Role of Amoxicillin in the Activation and Cytotoxicity of NK Against Dendritic Cells from Betalactam-Allergic Patients

Abstract

RATIONALE: Interaction with pathogenic or damaged-cell molecules produces cytotoxicity and lysis of target cells by NK lymphocytes in innate immunological reactions. However, the interaction with small molecular compounds like betalactams is unknown. NK may also crosstalk with dendritic cells (DC) and induce their maturation. This study analyses the effect of amoxicillin (AX) on NK and DC from patients with allergy to this drug.METHODS: NK lymphocytes and DC were isolated from patients with non-immediate reactions to AX and healthy donors. NK were cultured in presence of several stimuli (IL12+IL15 and/or AX). Cytotoxic NK phenotype (CD69, Perforin, Granzyme-B, TNFα and IFNγ) and cytotoxicity of immature DC (imDC) and AX-matured DC (mDC) with propidium iodide (PI) and anti-annexin was determined by flow cytometry. Cell-to-cell interaction between NK and DC was analyzed by transwell co-cultures.RESULTS: Phenotypic analysis showed a significant increase of CD69, IFNγ and Granzyme in NK IL12+IL15-cultured, in both patients and controls. With AX, it showed an increase of CD69 and Granzyme in allergic subjects only. When cytotoxicity was analyzed, an increase of annexin was found in imDC cocultured with NK previously exposed to IL12+IL15, in both patients and controls, and to AX only in patients. Cytotoxicity was also significatively increased when coculturing mDC with previously IL12+IL15-activated NK in patients and controls, but AX addition produced no variation. No cytotoxicity was observed when transwell cultures were used.CONCLUSIONS: Amoxicillin activates NK from patients with non-immediate reactions to this drug, increasing cytotoxicity against DC, especially imDC. NK-DC intercellular contact is necessary for this mechanism. RATIONALE: Interaction with pathogenic or damaged-cell molecules produces cytotoxicity and lysis of target cells by NK lymphocytes in innate immunological reactions. However, the interaction with small molecular compounds like betalactams is unknown. NK may also crosstalk with dendritic cells (DC) and induce their maturation. This study analyses the effect of amoxicillin (AX) on NK and DC from patients with allergy to this drug. METHODS: NK lymphocytes and DC were isolated from patients with non-immediate reactions to AX and healthy donors. NK were cultured in presence of several stimuli (IL12+IL15 and/or AX). Cytotoxic NK phenotype (CD69, Perforin, Granzyme-B, TNFα and IFNγ) and cytotoxicity of immature DC (imDC) and AX-matured DC (mDC) with propidium iodide (PI) and anti-annexin was determined by flow cytometry. Cell-to-cell interaction between NK and DC was analyzed by transwell co-cultures. RESULTS: Phenotypic analysis showed a significant increase of CD69, IFNγ and Granzyme in NK IL12+IL15-cultured, in both patients and controls. With AX, it showed an increase of CD69 and Granzyme in allergic subjects only. When cytotoxicity was analyzed, an increase of annexin was found in imDC cocultured with NK previously exposed to IL12+IL15, in both patients and controls, and to AX only in patients. Cytotoxicity was also significatively increased when coculturing mDC with previously IL12+IL15-activated NK in patients and controls, but AX addition produced no variation. No cytotoxicity was observed when transwell cultures were used. CONCLUSIONS: Amoxicillin activates NK from patients with non-immediate reactions to this drug, increasing cytotoxicity against DC, especially imDC. NK-DC intercellular contact is necessary for this mechanism.