In Vitro Enhancement of Mouse T Helper 2 Cell Sensitization to Ovalbumin Allergen by Carbon Black Nanoparticles

Agglomerated carbon black nanoparticles (CBNPs) administered via respiratory or subcutaneous routes have been shown to promote allergic sensitization to coadministered ovalbumin (OVA) protein in rodents. In the present study, we aimed to model and elucidate the mechanism of this adjuvanticity using an in vitro assay based on T cell sensitization to ovalbumin(323-339) peptide (OVA(p)). CBNP base particles of 22 and 39 nm were characterized and termed CBNP22 and CBNP39 powders. Splenic leukocytes derived from transgenic DO11.10 mice were exposed to suspensions of media alone, concanavalin A mitogen, CBNP agglomerates smaller than 220 nm, OVA(p) alone, OVA(p) + anti-CD28 costimulant, OVA(p) + cyclosporin A immunosuppressant, or OVA(p) + CBNPs. Samples were analyzed at 72 h post-exposure. Proliferation rate, a marker of cellular mitosis, was assessed. Polymerase chain reaction arrays were used to assess genes involved in allergic response pathways. The mitogen control, costimulatory control, and immunosuppressive control chemicals modified the T helper cell proliferation rate. CBNP22 mildly reduced proliferation at 12 mu g/ml, but CBNP39 did not. Gene expression analysis of cells treated with OVA(p) showed that coincubation with 12 mu g/ml CBNP22 enhanced gene expression of interleukin-4 (IL-4), IL-10, and IL-13, all allergy-associated Th2 cytokines. Coincubation of OVA(p) with 12 mu g/ml CBNP39 significantly enhanced IL-13 gene expression concurrent with downregulation of the Th1-associated transcription factor Stat4. IL-4 and IL-13 protein secretion reflected the mRNA trends. The changes were consistently higher in cells exposed to CBNP22 than CBNP39, suggesting that smaller particle size, higher surface area, and higher purity were associated with the direct adjuvant effect on Th2 cells in this genetically susceptible model of OVA allergy.