Marion T. Kasaian

IL-33 synergizes with IgE-dependent and IgE-independent agents to promote mast cell and basophil activation

ObjectiveMast cell and basophil activation contributes to inflammation, bronchoconstriction, and airway hyperresponsiveness in asthma. Because IL-33 expression is inflammation inducible, we investigated IL-33-mediated effects in concert with both IgE-mediated and IgE-independent stimulation.MethodsBecause the HMC-1 mast cell line can be activated by GPCR and RTK signaling, we studied the effects of IL-33 on these pathways. The IL-33- and SCF-stimulated HMC-1 cells were co-cultured with human lung fibroblasts and airway smooth muscle cells in a collagen gel contraction assay. IL-33 effects on IgE-mediated activation were studied in primary mast cells and basophils.ResultIL-33 synergized with adenosine, C5a, SCF, and NGF receptor activation. IL-33-stimulated and SCF-stimulated HMC-1 cells demonstrated enhanced collagen gel contraction when cultured with fibroblasts or smooth muscle cells. IL-33 also synergized with IgE receptor activation of primary human mast cells and basophils.ConclusionIL-33 amplifies inflammation in both IgE-independent and IgE-dependent responses.

IgE Generation and Mast Cell Effector Function in Mice Deficient in IL-4 and IL-13

IL-4 and IL-13 are potent cytokines that drive production of IgE, which is critical to the development of atopic disease. In this study, we directly compared IgE generation and IgE-dependent mast cell effector function in mouse strains lacking IL-4, IL-13, IL-4 + IL-13, or their common receptor component, IL-4Rα. Although serum IgE was undetectable under resting conditions in most animals deficient in one or both cytokines, peritoneal mast cells from mice lacking IL-4 or IL-13 had only partial reductions in surface IgE level. In contrast, peritoneal mast cells from IL-4/13−/− and IL-4Rα−/− animals were severely deficient in surface IgE, and showed no detectable degranulation following treatment with anti-IgE in vitro. Surprisingly, however, intradermal challenge with high concentrations of anti-IgE Ab induced an ear-swelling response in these strains, implying some capacity for IgE-mediated effector function in tissue mast cells. Furthermore, upon specific immunization with OVA, both IL-4/IL-13−/− and IL-4Rα−/− mice produced detectable levels of serum IgE and Ag-specific IgG1, and generated strong ear-swelling responses to intradermal administration of anti-IgE. These findings suggest that a mechanism for IgE production exists in vivo that is independent of IL-4 or IL-13.

AUTOIMMUNITY-PRONE B-1 (CD5 B) CELLS, NATURAL ANTIBODIES AND SELF RECOGNITION*

The delineation of distinct subsets committed to the production of antibodies with different antigen-binding activities supports the view of a compartmentalization and specialization of function in the B cell repertoire and is consistent with the hypothesis of a developmentally layered immune system; as originally proposed by Herzenberg and Herzenberg. On the basis of the data by Solvason and Kearney in the human fetus and our data in the adult, and in agreement with the findings of Herzenberg et al. and Hardy et al. in the mouse, we propose that the human B cell repertoire includes at least three distinct B cell subsets: B-1a cells, which develop from progenitors in the fetal splanchnic district, namely the omentum, and are maintained in adult life by virtue of their self-replenishing nature; B-1b cells, progenitors of which can be found in the splanchnic district and, perhaps, adult bone marrow; and, finally, B-2 cells, which arise in the fetal liver and are continuously replenished in adult life by progenitors in the bone marrow (Figure 5). The different B cells types are distinguished by their differential expression of surface CD5 and, perhaps, CD11b and CD14, their differential expression of CD5 mRNA, and the different classes and specificities of the Ig they produce (Figure 5). B-1 lymphocytes play a major role in autoimmunity and constitute the physiological equivalent of the neoplastic forms in various lymphoproliferative disorders, such as CLL and SLL, which are often associated with the production of monoclonal antibodies to self antigens. Human B-1a (CD5+ B) and B-1b (CD5- CD45RAlo B) cells are responsible for the production of natural (polyreactive and monoreactive) antibodies in the fetus, neonate, and adult, and can give rise to the autoantibody-producing cells characteristic of several autoimmune disease states. Our recent findings suggest that while in healthy subjects the majority of natural polyreactive antibodies is encoded in V genes in germline configuration, some polyreactive antibodies are encoded in somatically mutated V genes, in a fashion consistent with an antigen-driven process of selection of such mutations. The nature of the antigen(s) involved in these selection processes remains to be determined. Under possibly different circumstances, the application of an antigen-driven process of clonal selection to B-1a and/or B-1b cells, previously committed to natural antibody production, can result in the generation of monoreactive high affinity and possibly pathogenic autoantibodies (Figures 5A and 5B).(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin-13 Neutralization by Two Distinct Receptor Blocking Mechanisms Reduces Immunoglobulin E Responses and Lung Inflammation in Cynomolgus Monkeys

Interleukin (IL)-13 is a key cytokine driving allergic and asthmatic responses and contributes to airway inflammation in cynomolgus monkeys after segmental challenge with Ascaris suum antigen. IL-13 bioactivity is mediated by a heterodimeric receptor (IL-13Rα1/IL-4Rα) and can be inhibited in vitro by targeting IL-13 interaction with either chain. However, in cytokine systems, in vitro neutralization activity may not always predict inhibitory function in vivo. To address the efficacy of two different IL-13 neutralization mechanisms in a primate model of atopic disease, two humanized monoclonal antibodies to IL-13 were generated, with highly homologous properties, differing in epitope recognition. Ab01 blocks IL-13 interaction with IL-4Rα, and Ab02 blocks IL-13 interaction with IL-13Rα1. In a cynomolgus monkey model of IgE responses to A. suum antigen, both Ab01 and Ab02 effectively reduced serum titers of Ascaris-specific IgE and diminished ex vivo Ascaris-triggered basophil histamine release, assayed 8 weeks after a single administration of antibody. The two antibodies also produced comparable reductions in pulmonary inflammation after lung segmental challenge with Ascaris antigen. Increased serum levels of IL-13, lacking demonstrable biological activity, were seen postchallenge in animals given either anti-IL-13 antibody but not in control animals given human IgG of irrelevant specificity. These findings demonstrate a potent effect of IL-13 neutralization on IgE-mediated atopic responses in a primate system and show that IL-13 can be efficiently neutralized by targeting either the IL-4Rα-binding epitope or the IL-13Rα1-binding epitope.

Preclinical pharmacokinetics, interspecies scaling, and tissue distribution of humanized monoclonal anti-IL-13 antibodies with different IL-13 neutralization mechanisms

Numerous animal and in vitro studies suggest that neutralization of IL-13 is an attractive approach for therapeutic intervention in asthma. In this paper we describe preclinical pharmacokinetics (PK), interspecies scaling, and biodistribution of two humanized anti-IL-13 IgG1 monoclonal antibodies, Ab-01 and Ab-02, with different IL-13 neutralization mechanisms. PK parameters of Ab-01 and Ab-02 following IV or SC dosage to mouse, rat, cynomolgus monkey, and sheep, were similar. After IV administration, the elimination of anti-IL-13 antibodies was slow in all species tested and the serum clearance ranged from 0.13 mL/h/kg in monkeys to 0.81 mL/h/kg in mice. Both anti-IL-13 antibodies appeared to be confined primarily to the vascular space, as volume of distribution was relatively small (<120 mL/kg) in all species and tissue-to-serum concentration ratios (in mice and rats) were low (<0.5) in the tissues examined. The elimination half-life ranged from 3-6 days in mice to 14-17 days in monkey and sheep. In monkeys, PK parameters appeared to be approximately linear in the 1-100 mg/kg dose range. Following SC administration, the bioavailability of anti-IL-13 antibodies was 60-90% in all species tested. PK profile of Ab-02 in the model of acute airway inflammation (induced by Ascaris challenge) was, in general, similar to that in unchallenged monkeys; however, volume of distribution and clearance tended to decrease in Ascaris-challenged animals. Allometric scaling suggested that anti-IL-13 antibodies would likely to have a favorable PK profile, such as slow clearance and long terminal half-life, following IV or SC administration to humans.

Mast Cell-Dependent Contraction of Human Airway Smooth Muscle Cell-Containing Collagen Gels: Influence of Cytokines, Matrix Metalloproteases, and Serine Proteases

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.

CD5+ B lymphocytes

Summary A major proportion of CD5+ B lymphocytes are committed to the production of a discrete type of antibody that is polyreactive. Polyreactive antibodies appear to use selected V and, perhaps, D gene segments, mostly in unmutated configuration. They generally display relatively low affinity for different antigens and are often part of the primary response to foreign antigens. Polyreactive antibodies probably play a major role in vivo as a first line of defense against infectious agents not only by helping to temporarily limit the early stage of infection by directly binding to microorganisms, but also, owing to their RF-like activity, to amplify an ongoing IgG-mediated secondary response. In some cases, however, complete eradication of the invading pathogens may only be accomplished by the high affinity antibodies, mainly IgG, that appear late in the antibody response and are produced, in general, by cells that utilize a more diverse assortment of VH/VL gene combinations and/or have undergone an antigen-driven process of somatic point mutation and positive selection. Our experiments have clearly established that the “autoantibodies” normally produced by CD5+ B cells (polyreactive and low affinity) differ functionally from the autoantibodies that are characteristic of autoimmune diseases such as SLE, Hashimotos disease, and insulin-dependent diabetes mellitus (monoreactive and high affinity), which are consistently detectable only in autoimmune patients. As with cells that respond to an exogenous antigen, these cells are, in some cases, the progeny of (CD5–) B lymphocytes that underwent an antigen-driven process of somatic point mutation and positive selection. We also established, however, that under some circumstances (e.g., rheumatoid arthritis), monoreactive high affinity autoantibodies, i.e., RF, are produced by CD5+ B cells. Our preliminary experiments showed that the V genes expressed by these cells can be somatically mutated, consistent with an antigen-driven process of clonal selection. These findings do not support the view that CD5+ B cells are primordial cellular elements committed to production of germline antibodies, and suggest that they may accumulate somatic point mutation and play some role in the affinity maturation of an antibody response. CD5+ B lymphocytes may not be the only B cells capable of producing polyreactive antibodies. Studies in the mouse have revealed the existence of a cell population phenotypically similar to CD5+ B cells, but lacking the surface CD5 marker (4, 47). These CD5– B cells, named the “sister population” to reflect their putative similarity to CD5+ B cells, are thought to arise from a distinct B cell lineage (152). Although the ability of the murine “sister” (CD5–) B cells to produce antibodies similar to those made by CD5+ B lymphocytes has not been conclusively established (153, 154), we recently obtained evidence for the production of poly-reactive antibodies by an analogous (CD5–) “sister” B cell population in human peripheral blood (Kasaian et al., manuscript in preparation). The characterization of the human “sister” (CD5–) B cell population would represent a further step toward the definition of discrete functional and phenotypic layers of specialization and complexity within the B cell repertoire, as proposed by Herzenberg and Herzenberg (150). Many issues concerning the functional features of the “conventional” CD5+ B cell subset still remain to be addressed. Is there a role for CD5+ B cells in antigen presentation or in helping other B cells to produce antibodies (24)? What is the precise contribution of the self-replenishing and self-maintaining nature of CD5+ B cells to the establishment of human CD5+ B cell leukemias or lymphomas? Similar issues would most likely apply to the (CD5–) “sister” B cell subset. It is tempting to speculate that (CD5–) “sister” B cells may contain progenitors of the lymphocytes eventually producing high affinity, mainly IgG, autoantibodies in those autoimmune diseases, e.g., SLE, in which such potentially pathogenic autoantibody-producing cells have been shown to segregate mostly within the CD5– B cell compartment.

MMP dependence of fibroblast contraction and collagen production induced by human mast cell activation in a three-dimensional collagen lattice

Mast cell-fibroblast interactions may contribute to fibrosis in asthma and other disease states. Fibroblast contraction is known to be stimulated by coculture with the human mast cell line, HMC-1, or by mast cell-derived agents. Matrix metalloproteinases (MMPs) can also mediate contraction, but the MMP-dependence of mast cell-induced fibroblast contractility is not established, and the consequences of mast cell activation within the coculture system have not been fully explored. We demonstrate that activation of primary human mast cells (pHMC) with IgE receptor cross-linking, or activation of HMC-1 with C5a, enhanced contractility of human lung fibroblasts in a three-dimensional collagen lattice system. This enhanced contractility was inhibited by the pan-MMP antagonist, batimastat, and was transferrable in the conditioned medium of activated mast cells. Exogenously added MMPs promoted gel contraction by mediating the proteolytic activation of latent transforming growth factor-beta (TGF-beta). Consistent with this, fibroblast contraction induced by mast cell activation was enhanced by addition of excess latent TGF-beta to the cultures. Batimastat inhibited this response, suggesting that MMPs capable of activating latent TGF-beta were released following mast cell activation in coculture with fibroblasts. Collagen production was also stimulated by activated mast cells in an MMP-dependent manner. MMP-2 and MMP-3 content of the gels increased in the presence of activated mast cells, and inhibition of these enzymes blocked the contractile response. These findings demonstrate the MMP dependence of mast cell-induced fibroblast contraction and collagen production.

An IL-4/IL-13 Dual Antagonist Reduces Lung Inflammation, Airway Hyperresponsiveness, and IgE Production in Mice

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.

Pharmacokinetic and Pharmacodynamic Modeling of a Humanized Anti-IL-13 Antibody in Naive and Ascaris-Challenged Cynomolgus Monkeys

PurposeNeutralization of IL-13 is an attractive approach for treatment of asthma. In this report, we developed a novel PK–PD model that described the relationship between the circulating concentrations of total IL-13 and a neutralizing anti-IL-13 antibody (Ab-02) in the model of acute airway inflammation induced by Ascaris challenge to cynomolgus monkeys, as well as in naive monkeys.MethodsCynomolgus monkeys were administered a single intravenous or subcutaneous dose of Ab-02. Total IL-13 and Ab-02 concentrations were measured by immunoassays.ResultsModeling and simulations indicated that: (1) Ascaris challenge induced ∼ three-fold increase in circulating IL-13 concentrations, when compared to naive animals, consistent with the notion that Ascaris-induced airway inflammation was IL-13-mediated; (2) the transient increase in total IL-13 concentrations observed in both naive and Ascaris-challenged monkeys following Ab-02 administration was due to the increase in Ab-02-bound IL-13, while free IL-13 was decreased; and (3) the extent and duration of neutralization of circulating IL-13 were different in naive and Ascaris-challenged monkeys for the same Ab-02 dose regimen.ConclusionsThe PK–PD model presented in this report may be applied to study drug–ligand interactions when a free ligand cannot be directly assayed but total ligand concentrations are modulated by the drug administration.