No abstract

The present studies were carried out to identify surface molecules involved in the cytotoxic effector function of a human natural killer (NK) clone termed JT9. This clone represents a mature T lymphocyte (T3+T8+T11+) mediating NK-like activity. Using JT9 for immunization, one monoclonal antibody termed anti-NKTa was selected that blocked the cytotoxicity of the clone towards K562 cells. Reactivity of anti-NKTa antibody was assessed using a large panel of lymphoid and nonlymphoid cells including a variety of cloned cell lines with either cytotoxic T lymphocyte (CTL) or NK-like activity. Among all cells tested, only two individual clones, JT9 and JT10, were found to express NKTa antigen. JT10 was derived independently from the same individual as JT9 and also represents a mature T cell (T3+T8+T11+) mediating NK-like activity. Like the Ti structure on CTL clones, the molecule defined by anti-NKTa was shown to be membrane associated with T3 in co-modulation experiments. Moreover, anti-NKTa precipitated a 90 kD heterodimeric structure in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of 125I surface-labeled JT9 cells. The blocking capacity of anti-NKTa was evaluated in cytotoxicity assays using a panel of target cells. The influence of anti-T3 was tested in parallel and it was found that both anti-NKTa and anti-T3 blocked the cytotoxicity of the cloned cells against all targets. Given the potential role of 90 kD molecules as antigen-receptor structures, the specificity of the two NKTa+ NK clones was compared and found superimposable when assessed using 15 in vitro established cell lines. However, in contrast to conventional CTL clones, the expression of cytotoxicity by JT9 and JT10 was not dependent upon recognition of class I or class II major histocompatibility complex gene products on the target cells. In addition, the cytotoxicity of these T8+ NK active clones could not be blocked by anti-T8 antibodies. Taken together, the present data suggest that the specificity of one population of human NK active lymphocytes is determined by clonotypic structures. The NKTa determinant identified here appears to belong to the same family of molecules as Ti structures, previously identified on antigen-specific T lymphocytes.

IL-4 and IL-13 comprise promising targets for therapeutic interventions in asthma and other Th2-associated diseases, but agents targeting either IL-4 or IL-13 alone have shown limited efficacy in human clinical studies. Because these cytokines may involve redundant function, dual targeting holds promise for achieving greater efficacy. We describe a bifunctional therapeutic targeting IL-4 and IL-13, developed by a combination of specific binding domains. IL-4-targeted and IL-13-targeted single chain variable fragments were joined in an optimal configuration, using appropriate linker regions on a novel protein scaffold. The bifunctional IL-4/IL-13 antagonist displayed high affinity for both cytokines. It was a potent and efficient neutralizer of both murine IL-4 and murine IL-13 bioactivity in cytokine-responsive Ba/F3 cells, and exhibited a half-life of approximately 4.7 days in mice. In a murine model of ovalbumin-induced ear swelling, the bifunctional molecule blocked both the IL-4/IL-13-dependent early-phase response and the IL-4-dependent late-phase response. In the ovalbumin-induced lung inflammation model, the bifunctional IL-4/IL-13 antagonist reduced the IL-4-dependent rise in serum IgE titers, and reduced IL-13-dependent airway hyperresponsiveness, lung inflammation, mucin gene expression, and serum chitinase responses. Taken together, these findings demonstrate the effective dual blockade of IL-4 and IL-13 with a single agent, which resulted in the modulation of a more extensive range of endpoints than could be achieved by targeting either cytokine alone.

SummaryInterleukin-4 (IL-4) and IL-13 are critical drivers of immune activation and inflammation in ulcerative colitis, asthma and other diseases. Because these cytokines may have redundant function, dual targeting holds promise for achieving greater efficacy. We have recently described a bifunctional therapeutic targeting IL-4 and IL-13 developed on a novel protein scaffold, generated by combining specific binding domains in an optimal configuration using appropriate linker regions. In the current study, the bifunctional IL-4/IL-13 antagonist was evaluated in the murine oxazoloneinduced colitis model, which produces disease with features of ulcerative colitis. The bifunctional IL-4/IL-13 antagonist reduced body weight loss throughout the 7-day course of the model, and ameliorated the increased colon weight and decreased colon length that accompany disease. Colon tissue gene expression was modulated in accordance with the treatment effect. Concentrations of serum amyloid P were elevated in proportion to disease severity, making it an effective biomarker. Serum concentrations of the bifunctional IL-4/IL-13 antagonist were inversely proportional to disease severity, colon tissue expression of pro-inflammatory genes, and serum amyloid P concentration. Taken together, these results define a panel of biomarkers signifying engagement of the IL-4/IL-13 pathway, confirm the T helper type 2 nature of disease in this model, and demonstrate the effectiveness of dual cytokine blockade.

No abstract

In asthma, mast cells infiltrate the airway smooth muscle cell layer and secrete proinflammatory and profibrotic agents that contribute to airway remodeling. To study the effects of mast cell activation on smooth muscle cell-dependent matrix contraction, we developed coculture systems of human airway smooth muscle cells (HASM) with primary human mast cells derived from circulating progenitors or with the HMC-1 human mast cell line. Activation of primary human mast cells by IgE receptor cross-linking or activation of HMC-1 cells with C5a stimulated contraction of HASM-embedded collagen gels. Contractile activity could be transferred with conditioned medium from activated mast cells, implicating involvement of soluble factors. Cytokines and proteases are among the agents released by activated mast cells that may promote a contractile response. Both IL-13 and IL-6 enhanced contraction in this model and the activity of IL-13 was ablated under conditions leading to expression of the inhibitory receptor IL-13Rα2 on HASM. In addition to cytokines, matrix metalloproteinases (MMPs), and serine proteases induced matrix contraction. Inhibitor studies suggested that, although IL-13 could contribute to contraction driven by mast cell activation, MMPs were critical mediators of the response. Both MMP-1 and MMP-2 were strongly expressed in this system. Serine proteases also contributed to contraction induced by mast cell-activating agents and IL-13, most likely by mediating the proteolytic activation of MMPs. Hypercontractility is a hallmark of smooth muscle cells in the asthmatic lung. Our findings define novel mechanisms whereby mast cells may modulate HASM-driven contractile responses.

In obesity, IL-13 overcomes insulin resistance by promoting anti-inflammatory macrophage differentiation in adipose tissue. Endogenous IL-13 levels can be modulated by the IL-13 decoy receptor, IL-13Rα2, which inactivates and depletes the cytokine. In this study, we show that IL-13Rα2 is markedly elevated in adipose tissues of obese mice. Mice deficient in IL-13Rα2 had high expression of IL-13 response markers in adipose tissue, consistent with increased IL-13 activity at baseline. Moreover, exposure to the type 2 cytokine-inducing alarmin, IL-33, enhanced serum and tissue IL-13 concentrations and elevated tissue eosinophils, macrophages, and type 2 innate lymphoid cells. IL-33 also reduced body weight, fat mass, and fasting blood glucose levels. Strikingly, however, the IL-33-induced protection was greater in IL-13Rα2-deficient mice compared with wild-type littermates, and these changes were largely attenuated in mice lacking IL-13. Although IL-33 administration improved the metabolic profile in the context of a high fat diet, it also resulted in diarrhea and perianal irritation, which was enhanced in the IL-13Rα2-deficient mice. Weight loss in this group was associated with reduced food intake, which was likely related to the gastrointestinal effects. These findings outline both potentially advantageous and deleterious effects of a type 2-skewed immune response under conditions of metabolic stress, and identify IL-13Rα2 as a critical checkpoint in adipose tissues that limits the protective effects of the IL-33/IL-13 axis in obesity.

Proteomic profiles in serum and sputum distinguished controlled and poorly controlled asthmatics, and were maintained over time. Findings support a link between sputum neutrophil markers and loss of asthma control.