array of pathogens. In addition to identifying unique molecular patterns associated with various sections of pathogens, TLRs may also recognize a number of self proteins and endogenous nucleic acids. Several reports have indicated that inappropriate stimulation of the TLR pathway via endogenous or exogenous ligands in animal models or humans may lead to the induction and/or prolongation of autoimmune response and tissue injury.

Development of sandwich ELISA for detection and quantification of human and murine a disintegrin and metalloproteinase17.

ADAM17 (a disintegrin and metalloproteinase-containing protein 17) is a membrane-bound metalloproteinase, implicates in many physiological processes, including cell migration and proliferation. Of particular note, most of the studies so far are restricted on the analysis of ADAM17 mRNA levels. In this study we generated, utilizing hybridoma technology, three monoclonal antibodies (mAbs) (A 300, A 309 and A 318) against the extracellular domain of human ADAM17 to enable quantification of protein expression. The specificity of these mAbs against ADAM17 was tested by enzyme-linked immunoadsorbent assay (ELISA), flourescence-activated cell sorting (FACS) and western blotting. In order to quantify human and murine ADAM17 expression two pairs of these mAbs (biotinylated A 309 in combination with A 300 and biotinylated A 300 in combination with A 318), were used to develop sandwich ELISA. A panel of monoclonal antibodies was generated for first time to measure mouse ADAM17 with a sensitivty of 2 ng/ml. Such systems provide a useful tool to quantify protein levels of ADAM17 and are valuable tools for diagnostic purposes.

Benefits and burden of the maternally-mediated immunological imprinting.

The ontogenetic development of both the immune and the nervous system entirely depend on external environmental signals that induce a lifelong learning process. The resulting collective immunological knowledge about the external world is transmitted in an epi-genetic fashion to the offspring, but only from the maternal and not the paternal side, with maternal IgG as the main transgenerational vector. As products of thymus-dependent responses, maternal IgG have undergone immune maturation by somatic hypermutations and are, therefore, acquired immunological phenotypes representing a great deal of the mother's immunological experience. During a limited neonatal imprinting period, maternal antibodies induce T cell-dependent idiotypic responses. These exert up to life-long determinative influences which may even be dominant over seemingly genetic predispositions. Such long-term immunological imprinting effects can be detected as (a) selection of the adult T and B cell repertoires, (b) anti-microbial protection by antigen-reactive antibodies (idiotypes) and anti-idiotypes, (c) allergen-specific suppression of IgE responsiveness by allergen-reactive IgG idiotype or corresponding anti-idiotype and (d) induction of autoimmune diseases by maternally-derived autoantibodies. Hence, immunological imprinting by maternal IgG antibodies will mostly be beneficial, but in case of autoantibodies can also be a burden for the initial development of the nascent immune system.