Identification of Microcystis aeruginosa Peptides Responsible for Allergic Sensitization and Characterization of Functional Interactions between Cyanobacterial Toxins and Immunogenic Peptides.

BACKGROUND: The cyanobacterium species Microcystis aeruginosa produces microcystin and an array of diverse metabolites believed responsible for their toxicity and/or immunogenicity. Previously, chronic rhinitis patients were demonstrated to elicit a specific IgE response to nontoxic strains of M. aeruginosa by skin-prick testing, indicating that cyanobacteria allergenicity resides in a non-toxin-producing component of the organism. OBJECTIVES: We sought to identify and characterize M. aeruginosa peptide(s) responsible for allergic sensitization in susceptible individuals, and we investigated the functional interactions between cyanobacterial toxins and their coexpressed immunogenic peptides. METHODS: Sera from patients and extracts from M. aeruginosa toxic [MC(+)] and nontoxic [MC(-)] strains were used to test IgE-specific reactivity by direct and indirect ELISAs; 2D gel electrophoresis, followed by immunoblots and mass spectrometry (MS), was performed to identify the relevant sensitizing peptides. Cytotoxicity and mediator release assays were performed using the MC(+) and MC(-) lysates. RESULTS: We found specific IgE to be increased more in response to the MC(-) strain than the MC(+) strain. This response was inhibited by preincubation of MC(-) lysate with increasing concentrations of microcystin. MS revealed that phycocyanin and the core-membrane linker peptide are the responsible allergens, and MC(-) extracts containing these proteins induced ß-hexosaminidase release in rat basophil leukemia cells. CONCLUSIONS: Phycobiliprotein complexes in M. aeruginosa have been identified as the relevant sensitizing proteins. Our finding that allergenicity is inhibited in a dose-dependent manner by microcystin toxin suggests that further investigation is warranted to understand the interplay between immunogenicity and toxicity of cyanobacteria under diverse environmental conditions. CITATION: Geh EN, Ghosh D, McKell M, de la Cruz AA, Stelma G, Bernstein JA. 2015. Identification of Microcystis aeruginosa peptides responsible for allergic sensitization and characterization of functional interactions between cyanobacterial toxins and immunogenic peptides. Environ Health Perspect 123:1159-1166; http://dx.doi.org/10.1289/ehp.1409065.

Characterization of memory B cells responsible for affinity maturation of anti- (4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies.

We searched for memory B cells responsible for high-affinity anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody production by C57BL/6 mice immunized with NP-chicken γ-globulin (CGG), using flow cytometry. We first prepared transfectants expressing B-cell antigen receptor (BCR) of known affinity as a memory B-cell model as well as NP-allophycocyanin (APC) of different NP valences, NP(lo), NP(med) and NP(hi). We then used the latter as probes capable of distinguishing BCR affinities: NP(lo)-APC bound to BCRs with an affinity higher than 3.4 × 10(6) M(-1), while NP(med)-APC bound to those with a higher than germline affinity. B cells capable of binding to NP(lo)-APC appeared in spleens on day 14 post-immunization, and harbored Tyr95 (Tyr95 type) as well as a mutation from Trp33 to Leu. B cells with BCRs harboring Gly95 (Gly95 type) appeared only in the NP(med)-APC-binding fraction on day 56 and in the NP(lo)-APC-binding fraction on day 77, indicating that this long duration was necessary for Gly95 type B cells to acquire high affinity and to become a member of the group of memory B cells with high affinity. Administration of NP-CGG on day 77 caused little change in the proportion of the Gly95 type in NP(lo)-APC-binding B cells in the following 2 weeks but brought about an increase in the number of high-affinity antibody-secreting cells (ASC), suggesting that the memory B-cell compartment established was maintained at a later stage and supplied high-affinity ASCs. The relationship between these Gly95 type memory B cells and ASCs is discussed.

Different B cell populations mediate early and late memory during an endogenous immune response.

Memory B cells formed in response to microbial antigens provide immunity to later infections; however, the inability to detect rare endogenous antigen-specific cells limits current understanding of this process. Using an antigen-based technique to enrich these cells, we found that immunization with a model protein generated B memory cells that expressed isotype-switched immunoglobulins (swIg) or retained IgM. The more numerous IgM(+) cells were longer lived than the swIg(+) cells. However, swIg(+) memory cells dominated the secondary response because of the capacity to become activated in the presence of neutralizing serum immunoglobulin. Thus, we propose that memory relies on swIg(+) cells until they disappear and serum immunoglobulin falls to a low level, in which case memory resides with durable IgM(+) reserves.

Intraclonal competition inhibits the formation of high-affinity antibody-secreting cells.

Protective immunity requires a diverse, polyclonal B cell repertoire. We demonstrate that affinity maturation of the humoral response to a hapten is impaired when preexisting clonally restricted cells recognizing the hapten are dominant in the B cell repertoire. B1-8i(+/-) mice, which feature a high frequency of B cells with nitrophenyl (NP)-binding specificity, respond to NP-haptenated proteins with the production of NP-specific Abs, but affinity maturation is impaired due to insufficient generation of high-affinity Ab-producing cells. We manipulated the frequency of NP-specific B cells by adoptive transfer of B1-8 B cells into naive, wild-type recipients. Remarkably, when 10(4) B1-8 B cells were transferred, these cells supported efficient affinity maturation and plasma cell differentiation. In contrast, when 10(6) B1-8 cells were transferred, affinity maturation did not occur. These data indicate that restricting the frequency of clonally related B cells is required to support affinity maturation.

Preparation of nitrocellulose (NC) immuno-affinity membrane for purification of rAPC antibody.

In this study, recombinant allophycocyanin (rAPC) with a purity of 98% was transferred from a gel to a nitrocellulose (NC) membrane to develop a simple and efficient immuno-affinity membrane. Atomic force microscopy (AFM) was used to investigate the surface topography of the affinity membrane and its characterization indicated that rAPC easily forms trimers or hexamers on the membrane surface on use of the given transfer method. The hydrodynamic radius (R(h)) of the rAPC aggregation was equal to 103 nm or 365 nm according to dynamic light scattering (DLS), which was in agreement with the result obtained by AFM. Based on the specific immunological reaction of antigen and antibody, anti-APC antibodies were purified from rabbit polyclonal serum in a single step. The amount of absorbed antibody was 5.79 mg/g membrane according to analysis by ELISA methods. The purity of antibodies was up to 98% according to SDS-PAGE. The adsorption-desorption cycle of rAPC was repeated six times using the same immuno-affinity membrane, and there was no significant loss in adsorption capacity. The method provides a novel and efficient immunological affinity membrane for the purification of antibodies.

Identification of antigen-capturing cells as basophils.

Binding of intact Ag is a hallmark of Ag-specific B cells. Apart from B cells, a small number of non-B cells can bind Ag with comparable efficacy as B cells and are found in the peripheral blood, spleen, and bone marrow of mice. This population has been observed for a long time and recently named "Ag-capturing cells." Their identity remained enigmatic. In this study, we show that these cells are basophilic granulocytes. Their ability to capture Ags is dependent on surface IgE receptors and on Ag-specific plasma IgE molecules appearing after immunization. Several surface markers including surface bound IgE, IL-3R, CD45, CD16/32, and the chemokine receptor CCR2 were used to clearly identify these cells. Cross-linkage of surface Igs results in the release of large amounts of IL-4 and IL-6. The data identify basophils as Ag-capturing cells and support the concept of basophils as important regulators of humoral immune responses.

Distribution of Synechococcus sp. and Synechococcus bacillaris in the waters of the Straits of Magellan (April 1995-early austral autumn).

During the last oceanographic cruise carried out in the Straits of Magellan (April 1995), a serological approach was used in order to determine the distribution and composition of the picophytoplankton community with respect to two cyanobacteria species, Synechococcus sp. and bacillaris, characterized respectively by phycoerythrin and phycocyanin as the main accessory photosynthetic pigment. In the period examined, the Straits were characterized by generally low concentrations of total picophytoplankton (10(5)-10(6) cells/l). The qualitative composition of the community showed the prevalence of the species Synechococcus sp. in the Pacific basin, whereas S. bacillaris appears to be predominant in the central area. The immunofluorescence method proved to be effective in the study of the diversity of these microorganisms in aquatic environments.