Marilyn M. Huston

Production of IL-5 and granulocyte-macrophage colony-stimulating factor by naive human mast cells activated by high-affinity IgE receptor ligation

BACKGROUND The late-phase allergic reaction is an eosinophilic inflammatory response that begins several hours after allergen exposure, may persist for 24 hours, and is an important pathogenic mechanism in allergic disease. OBJECTIVE Cultured naive human mast cells were used to investigate whether mast cells are a direct source of the eosinophil-promoting cytokines IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF). METHODS Naive human mast cells were derived from bone marrow mononuclear cells cultured in the presence of stem-cell factor. Cytokine message and protein production in response to high-affinity IgE receptor ligation of cultured mast cells were measured by semiquantitative polymerase chain reaction and ELISA, respectively. RESULTS IL-5, IL-3, and GM-CSF messenger RNA increased within 2 hours of mast cell activation, with IL-5 and GM-CSF message remaining elevated for 24 hours, whereas IL-3 mRNA rapidly declined. IL-5 and GM-CSF protein were measurable 4 to 6 hours after stimulation and peaked by 24 and 12 hours, respectively. IL-3 protein was not detectable. CONCLUSION These findings demonstrate that naive mast cells do not constitutively produce IL-5 or GM-CSF protein but are a major source of these eosinophilotropic cytokines on high-affinity IgE receptor ligation.

Enhanced detection of human IL-5 in biological fluids utilizing murine monoclonal antibodies which delineate distinct neutralizing epitopes.

Interleukin 5 (IL-5) is a homodimeric cytokine arranged in a head-to-tail configuration covalently linked by two disulfide bonds. IL-5 has pleiotropic effects on murine and human leukocytes and has been implicated in the pathogenesis of many inflammatory disorders. To facilitate the study of functionally relevant IL-5 domains involved in receptor binding and to develop a highly sensitive and specific ELISA capable of detecting IL-5 in biological fluids, a library of murine anti-human IL-5 (hIL-5) mAb was generated to baculovirus expressed recombinant hIL-5 (rhIL-5). Fifteen subclones of seven hybridomas were characterized. All mAb bound hIL-5, but not murine IL-5 (mIL-5), and neutralized hIL-5 biological activity in the BCl1 proliferation assay. By competitive ELISA, the mAb were divided into two binding groups. Utilizing comparative analysis with TRFK-5, a rat anti-mIL-5 mAb crossreactive with hIL-5, at least three hIL-5 neutralizing epitopes were defined. By ELISA and Western analysis, each epitope was shown to be present as a conformationally identical pair on the hIL-5 dimer. Various combinations of mAb in sandwich ELISA were used to predict the relative proximity of each epitope pair. Utilizing mAb binding characteristics, highly sensitive and specific sandwich ELISA were developed with a minimum detection limit of 6.25 pg hIL-5/ml (P < 0.05). Quantitation of hIL-5 in both serum and bronchoalveolar lavage (BAL) fluid demonstrated the utility of these anti-hIL-5 mAb for investigating the role of hIL-5 in inflammation. These mAb should also serve as useful reagents for epitope mapping of functional hIL-5 domains.

Maternally transmitted antigen.

Publisher Summary This chapter summarizes the evidence regarding the genetic mechanisms of Mta transmission and the class I nature of the surface antigen. By far the most intriguing aspect of maternally transmitted antigen (Mta) is its strict maternal inheritance. It is because of unawareness of any other cell surface antigen in metazoa that is inherited in this manner. Although the conclusive experiments have yet to be completed, it is thought that the evidence for mitochondrial control of Mta phenotype is rather persuasive. Mta is defined by cytotoxic T-lymphocyte (CTL) recognition; to date no antibodies to Mta have been reported. Mta disparity also induces skin graft rejection as well as weak secondary mixed lyinphocyte responses. Restriction enzyme polymorphism analysis and transfer of mitochondria via cell fusion indicate that the genetic element controlling the Mta polymorphism is probably located within mitochondria.

Abnormal response to IL-5 in B-cell chronic lymphocytic leukemia

In B-cell chronic lymphocytic leukemia, neoplastic B-lymphocytes are arrested in development. Since interleukins are essential for B-cell differentiation, we examined whether B-CLL cells were capable of responding normally to interleukins. Purified B-lymphocytes from B-CLL patients and controls were compared for their ability to proliferate and differentiate after stimulation with MCAT or SAC plus rhIL-2 or rhIL-5. When rhIL-5 was added to MCAT-stimulated cells, 8 of 10 controls showed a substantial increase in IgM production, compared with only 1 of 10 B-CLL patients. Lack of IL-5 responsiveness could provide insight into the arrested B-lymphocyte development of some B-CLL patients.