Graham A. Mackay

Comparison of IgE and IgG antibody-dependent cytotoxicity in vitro and in a SCID mouse xenograft model of ovarian carcinoma.

Allergic reactions are mediated by IgE antibodies bound to high‐affinity receptors on mast cells in peripheral tissues and are characterized by their immediacy and hypersensitivity. These properties could also be advantageous in immunotherapy against cancer growth in peripheral tissues. We have constructed chimeric IgE and IgG1 antibodies with murine V regions and human C regions corresponding to the MOv18 monoclonal antibody against the human ovarian tumor‐associated antigen, folate binding protein. The antibodies exhibited the expected binding affinities for antigen and Fc receptors, and effector activities with human basophils and platelets in vitro. The protective activities of MOv18‐IgE and MOv18‐IgG1 were compared in a SCID mouse xenograft model of ovarian carcinoma. The beneficial effects of MOv18‐IgE were greater and of longer duration than those of MOv18‐IgG1. Our results suggest that the allergic reaction could be harnessed for the suppression of ovarian tumors.

Structure based design and characterization of peptides that inhibit IgE binding to its high-affinity receptor

We have designed synthetic peptide inhibitors of the interaction between IgE and its high affinity receptor, FcεRI. The structure of the second domain of CD2 was used as a modelling template for the second α-chain domain of FcεRI, the C-C′ loop of which has been implicated in the interaction with IgE. An L-amino acid peptide and a retro-enantiomeric D-amino acid peptide were designed to mimic the conformation of the C-C′ region. Both peptides were cyclized by disulphide bond formation between terminal cysteine residues, and show mirror image symmetry by circular dichroism analysis. The C-C′ peptide mimics act as competitive inhibitors of lgE binding. The cyclic L- and retro D-peptides exhibited K Ds of approximately 3 μM and 11 μM, respectively, for IgE. Further, the peptides inhibit IgE-mediated mast cell degranulation, an in vitro model of an allergic response.

The Src kinase Lyn is a negative regulator of mast cell proliferation

Previous investigators have reported that deletion of the protein tyrosine kinase Lyn alters mast cell (MC) signaling responses but does not affect or reduces the cytokine‐mediated proliferation of mouse bonemarrow‐derived MC (BMMC) precursors and of mature MC. We observed that Lyn‐deficient mice have more peritoneal MC than wild‐type (WT) mice. Studies to explore this unexpected result showed that Lyn−/− BM cells expand faster than WT cells in response to interleukin (IL)‐3 and stem‐cell factor over the 4–5 weeks required to produce a >95% pure population of granular, receptor with high affinity for immunoglobulin E‐positive BMMC. Furthermore, differentiated Lyn−/− BMMC continue to proliferate more rapidly than WT BMMC and undergo less apoptosis in response to cytokine withdrawal. Additionally, Lyn−/− BMMC support greater IL‐3‐mediated phosphorylation of the prosurvival kinase, Akt, and the proliferative kinase, extracellular‐regulated kinase 1/2. These results identify Lyn as a negative regulator of murine MC survival and proliferation.

Basis of the 1:1 stoichiometry of the high affinity receptor Fc epsilon RI-IgE complex.

Abstract A soluble fragment of the high-affinity IgE receptor FcεRI α-chain (sFcεRIα) binds to the Fc fragment of IgE (IgE-Fc) as a 1:1 complex. IgE-Fc consists of a dimer of the Cε2, Cε3 and Cε4 domains of the ε-heavy chain of IgE. This region of IgE has been modelled on the crystal structure of the Fc region of IgG1, which exhibits twofold rotational symmetry. This implies that IgE should be divalent with respect to its ligands. X-ray scattering studies reveal however that the twofold rotational symmetry of IgE-Fc is perturbed by a bend in the linker region between the Cε2 and Cε3 domains. The 1:1 stoichiometry could then arise from the conformational asymmetry or from steric occlusion of one of the sites by the overhanging Cε2 domains. To test this hypothesis we have expressed a recombinant ε-chain fragment containing Cε3 and Cε4. This product, Fcε3–4, is secreted from cells as a disulphide linked dimer and binds with higher affinity than either IgE or IgE-Fc to cell surface FcεRI. Titration experiments, together with molecular mass measurements of the Fcε3–4/sFcεRIα complex, reveal that Fcε3–4 binds only a single receptor molecule. This excludes the possibility that steric hindrance by Cε2 accounts for the unexpected stoichiometry.

The MS4A family: counting past 1, 2 and 3

The MS4A (membrane‐spanning 4‐domain family, subfamily A) family of proteins contains some well‐known members including MS4A1 (CD20), MS4A2 (FcɛRIβ) and MS4A3 (HTm4). These three MS4A family members are expressed on the cell surface of specific leukocyte subsets and have been well characterized as having key roles in regulating cell activation, growth and development. However, beyond MS4A1‐3 there are a large number of related molecules (18 to date in humans) where our understanding of their biological roles is at a relatively nascent stage. This review examines the larger MS4A family focusing on their structure, expression, regulation and characterized and/or emerging biological roles. Our own work on one family member MS4A8B, and its possible role in epithelial cell regulation, is also highlighted.

Functional Expression of IgG-Fc Receptors in Human Airway Smooth Muscle Cells

IgE-Fc receptors and IgG-Fc receptors are expressed on hematopoietic cells, but some evidence suggests that these receptors are also found on nonhematopoietic cells, including human airway smooth muscle (hASM) cells. Our study characterizes the expression of IgE-Fc receptors (FcεRI/CD23) and IgG-Fc receptors (FcγRs-I, -II, and -III) in cultured hASM cells by flow cytometry and Western blotting, and the functional activity of receptors was determined through quantification of cell proliferation and released cytokines. Expression of Fc receptor-linked intracellular signaling proteins and phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1/2 and p38(MAPK) in hASM cells was examined by Western blotting. Expression of FcεRI and CD23 was not detectable in hASM cells. However, FcγRI and FcγRII were shown to be expressed on these cells. Specific antibodies, validated using transfected cell lines, revealed that the inhibitory IgG receptor, FcγRIIb, was the most abundant Fc receptor subtype expressed. Although cross-linking FcγR with heat-aggregated γ globulin (HAGG) did not induce detectable cell stimulation, pretreating hASM cells with HAGG significantly inhibited IL-1α-induced increases in cytokine levels and basic fibroblast growth factor-induced cell proliferation. This inhibitory effect of HAGG was abrogated by preincubation of cells with an anti-FcγRIIb antigen-binding fragment (Fab). Expression of proteins involved in the canonical FcγRIIb inhibitory signaling pathway was established in hASM cells. Pretreatment of hASM cells with HAGG significantly inhibited IL-1α- and basic fibroblast growth factor-induced extracellular signal-regulated kinase 1/2 and p38(MAPK) phosphorylation. This study identifies functional expression of FcγRIIb in hASM cells, with the potential to suppress their remodeling and immunomodulatory roles.

Plasminogen-Stimulated Inflammatory Cytokine Production by Airway Smooth Muscle Cells Is Regulated by Annexin A2

Plasminogen has a role in airway inflammation. Airway smooth muscle (ASM) cells cleave plasminogen into plasmin, a protease with proinflammatory activity. In this study, the effect of plasminogen on cytokine production by human ASM cells was investigated in vitro. Levels of IL-6 and IL-8 in the medium of ASM cells were increased by incubation with plasminogen (5-50 μg/ml) for 24 hours (P < 0.05; n = 6-9), corresponding to changes in the levels of cytokine mRNA at 4 hours. The effects of plasminogen were attenuated by α2-antiplasmin (1 μg/ml), a plasmin inhibitor (P < 0.05; n = 6-12). Exogenous plasmin (5-15 mU/ml) also stimulated cytokine production (P < 0.05; n = 6-8) in a manner sensitive to serine-protease inhibition by aprotinin (10 KIU/ml). Plasminogen-stimulated cytokine production was increased in cells pretreated with basic fibroblast growth factor (300 pM) in a manner associated with increases in urokinase plasminogen activator expression and plasmin formation. The knockdown of annexin A2, a component of the putative plasminogen receptor comprised of annexin A2 and S100A10, attenuated plasminogen conversion into plasmin and plasmin-stimulated cytokine production by ASM cells. Moreover, a role for annexin A2 in airway inflammation was demonstrated in annexin A2-/- mice in which antigen-induced increases in inflammatory cell number and IL-6 levels in the bronchoalveolar lavage fluid were reduced (P < 0.01; n = 10-14). In conclusion, plasminogen stimulates ASM cytokine production in a manner regulated by annexin A2. Our study shows for the first time that targeting annexin A2-mediated signaling may provide a novel therapeutic approach to the treatment of airway inflammation in diseases such as chronic asthma.

Human mast cell line-1 (HMC-1) cells transfected with FcεRIα are sensitive to IgE/antigen-mediated stimulation demonstrating selectivity towards cytokine production

Mast cells play important roles in allergic and inflammatory diseases. Efforts to better understand human mast cell activation and develop novel inhibitory agents have been hampered by the lack of suitable human mast cell lines. The HMC-1 mast cell line has been extensively used, but lacks native expression of the human high-affinity IgE receptor FcεRI limiting its applications. We have stably transfected HMC-1 cells with the IgE-binding α-subunit of FcεRI to generate HMCα cells that are antigen-responsive. We have used flow cytometry, cell signaling assays, pharmacological pathway inhibitors and cell functional assays to characterize the properties of HMCα cells. IgE/antigen responses were compared with those of the adenosine receptor agonist NECA. Surface expression of FcεRI in HMCα cells was demonstrated and was enhanced by prior sensitization with IgE. Activation of HMCα cells with IgE/antigen did not produce degranulation, but did lead to release of numerous cytokines. Whilst there was no measurable increase of intracellular Ca(2+) or marked general changes in protein tyrosine phosphorylation, IgE/antigen stimulation of HMCα cells enhanced phosphorylation of p38(MAPK) and Erk. Inhibitors of these pathways, as well as the src kinase inhibitor PP2, attenuated IgE/antigen-induced cytokine release. In summary, we have generated and characterized HMCα cells and show that they are a useful and relevant human mast cell model to examine FcεRI stabilization, signaling and mediator release. We envisage that HMCα cells will have utility in understanding the importance of mast cells in human allergic disease and in assessing the activity of novel anti-allergic compounds.

A comparison of rat peritoneal mast cells purified using Percoll and path-O-cyte 4

Rat peritoneal mast cells (RPMC) purified through Percoll and a commercially available 35% bovine serum albumin (BSA) solution (Path-O-Cyte 4) are compared over a range of criteria with mixed peritoneal cell suspensions. Both the purity and spontaneous histamine release were slightly better in RPMC isolated through Percoll and histamine recovery was considerably greater using this method as opposed to Path-O-Cyte 4. The responsitivity of cells isolated by either method to compound 48/80 and ionomycin was comparable with that seen in non-purified RPMC. However, the histamine release induced by anti-rat IgE and concanavalin A was reduced in the purified cells especially in those isolated through BSA. Histamine release induced by IgE-directed ligands was potently inhibited by theophylline, isobutyl methyl xanthine, disodium cromoglycate and nedocromil sodium in both non-purified and Percoll-purified RPMC. In total, this study has shown that highly purified and viable RPMC can be obtained through Percoll purification. This method was seen to be generally superior to that of Path-O-Cyte 4.

Secreted factors from human mast cells trigger inflammatory cytokine production by human airway smooth muscle cells.

Background: A notable feature of allergic asthma is the infiltration of mast cells into smooth muscle in the human airway. Thus, mast cells and human airway smooth muscle (hASM) cells are likely to exhibit mutual functional modulation via direct cell-cell contact or through released factors. This study examined mast cell modulation of hASM cell cytokine release. Methods: The mast cell line HMCα was used to model mast cell function. hASM cells were either co-cultured directly with resting or IgE/antigen-stimulated HMCα cells or treated with HMCα-conditioned media to examine the impact on cytokine release. The activation pathways triggered in hASM cells by the mast cell-derived factors were examined through the use of selective inhibitors and by Western blotting. Results: HMCα cells, or their conditioned media, induced the expression of cytokines (IL-8 and IL-6) by hASM cells at both the mRNA and the protein level. Cytokine expression in hASM cells was greatly amplified when HMCα cells were IgE/antigen-activated. The effects of the conditioned media were not mediated by the chemokines MCP-1 and MIP-1α or by exosomes. While the mast cell-derived factor(s) increased p38MAPK phosphorylation in hASM cells, cytokine production was not inhibited by the p38MAPK inhibitor SB203580. hASM cell production of IL-8 induced by HMCα condition media but not IL-6 was, however, attenuated by the Src tyrosine kinase inhibitor PP2. Conclusions: Our study shows that the release of soluble mediators by activated mast cells can stimulate hASM cells to elicit production of proinflammatory cytokines that may then exacerbate airway inflammation in asthma.