Antibody Array-Generated Profiles of Cytokine Release from THP-1 Leukemic Monocytes Exposed to Different Amphotericin B Formulations

Abstract

Cytokine antibody arrays were used to establish the profiles of cytokine release from THP-1 monocytes exposed to different amphotericin B (AMB) drug delivery systems. Fungizone (FZ) and Amphotec (ABCD) caused the release of significantly more inflammatory molecules and the release of inflammatory molecules at higher levels than either AmBisome (L-AMB) or Abelcet (ABLC) after 6 h of treatment. Specifically, tumor necrosis factor alpha (TNF-␣), interleukin-8 (IL-8), GRO-(␣␤␥), monocyte chemoattractant protein-1 (MCP-1), RANTES, IL-10, and IL-6 were detected and semiquantified with a chemiluminscence imaging system. TNF-␣, IL-8, and MCP-1 were the most predominant; however, little if any TNF-␣ was present in ABLC-or L-AMB-treated cultures. The TNF-␣ and IL-8 levels determined by quantitative enzyme-linked immunosorbent assay correlated with the relative cytokine levels measured by using the antibody arrays. Although the viabilities of THP-l monocytes in all AMB-treated cultures were similar by trypan blue exclusion, the amount of lactic dehydrogenase released was significantly larger in FZ-and ABCD-treated cultures than in L-AMBand ABLC-treated cultures, indicating more membrane perturbations with those formulations. Membrane cation channel formation was also measured in model cholesterol-containing large unilamellar vesicles to directly assess the ion channel formation ability of the system. Only FZ and ABCD induced significant ion currents at concentrations less than 1.5 ؋ 10 ؊5 M. These results may help provide rationales for the immediate cytokine-mediated side effects observed with FZ and ABCD and the reduced side effects observed with L-AMB and ABLC.Amphotericin B (AMB) is a polyene antifungal antibiotic and for a number of years has been the "gold standard" for the treatment of systemic fungal infections. Three new lipid-based formulations, Abelcet (ABLC), Ambisome (L-AMB), and Amphotec (ABCD), have been developed in an attempt to obtain improved efficacies, therapeutic indices, and tolerabilities compared to those of the traditional AMB formulation, Fungizone (FZ). In clinical trials, improvements in renal tolerability were shown for all three lipid formulations; however, the levels of acute toxicity, including fever, chills, hypotension, and nausea, vary with each formulation (8, 23). L-AMB has been shown to promote the least adverse effects, while immediate reactions to ABCD are often as frequent and severe as those to FZ (23). Clinical toxicity has been associated with increases in plasma tumor necrosis factor alpha (TNF-␣) and interleukin-6 (IL-6) levels during infusion of the drug (1), while in vitro studies with the human THP-1 monocytic cell line have further identified and characterized a number of proinflammatory cytokines that are induced and released following exposure to FZ (5,19,20). Because the clinical effects of the four AMB formulations are different, we hypothesized that each AMB formulation would produce a different cytokine profile. Using recently developed antibody arrays (13, 27) and t...

Differential Expression of Cytokines and Chemokines in Human Monocytes Induced by Lipid Formulations of Amphotericin B

“…To the best of our knowledge, this is the first report of the effects of amphotericin B lipid formulations on gene expression of MIP-1␤. Previous observations on gene expression of MCP-1 in DAMB-treated MNCs or THP-1 cell lines (29) and on MIP-1␤ expression after THP-1 exposure to the four amphotericin B formulations (39) are consistent with our studies. Collectively these data suggest that DAMB and ABCD formulations may augment host response through MIP-1␤ release.…”

“…Taking into consideration the adverse infusion-related reactions following DAMB administration, an imbalance in the protein synthesis of IL-1␤ and IL-1ra could indeed be responsible for the observed toxic adverse effects. DAMB-mediated up-regulation of IL-1␤ protein synthesis has previously been reported for THP-1 cells after incubation of DAMB at 5 g/ml for 2 and 6 h, respectively (26,39). However, stimulation of MNCs with 1 g of DAMB per ml for 24 h did not result in IL-1␤ synthesis (40) suggesting an optimum time window for IL-1␤ expression between 0.5 and 6 h. Keeping in mind the different conditions used, results from the present study and the previously mentioned reports are essentially in agreement with clinical data, where DAMB-induced IL-1␤ synthesis occurs between 2 and 6 h, which coincides with the time interval that adverse reactions are observed in humans (11).…”

“…In vitro, amphotericin B-responsive cytokines and chemokines have been identified to be expressed either in THP-1 cells, a leukemic monocytic cell line (26)(27)(28)(29)39), or in human peripheral blood mononuclear cells (PBMCs) (40). Little is known, however, about the cytokine gene expression in primary human monocytes in response to lipid formulations of amphotericin B.…”

Assessment of Effective Renal Plasma Flow, Enzymuria, and Cytokine Release in Healthy Volunteers Receiving a Single Dose of Amphotericin B Desoxycholate

“…These data have predominantly been generated through in vitro exposure of monocytes to AmB (15). In addition, induction of cytokine release from THP-1 leukemic monocytes to different amphotericin B formulations has yielded similar results (23). Antibody array analysis in this previous study revealed that AmB has the greatest effect on TNF-␣ with no effect on IL-1 (23).…”

Microarray Technology as a Universal Tool for High-Throughput Analysis of Biological Systems

“…They determined the level of 25 plasma cytokines and were able to elucidate different cytokine profiles between patients with or without extraglandular manifestations [173]. Turtinen and coworkers studied the effects of different amphotericin B formulations on cytokine release from THP-1 leukemic monocytes and showed that TNF-alpha and IL-8 levels correlated well between the antibody microarray and quantitative ELISA measurements [174].…”