Abstract
β-Lactam antibiotics provide the cornerstone of treatment and reduce the rate of decline in lung function in patients with cystic fibrosis, but their use is limited by a high frequency of delayed-type allergic reactions. The objective of this study was to use cloned T-cells expressing a single T-cell receptor from five piperacillin-hypersensitive patients to characterize both the cellular pathophysiology of the reaction and antigen specificity to define the mechanism of activation of T-cells by piperacillin. More than 400 piperacillin-responsive CD4+, CD4+CD8+, or CD8+ T-cell clones were generated from lymphocyte transformation test and ELIspot-positive patients. The T-cell response (proliferation, T helper 2 cytokine secretion, and cytotoxicity) to piperacillin was concentration-dependent and highly specific. Enzyme-linked immunosorbent assay, gel electrophoresis, and mass spectrometry revealed that piperacillin bound exclusively to albumin in T-cell culture. Irreversible piperacillin binding at Lys 190, 195, 199, 432, and 541 on albumin and the stimulation of T-cells depended on incubation time. A synthetic piperacillin albumin conjugate stimulated T-cell receptors via a major histocompatibility complex- and processing-dependent pathway. Flucloxacillin competes for the same Lys residues on albumin as piperacillin, but the resulting conjugate does not stimulate T-cells, indicating that binding of the β-lactam hapten in peptide conjugates confers structural specificity on the activation of the T-cell receptors expressed on drug-specific clones. Collectively, these data describe the cellular processes that underlie the structural specificity of piperacillin antigen binding in hypersensitive patients with cystic fibrosis.
Footnotes
This work was funded by the Wellcome Trust [Grant 078598/Z/05/Z] as part of the Centre for Drug Safety Science supported by the Medical Research Council [Grant G0700654]. S.E.-G. and A.E. are Ph.D. students funded by the Egyptian government. M.M.M. is a Ph.D. student funded by the Saudi Arabian government.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS:
- CF
- cystic fibrosis
- PBMC
- peripheral blood mononuclear cells
- IFN
- interferon
- IL
- interleukin
- TNF
- tumor necrosis factor
- MIP
- macrophage inflammatory protein
- ELISA
- enzyme-linked immunosorbent assay
- TST
- Tris/saline/Tween
- OD
- optical density
- MRM
- multiple reaction monitoring
- amu
- atomic mass units: MHC, major histocompatibility complex
- Th2
- T helper 2.
- Received December 8, 2011.
- Accepted February 24, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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