We first determined that either IgE or IgG1 cross-linking could promote IL-3 mRNA expression by bone-marrowCderived murine basophils in vitro (Fig

We first determined that either IgE or IgG1 cross-linking could promote IL-3 mRNA expression by bone-marrowCderived murine basophils in vitro (Fig. rejection following challenge infection. Results Isotype-Switched Antibodies Support Helminth-Induced Basophil Expansion. We previously reported a crucial role for isotype switched antibodies in providing protective immunity against the enteric helminth (18). To further assess the role of such antibodies, we used AID?/? mice, which contain normal numbers of B cells and IgM-secreting plasma cells, but have lost the ability to undergo isotype class switching or somatic hypermutation (19). Differential cell counting of Diff Quik-stained blood smears (Fig. S1) from C57BL/6 or AID?/? mice revealed comparable increases in circulating eosinophils following helminth infection (Fig. 1and the percentage of basophils in the blood was determined by flow cytometry. (with or without immune serum supplementation, and Resiniferatoxin the percentage of basophils in the blood was determined by flow cytometry. All data are shown as the combined mean SEM of individual mice (= 3C7 mice per group) from one experiment and are representative of at least two independent experiments. Although the variance between AID?/? and JHT?/? cannot be readily explained, one obvious difference between these mice is the ability of AID?/? but not JHT?/? mice to make IgM. Analysis of serum from AID?/? mice indicated that they could also generate IgM specific for antigens (Fig. S2). Thus, the B-cellCdeficient JHT?/? mice are likely to exhibit increased Rabbit Polyclonal to CATD (L chain, Cleaved-Gly65) and/or prolonged levels of circulating antigens compared with either wild type or AID?/? mice and this antigen may act independently of antibodies to promote the late basophil response observed in these mice. To determine the presence of soluble antigen, we took advantage of the recently generated monoclonal antibodies exhibiting specificity against excretoryCsecretory (HES) proteins (21). As expected, increased quantities of soluble helminth antigens were detected in the serum of B-cellCdeficient, MT mice, compared with C57BL/6 mice (Fig. S2). That antibodies were resulting in enhanced antigen clearance by forming immune complexes was additionally demonstrated by the finding that heat treatment of wild-type serum results in increased levels of soluble antigen (Fig. S2). Promotes Basophil Expansion Within the Bone Marrow and Spleen. We next set out to determine whether antibodies could promote basophil proliferation and maturation from progenitor cells or survival within the peripheral tissues. A kinetic analysis of various tissues from and and was determined for (and infection for (= 4C5 mice per group) from one experiment and are representative of two independent experiments. Open in a separate window Fig. 3. and the percentage of blood basophils was determined by flow cytometry. (and and and the percentage of blood basophils was determined by flow cytometry. (and the percentage of blood basophils was determined by flow cytometry. ((Fig. 3infection resulted in enhanced IL-3 production by in vitro restimulated cells from both organs, peaking at day 10 postinfection (Fig. 3 and and and infection (Fig. 3and and (Fig. S6) (18). We therefore determined whether antibody supplementation of Resiniferatoxin IL-4R?/? mice could restore basophilia in these mice. Supplementation of immune serum in IL-4R?/? mice completely restored and and additionally injected with 100 L of na? ve or immune serum from C57BL/6 mice every second day from day 0 of infection, and the percentage of basophils in the blood was determined by flow cytometry. (= 3C5 mice per group) from one experiment and are representative of at least two independent experiments. These data indicated that antibodies promote basophil mobilization even in the absence of T-cellCderived IL-3, despite basophil mobilization being an IL-3Cdependent process. IgE stimulation of murine mast cells (24) or human basophils (25) can result in IL-3 production and proliferation in vitro, raising the possibility that antibodies act to elicit autocrine IL-3 production by basophils in vivo following helminth infection. We first determined Resiniferatoxin that either IgE or IgG1 cross-linking could promote IL-3 Resiniferatoxin mRNA expression by bone-marrowCderived murine basophils in vitro (Fig. S7). We then investigated whether a similar process occurred in vivo. As basophils express high levels of IL-3R, we reasoned that ELISA-based detection of IL-3 production by these cells may not be possible, and instead analyzed IL-3 production by intracellular cytokine staining of spleen and bone marrow cells enriched for CD49b and restimulated with phorbolC12-myristatC13-acetate (PMA) plus ionomycin in the presence of monensin to prevent IL-3 export from the endoplasmic reticulum. No increase in IL-3 production by bone marrow cells could be observed using this approach; however, a small population of IL-3+ cells was observed in.

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