Felix A. Montero-Julian

The IL-6-Soluble IL-6Rα Autocrine Loop of Endothelial Activation as an Intermediate Between Acute and Chronic Inflammation: an Experimental Model Involving Thrombin

Thrombin is a procoagulant and proinflammatory molecule in vivo. In vitro, thrombin has been shown to induce endothelial activation, notably IL-8 secretion and adhesion molecule expression. In this study, we showed that thrombin may induce a new cascade leading from acute to chronic inflammation. Thrombin was able to induce the production of both IL-6 and monocyte chemotactic protein-1 (MCP-1) by HUVEC independently of IL-1αβ and TNF-α. Addition of physiological concentrations of exogenous soluble IL-6Rα (sIL-6Rα) to thrombin-activated HUVEC was sufficient to increase the amounts of MCP-1 produced, but not those of IL-8. These effects could be blocked by anti-IL-6 or anti-sIL-6Rα blocking mAb, demonstrating the existence of an autocrine loop of MCP-1 secretion, involving the IL-6/IL-6Rα/gp130 complex on HUVEC. In addition, we identified IL-8-activated neutrophils as a potential source of sIL-6Rα because IL-8 induced IL-6Rα shedding from the neutrophil membranes and increased in parallel sIL-6Rα concentrations in neutrophil supernatants. Furthermore, addition of neutrophils to thrombin-activated HUVEC significantly increased MCP-1 secretion, which could be decreased by blocking IL-6. Thus, thrombin-activated endothelium may induce a cascade of events characterized by IL-8 secretion, neutrophil local infiltration, and the release of IL-6Rα from neutrophil membranes. sIL-6Rα may then complex with IL-6 and increase the amount of MCP-1 produced by thrombin-activated endothelium, favoring monocyte infiltration, and the transformation of acute into chronic inflammation.

Chemotactic agents induce IL-6Rα shedding from polymorphonuclear cells: involvement of a metalloproteinase of the TNF-α-converting enzyme (TACE) type

Interleukin (IL)‐6 transsignaling plays a pivotal role in the shift from neutrophil to monocyte recruitment at the inflammatory site. Release of neutrophil IL‐6 receptor‐α (IL‐6Rα, CD126) in its soluble form is a key step of IL‐6 transsignaling, however, its physiological inducers are poorly known. Here, we observed that the neutrophil chemoattractants IL‐8, C5a complement fraction, platelet activating factor, leukotriene B4 and N‐formyl‐methionyl‐leucyl‐phenylalanine rapidly decreased IL‐6Rα membrane expression and increased soluble IL‐6Rα concentrations in the neutrophil supernatants, consistent with a shedding process. IL‐6Rα shedding involved a TNF‐α‐converting enzyme‐type metalloproteinase since it was partly decreased in the presence of a specific inhibitor, but not cathepsin G since PMSF or α1 antichymotrypsin had no effect. Neutrophil IL‐6Rα shedding may be a common feature of neutrophilic infiltrates in various inflammatory situations, allowing IL‐6 transsignaling, decreasing neutrophil infiltration and in the meantime favoring monocyte recruitment, thus the initiation of an immune response and subsequently the resolution of inflammation.

Kinetic evidence for a ligand-binding-induced conformational transition in the T cell receptor.

Thermodynamics and kinetics of the interaction between T cell receptor specific for cytomegalovirus peptide (TCRCMV) and its specific ligand, pp65–HLA-A*0201 complex, were studied by surface plasmon resonance and stopped-flow methods. In the latter measurements, fluorescence resonance energy transfer (FRET) between fluorescently labeled reactants was used. Thermodynamic data derived from surface plasmon resonance measurements suggest that the complex formation is driven by both favorable enthalpy and entropy. Two reaction phases were resolved by the stopped-flow measurements. The rate constant of the first step was calculated to be close to the diffusion-controlled limit rate (3·105 to 106 M−1·s−1), whereas the second steps reaction rate was found to be concentration independent and relatively slow (2–4 s−1 at 25°C). These findings strongly suggest that the interactions between the TCR and its ligand, the peptide–MHC complex, proceed by a two-step mechanism, in which the second step is an induced-fit process, rate determining for antigen recognition by TCR.

Immunoassay for functional human soluble interleukin-6 receptor in plasma based on ligand/receptor interactions

Soluble forms of most cytokine receptors, able to bind effectively to their respective ligands, have now been described. A soluble interleukin-6-binding molecule derived from the gp80 component of the multichain IL-6 receptor can be detected in biological fluids, and can act as an agonist of IL-6 activity. The clinical significance of the soluble receptor levels still remains to be explored. We took advantage of the characterization of an anti-IL-6 monoclonal antibody and of an anti-IL-6R monoclonal antibody that both bound to IL-6/IL-6R complexes to design an immunometric assay for the measurement of soluble IL-6R complexed to IL-6. This reaction scheme was designated as ELIA (enzyme-ligand immunoassay). When exogeneous IL-6 was added in excess to an sIL-6R containing sample, all sIL-6R was present in a complexed form. Thus, the reaction scheme could also be used to determine total sIL-6R concentrations. A recombinant sIL-6R standard was prepared from the supernatant of murine thymoma cells transfected with a gene coding for an extracellular portion of the IL-6 receptor. The assay permitted the precise and reproducible measurement of sIL-6R in serum or plasma. This approach is of general relevance for the determination of soluble cytokine receptors in biological fluids, provided that adequate anti-cytokine and anti-receptor antibodies are available.

Distinct orientation of the alloreactive monoclonal CD8 T cell activation program by three different peptide/ MHC complexes

We have characterized three different programs of activation for alloreactive CD8 T cells expressing the BM3.3 TCR, their elicitation depending on the characteristics of the stimulating peptide/MHC complex. The high‐affinity interaction between the TCR and the Kb‐associated endogenous peptide pBM1 (INFDFNTI) induced a complete differentiation program into effector cells correlated with sustained ERK activation. The Kbm8 variant elicited a partial activation program with delayed T cell proliferation, poor CTL activity and undetectable ERK phosphorylation; this resulted from a low‐avidity interaction of TCR BM3.3 with a newly identified endogenous peptide, pBM8 (SQYYYNSL). Interestingly, mismatched pBM1/Kbm8 complexes induced a split response in BM3.3 T cells, with total reconstitution of T cell proliferation but defective generation of CTL activity that was correlated with strong but shortened ERK phosphorylation. Crystal structures highlight the molecular basis for the higher stability of pBM8/Kbm8 compared to pBM1/Kbm8 complexes that exist in two conformers. This study illustrates the importance of the stability of both peptide/MHC and peptide/MHC‐TCR interactions for induction of sustained signaling required to induce optimal CTL effector functions. Subtle allelic structural variations, amplified by peptide selection, may thus orient distinct outcomes of alloreactive TCR‐based therapies.

Monoclonal antibodies against the human interleukin-11 receptor alpha-chain (IL-11Rα) and their use in studies of human mononuclear cells

Abstract A panel of 14 hybridoma cell lines secreting monoclonal antibodies against the human interleukin-11 receptor alpha chain (hIL-11Rα) was obtained using two different approaches. Two antibodies were raised against peptides of the N- and C-terminal sequences, respectively, of the extracellular part of the hIL-11Rα. Another group of 12 antibodies was generated against a hybrid protein consisting of the extracellular part of the hIL-11Rα fused to mature full-length human IL-2. All these antibodies recognized native hIL-11Rα and most also recognized the denatured receptor on immunoblots after SDS–PAGE. Four different epitopes were identified on the extracellular part of the hIL-11Rα. One epitope, defined by the E27 antibody, is located at the N-terminus and the other three epitopes are clustered in the membrane-proximal, C-terminal region. The antibodies defining epitopes I and II recognized membrane-bound hIL-11Rα expressed in gp130/hIL-11Rα-co-transfected Ba/F3 cells. The E27 antibody cross-reacted with murine IL-11Rα, in agreement with the fact that the N-terminal region is highly conserved between species. The other 13 antibodies all recognized a region between amino acids 319 and 363, which is the membrane-proximal part of the hIL-11Rα. This region, which is less conserved between mouse and human, is shown here to be an immunodominant region. Anti-IL-11Rα monoclonal antibodies, which have not been described previously enabled us to explore the expression and tissue distribution of IL-11Rα on human peripheral blood mononuclear cells and cell lines. The antibodies provide powerful tools for the study of the regulation and function of the receptor.

Pattern of DAP12 Expression in Leukocytes from Both Healthy and Systemic Lupus Erythematosus Patients

DAP12 is an ITAM-bearing transmembrane adaptor originally identified on the surface of Natural Killer cells. A broad expression among other immune cells was later found in myeloid and lymphoid cells. However, data on DAP12 expression pattern rely only on immunoblot and microarray analysis. Here, we describe the generation and the characterization of an anti-DAP12 monoclonal antibody. Using this novel reagent, we show that DAP12 expression is restricted to innate immune cells in basal condition. Since a decreased expression of DAP12 has been suggested in NK cells of systemic lupus erythematosus patients, we have further investigated the NK cell receptor repertoire and leukocyte expression of DAP12 in these patients and no major changes were detectable when compared to controls.

Structural and kinetic basis for low affinity cross-reactivity in T cell allorecognition

The alloreactive BM3.3TCR interacts with high affinity with H‐2Kb loaded with the endogenous peptide pBM1 (INFDFNTI), and shows low affinity cross‐reactivity for H‐2Kb loadedwith a viral peptide VSV8 (RGYVYQGL), CTL activity requiring 103‐fold higher peptide concentration and being highly sensitive to inhibition by anti‐CD8 monoclonal antibody. VSV8 peptides substituted with pBM1/TCR contact residues (N6 and T7) retained low affinity characteristics and among pBM1 peptides substituted with residues Q6 and/or G7 present in VSV8, only pBM1(G7) was recognized, albeit with characteristics akin to those of VSV8. Despite the difference in KD values and the faster dissociation rate of multimeric VSV8/H‐2Kb as compared to pBM1/H‐2Kb complexes, similar TCR occupancy could be achieved with both multimers either at 4 or 37°C. Only TCR engagement with pBM1/H‐2Kb, however, resulted in early (Ca2+ flux) and late(CD69 expression) activation events in naive BM3.3TCR CD8 T cells. CD8 coreceptor, essential for binding of the weak agonists, was dispensable for binding of pBM1/H‐2Kb multimers and their induction of signaling in naive T cells. Hence, high number of TCR and coreceptor engagement by weak agonists fail to substitute for strong agonist TCR engagement that can be coreceptor‐independent and involve a limited number of TCR.