Identification and categorization of inducible mast cell genes in a subtraction library.

Mast cells play an important role in allergic inflammation by releasing inducible proinflammatory cytokines. While many inducible genes have been identified, we hypothesized that a significant number remain to be identified. We thus constructed an activation-specific mast cell subtraction library to establish a profile of induced genes in mast cells following allergic stimulation. To date, we have sequenced 150 cDNA clones. Among them, we have isolated 22 known genes whose expression has not been reported in mast cells, and an additional 26 cDNA clones which do not have significant homology to known genes in the Genbank database. We next selected 10 cDNA clones with strong signals by differential plaque hybridization. Of these cDNA clones, five genes were induced in mast cells upon Fc epsilon RI-mediated stimulation. They are cofilin, annexinVI, interferon (IFN)-beta, serglycin, and a novel inducible mast cell (IMC) gene, IMC-415. Characterization and relevant studies of this novel gene and other inducible known genes in mast cells will provide insight into the functions of mast cells in mammalian biology.

Identification of a novel hydroxyproline-rich glycoprotein as the major allergen in Parthenium pollen.

BACKGROUND: The airborne pollen of the Compositae weed, Parthenium hysterophorus, is a major cause of allergic rhinitis in the Indian subcontinent and in certain parts of the southern United States and western Australia. Earlier studies have identified a 31 kd protein as the major allergen in Parthenium pollen. OBJECTIVE: This study was undertaken to carry out the purification, immunochemical characterization, sequencing, and epitope analysis of this major allergen, designated as Par h I. METHODS: The IgE-binding activity of the allergen was evaluated by immunoblot and inhibition ELISAs. Pronase digestion, periodate oxidation, and chemical deglycosylation were performed to determine the role of peptide and carbohydrate components of the allergen in IgE binding. RESULTS: The data provide evidence for the involvement of carbohydrate moieties on Par h 1 in its IgE-binding ability. The N-terminal 91 amino acid sequence of Par h 1 shows 81% identity with a protein from sunflower anther, and the hydroxyproline-rich region of Par h 1 is 30% to 40% identical to similar stretches in extensins, a class of hydroxyproline-rich cell wall glycoproteins from different plant species. IgE antibodies in the sera of individuals allergic to Parthenium cross-reacted with a 50 kd hydroxyproline-arabinose-rich extensin precursor from potato tuber, and this binding was periodate-sensitive. CONCLUSIONS: It appears that a group of soluble plant glycoproteins, which are related to the ubiquitous extensins, have certain carbohydrate-containing IgE-binding epitopes that may contribute to allergenic cross-reactivity among specific pollens and foods.

The use of murine polyclonal anti-idiotypic antibodies as surrogate allergens in the diagnosis of Parthenium hypersensitivity.

BACKGROUND: Anti-idiotypic antibodies (Ab-2), which are the mirror images of idiotypic antibodies (Ab-1), may be useful as diagnostic reagents and for use as immunogen to induce antigen-specific immune responses. METHODS AND RESULTS: To explore the biologic potential of Ab-2 as diagnostic reagents in allergic diseases, murine mouse (m) Ab-2 were raised by immunizing Balb/c mice with affinity purified rabbit (r) Ab-1 specific for the pollen of Parthenium hysterophorus, an allergenic weed that grows wild on the Indian subcontinent and in Australia, Mexico, and the southern United States. Affinity purified Parthenium-specific human (h)AB-1 could successfully inhibit the binding of mAb-2 to immobilized rAb-1. Further, Balb/c mice immunized with mAb-2 induced Parthenium-specific anti-anti-idiotypic IgE and IgG antibodies. Specificity of the Ab-2 was confirmed by the ability of Parthenium pollen extracts to inhibit the binding of allergen-specific IgE and IgG Ab-1 in the sera of patients with rhinitis to immobilized mAb-2. Parthenium-sensitive patients with rhinitis who had positive results on skin prick tests to Parthenium pollen extracts also responded with a positive skin reaction to mAb-2. CONCLUSION: Our data demonstrate that Parthenium-specific mAb-2 may be of value as surrogate allergens in allergen standardization and for in vitro diagnosis.

Immortalization of mouse bone marrow-derived mast cells with Ad12-SV40 virus.

Mast cells arise in cultures of murine bone marrow in medium supplemented with interleukin-3 (IL-3). In the present study, we report the development of long-term mast cell lines from murine bone-marrow-derived cultured mast cells (BMCMC) following inoculation with adenovirus 12-simian virus 40 (Ad12-SV40) hybrid virus. One culture of Ad12-SV40 immortalized BMCMC (designated as MCP-5) was selected for further analysis. These transformed cells appear similar in morphology and histochemistry to the primary BMCMC from which they are derived and did not shed infectious virus into the culture supernatants. In addition, these cells synthesize predominantly chondroitin sulfate proteoglycans and contain histamine which is released following a physiologic stimulus. Limiting-dilution single-cell cloning produced five independent mast cell lines (MCP-5.1 to MCP-5.5). Southern blot analysis of genomic DNA isolated from these single-cell clones demonstrates different patterns of viral integration in all the five clones. All clones retain responsiveness to an exogenous source of IL-3 for growth and proliferation. Each single-cell clone also demonstrates a unique pattern of cytokine gene expression in response to calcium ionophore A23187 and phorbol-12-myristate-13-acetate. This suggests that within a culture of BMCMC there are differences in cytokine gene expression that vary from one cell to another. The availability of immortalized mast cell lines derived from murine bone marrow which retain their growth factor responsiveness and the ability to respond to degranulating stimuli should facilitate future studies of mast cell biology.

Mast cells chemotax to laminin with enhancement after IgE-mediated activation.

The increase of mast cells at sites of tissue inflammation suggests the production of local factors chemotactic for mast cells. In this report, we demonstrate that the murine mast cell line PT18 and primary mouse bone marrow-derived mast cells chemotax to the basement membrane glycoprotein laminin, and that the synthetic laminin A chain-derived peptide, PA22-2, represents a region of laminin that contains a major chemoattractant site. Mast cell chemotaxis to laminin is enhanced after activation of mast cells by the calcium ionophore, A23187, or PMA and by sensitization of the cells with IgE followed by exposure to antigen. Chemotaxis is not increased in the presence of IL-3 and is independent of mast cell degranulation, as histamine release did not occur when cells were activated with PMA. Mast cell chemotaxis to laminin and its enhancement by IgE-dependent mast cell activation provides a mechanism by which these cells may be attracted to sites of tissue injury. Such activity may be particularly relevant in the response of host tissues to inflammation accompanying parasitic infestations, allergic reactions, and wound healing.