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Fc gamma receptors (FcγR) are involved in multiple aspects of immune cell regulation, are central to the success of monoclonal antibody (mAb) therapeutics and underpin the pathology of several autoimmune diseases. However, reliable assays capable of accurately measuring FcγR interactions with their physiological ligands, immunoglobulin G (IgG) immune complexes (IC), are limited. A method to study and detect IC interactions with FcγRs was therefore developed. This method, designed to model the signalling pathway of the inhibitory FcγRIIB (CD32B), utilised NanoLuc® Binary Interaction Technology (NanoBiT™) to measure recruitment of the Src homology 2 (SH2) domain-containing inositol phosphatase 1 (SHIP-1) to the immunoreceptor tyrosine-based inhibitory motif (ITIM) of this receptor. Such recruitment required prior crosslinking of an immunoreceptor tyrosine-based activation motif (ITAM)-containing activatory receptor, and evoked luciferase activity in discrete clusters at the cell surface, recapitulating the known biology of CD32B signalling. The assay detected varying forms of experimental IC, including heat-aggregated IgG, Rituximab:anti-idiotype complexes and anti-trinitrophenol (TNP)-TNP complexes in a sensitive manner (≤1µg/ml), and discriminated between complexes of varying size and isotype. Proof-of-concept for the detection of circulating ICs in autoimmune disease was provided, as responses to sera from patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) were detected in small pilot studies. Finally, the method was translated to a stable cell line system. In conclusion, a rapid and robust method for the detection of IC was developed, which has numerous potential applications including the monitoring of IC in autoimmune diseases and the study of underlying FcγR biology.

Following the success of rituximab, 2 other anti-CD20 monoclonal antibodies (mAbs), ofatumumab and obinutuzumab, have entered clinical use. Ofatumumab has enhanced capacity for complement-dependent cytotoxicity, whereas obinutuzumab, a type II mAb, lacks the ability to redistribute into lipid rafts and is glycoengineered for augmented antibody-dependent cellular cytotoxicity (ADCC). We previously showed that type I mAbs such as rituximab have a propensity to undergo enhanced antigenic modulation compared with type II. Here we assessed the key effector mechanisms affected, comparing type I and II antibodies of various isotypes in ADCC and antibody-dependent cellular-phagocytosis (ADCP) assays. Rituximab and ofatumumab depleted both normal and leukemic human CD20-expressing B cells in the mouse less effectively than glycoengineered and wild-type forms of obinutuzumab, particularly when human immunoglobulin G1 (hIgG1) mAbs were compared. In contrast to mouse IgG2a, hIgG1 mAbs were ineffective in ADCC assays with murine natural killer cells as effectors, whereas ADCP was equivalent for mouse IgG2a and hIgG1. However, rituximab's ability to elicit both ADCC and ADCP was reduced by antigenic modulation, whereas type II antibodies remained unaffected. These data demonstrate that ADCP and ADCC are impaired by antigenic modulation and that ADCP is the main effector function employed in vivo.

CD30 is an unusual member of the TNF receptor superfamily with a duplicated segment within its extracellular domain that may function as an additional ligand binding site. The extracellular domain of CD30 is shed from cells and soluble CD30 levels are elevated in certain disease states. Soluble CD30 may bind to its ligand, CD153, with high affinity and block its interaction with membrane-bound CD30. We have generated soluble recombinant forms of CD30 and CD153 in order to characterize their interaction and assess their biological activity. Soluble trimeric CD153 bound to membrane-anchored CD30 with a relatively high affinity (K(D)=23 nM) and was effective in triggering cell death and TNF-alpha production in the presence of cross-linking antibodies. The affinity and kinetics of the interaction between soluble CD30 and CD153 were determined using the BIAcore biosensor. In contrast to other members of the TNF receptor superfamily, soluble monomeric CD30 bound to its ligand with high affinity (K(D)=4.5 nM) and prevented the interaction of cellular CD153 with immobilized CD30. Furthermore, soluble CD30 blocked cell death signals generated by cell surface-expressed CD30 effectively. These data suggest that soluble CD30 may have biological functions in vivo.

Syndecan-1 is a cell surface proteoglycan that is expressed on human myeloma cells and is thought to act as a co-receptor for certain extracellular matrix proteins and growth factors. The ectodomain of syndecan-1 is thought to be shed from the surface of myeloma cells, although the exact mechanism of release remains unclear. In this study, we used a panel of inhibitors to identify the class of proteinase responsible for shedding the soluble syndecan-1 ectodomain from human myeloma cells. Using enzyme-linked immunosorbent assay, flow cytometry and immunocytochemistry, we demonstrated that myeloma cell lines expressed syndecan-1 on their surface and that this was shed constitutively, but to a varying extent. In addition, phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, stimulated a marked loss of cell surface syndecan-1 from each of the cell lines and this was associated with a corresponding increase in soluble syndecan-1. Inhibitors of serine and cysteine proteinases, and matrix-type metalloproteinases, did not inhibit constitutive or PMA-stimulated syndecan-1 shedding from JJN3 and RPMI 8226 cells. However, BB-94, a hydroxamate-based, broad-spectrum, metalloproteinase inhibitor, substantially suppressed constitutive and PMA-stimulated syndecan-1 loss from myeloma cells. These data indicate that a non-matrix-type metalloproteinase is responsible for syndecan-1 shedding from the surface of myeloma cells.

Interleukin-6 (IL-6) is the major growth factor for human myeloma cells, exerting its effect through the IL-6 receptor (IL-6R). A soluble form of IL-6R (sIL-6R) has been identified, which increases the sensitivity of myeloma cells to IL-6. In patients with multiple myeloma (MM), serum concentrations of sIL-6R are elevated and associated with poor prognosis. The present study was undertaken to determine whether proteolytic cleavage of IL-6R could contribute to sIL-6R release from human myeloma cells, and also to identify the class of proteinase responsible for this event. Human myeloma cell lines were shown to express IL-6R upon their surface and also to release sIL-6R into culture supernatants. In addition, phorbol 12-myristate 13-acetate (PMA) stimulated a loss of IL-6R from the cell surface, with a corresponding increase in the concentration of sIL-6R in the supernatant. Inhibitors of serine and cysteine proteinases, and tissue inhibitor of metalloproteinase (TIMP) -1 and TIMP-2, were shown to have no effect on the magnitude of sIL-6R release. In contrast, TIMP-3 and a hydroxamate-based metalloproteinase inhibitor (BB-94), inhibited both constitutive and PMA-induced release of sIL-6R. Myeloma cells freshly isolated from the bone marrow of a patient with MM were also shown to express IL-6R upon their surface, and to shed this receptor in response to PMA. These data demonstrate that increased proteolytic cleavage of IL-6R, mediated by a non-matrix-type metalloproteinase, is likely to contribute to the elevated concentrations of sIL-6R found in the serum of patients with MM. Inhibition of sIL-6R release by hydroxamate-based metalloproteinase inhibitors may represent a novel therapeutic approach to the treatment of MM.

The platelet reactivities of two simple collagen-like synthetic peptides, Gly-Lys-Hyp-(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly and Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly, were investigated. Both peptides adopted a stable triple-helical conformation in solution. Following cross-linking, both peptides proved to be highly platelet-aggregatory, more active than collagen fibres, inducing aggregation at concentrations as low as 20 ng/ml. These peptides formed microaggregates in solution, and cross-linking was thought to stabilize these structures, allowing expression of their platelet reactivity at 37 degrees C. Like collagen fibres, the peptides caused platelet secretion and release of arachidonate from platelet membrane lipids as well as activation of integrin alpha IIb beta 3 culminating in aggregation. Monoclonal antibodies directed against the integrin alpha 2 beta 1 failed to prevent aggregation release of arachidonate or platelet adhesion to the peptides. Our results indicate that collagen can activate platelets by a mechanism that is independent of integrin alpha 2 beta 1 and for which collagen tertiary and quaternary structures are sufficient alone for activity without the involvement of highly specific cell-recognition sequences.

SummaryWe have investigated the effects of the tyrosine kinase inhibitors, genistein and methyl 2,5-dihydroxycinnamate, on [3H]arachidonic acid release from human platelets. Both tyrosine kinase inhibitors blocked, in a dose-dependent manner, the release of arachidonic acid stimulated by thrombin or suspensions of collagen fibres. Blockade by the tyrosine kinase inhibitors occurred early in the arachidonate release time course. Both genistein and methyl 2,5-dihydroxycinnamate also inhibited tyrosine phosphorylation in platelets. The inhibitors were specific in that they did not affect protein kinase C activity, ATP levels or mobilization of Ca2+ from internal stores. These findings suggest a role for tyrosine kinase activity in the regulation of phospholipase A2 in platelets stimulated by the physiological ligands, thrombin and collagen

Careful control of surface chemistry results in strong surface enhanced resonance Raman scattering from dye-labelled oligonucleotides assembled on nanostructured gold surfaces, releasing their potential as reliable enhancing surfaces.

Platelets respond to low levels of agonists such as collagen or thrombin with primary signals that, although small, are functionally significant because they are amplified by autocrine and paracrine pathways such as the secretion of ADP and ATP from the dense granules. An important mediator of the action of low-dose collagen, especially, which is not dependent on granule secretion, is thromboxane A2, a prostanoid that is a potent platelet agonist in its own right, binding to G-protein-linked receptors on the cell surface to stimulate calcium signals via phospholipase Cβ. The precursor of the prostanoids is arachidonic acid (AA), liberated from platelet membrane lipids by the action of phospholipase A2. AA is processed in two stages by cyclooxygenase to yield prostaglandin (PG) H2. This process is aspirin-sensitive, as the drug irreversibly acetylates and inhibits cyclooxygenase, providing the basis for the old and effective antithrombotic use for aspirin, which itself underscores the involvement of collagen in arterial thrombosis. Thromboxane synthase then consumes a proportion of PGH2 to generate thromboxane (Tx) A2. Other products of platelet arachidonate metabolism include PGD2, which exercises an inhibitory role on platelet function as a consequence of its capacity to activate adenylate cyclase, like its homolog, PGI2 (prostacyclin). PGI2 in circulation, however, is largely produced by the vascular endothelial cells rather than the platelets. This chapter will deal with the measurement of the activity of the prostanoid pathway, detailing a method to measure AA release from metabolically labeled platelets, a thinlayer chromatography procedure for AA-containing lipids, and an ELISA assay for TxA2. Together, these methods provide a means of measuring the activity of the thromboxane pathway at its initiation (phospholipase A2 activity), intermediate metabolism (cyclooxygenase activity), and endpoint (thromboxane production). A plateletpreparation section provides details which are applicable to each of these methods.

CD30 is an unusual member of the TNF receptor superfamily with a duplicated segment within its extracellular domain that may function as an additional ligand binding site. The extracellular domain of CD30 is shed from cells and soluble CD30 levels are elevated in certain disease states. Soluble CD30 may bind to its ligand, CD153, with high affinity and block its interaction with membrane-bound CD30. We have generated soluble recombinant forms of CD30 and CD153 in order to characterize their interaction and assess their biological activity. Soluble trimeric CD153 bound to membrane-anchored CD30 with a relatively high affinity (K(D)=23 nM) and was effective in triggering cell death and TNF-alpha production in the presence of cross-linking antibodies. The affinity and kinetics of the interaction between soluble CD30 and CD153 were determined using the BIAcore biosensor. In contrast to other members of the TNF receptor superfamily, soluble monomeric CD30 bound to its ligand with high affinity (K(D)=4.5 nM) and prevented the interaction of cellular CD153 with immobilized CD30. Furthermore, soluble CD30 blocked cell death signals generated by cell surface-expressed CD30 effectively. These data suggest that soluble CD30 may have biological functions in vivo.

Collagen fibers or a glycoprotein VI-specific collagen-related peptide (CRP-XL) stimulated tyrosine phosphorylation of the focal adhesion kinase, p125(fak) (FAK), in human platelets. An integrin alpha(2)beta(1)-specific triple-helical peptide ligand, containing the sequence GFOGER (single-letter nomenclature, O = Hyp) was without effect. Antibodies to the alpha(2) and beta(1) integrin subunits did not inhibit platelet FAK tyrosine phosphorylation caused by either collagen fibers or CRP-XL. Tyrosine phosphorylation of FAK caused by CRP-XL or thrombin, but not that caused by collagen fibers, was partially inhibited by GR144053F, an antagonist of integrin alpha(IIb)beta(3). The intracellular Ca(2+) chelator, BAPTA, and the protein kinase C inhibitor, Ro31-8220, were each highly effective inhibitors of the FAK tyrosine phosphorylation caused by collagen or CRP-XL. These data suggest that, in human platelets, 1) occupation or clustering of the integrin alpha(2)beta(1) is neither sufficient nor necessary for activation of FAK, 2) the fibrinogen receptor alpha(IIb)beta(3) is not required for activation of FAK by collagen fibers, and 3) both intracellular Ca(2+) and protein kinase C activity are essential intermediaries of FAK activation.

Cytokines and local growth factors are important soluble mediators that play a fundamental role in regulating the growth and differentiated function of many cell types. They mediate their effects by binding to membrane-bound receptors which initiates a complex sequence of signaling events that leads to a cellular response. The magnitude of this effect reflects the complex balance that exists between the different components of the cytokine system. These include the cytokine itself, the membrane-bound receptor, and the presence or absence of soluble receptor and/or receptor antagonists.

Introduction Interleukin-6 (IL-6) is a cytokine that was originally identified as a factor that could induce B lymphocytes to differentiate into immunoglobulin-producing plasma cells [ 11. Subsequent studies have shown IL-6 to be a multifunctional cytokine that is produced by, and can affect, a range of different cells [2,3]. IL-6 is the original member of a family of cytokines that now include leukaemia inhibitory factor, interleukin 1 1, oncostatin M, ciliary neurotropic factor and cardiotrophin 1. The receptors for these molecules share a common signal transducing component, which accounts for their overlapping biological activities.

Synthetic collagen-like peptides, of general structure [Gly-Pro-HyP]n, adopt the triple-helical structure which is essential for the platelet-reactivity of native collagens. These peptides are potent activators of platelets, stimulating platelet aggregation at much lower dose than collagen fibres, but, unlike collagen fibres, they are not recognised by the integrin alpha 2 beta 1. We have examined the ability of the synthetic peptides to activate the various signalling pathways which regulate human platelet function. The peptides are potent activators of Ca2+ mobilisation and of protein kinase C, and they stimulate tyrosine phosphorylation of some substrates preferentially. However, in contrast with native type I collagen fibres, they are unable to inhibit platelet adenylate cyclase. This suggests a mode of action for the synthetic peptides which substantially overlaps, but which is not entirely identical with, that of native collagen.