Phenotype and cytokine expression profiles of ovine dendritic cells migrating to lymph nodes

Abstract

Dendritic cells (DCs) function at the interphases of innate and adaptive immunity. The ability ofDCs to control the responses ofeffector T cells, B cells and NK cells are due, in part, to cell contacts and DC cytokines. Different DC subsets exist, that appear to have different biological roles in vivo. Cannulation of the 'pseudoafferent' lymphatics is one of the few methods of obtaining differentiated DCs ex vivo. Lymph DCs are not a homogenous population, and this study tested the hypothesis that discrete populations exist in vivo, that differentially express immunomodulatoiy molecules. Two lymph DC populations were defined by expression of signal regulatory protein-a (SIRPa). SIRPa+ DCs were CD58+, CD1 la+, WC6+, CD45RA", expressed CD16, CD14, CD206, CD4 and CD8 at low levels or on minor sub-populations, and as a single population expressed higher levels of CDllc, CD40, CD80, CD86 and MHC II than did SIRPa DCs. SIRPa" DCs were CD16", CD14", CD206", CD4", CD8", CD1 la+ and displayed heterogeneity of expression of CD58, CD1 lc, WC6 and CD45RA. Importantly, freshly isolated SIRPa+ DCs expressed IL-12 p40 and IL-18 mRNA, whereas SIRPa" DCs were negative for these transcripts. Neither IL-6 nor IL-10 mRNA were detected by PCR in the DC populations. Differential expression of molecules involved in antigen uptake (CD14, CD16, CD206), interactions with lymphocytes (CD54, CDlla, CD40) and antigen presentation and T cell activation (MHC II, CD80, CD86, IL-12, IL-18), suggests that the lymph DC populations may have different funtions in vivo. This knowledge would support therapeutic strategies to target particular DC populations in vivo, to enhance, or to suppress their natural role in the immune responseIn order to perform multiple cytokine analysis on the lymph DCs, a multiprobe RNase protection assay (RPA) was developed for eleven cytokines. First, sheep interleukin-18 (IL-18) was cloned and sequenced. By inducing the production of interferon gamma, IL-18 is an important regulator of T cell immunity. This was the first description ofIL-18 expression patterns in sheep. Second, standardisation analysis showed that the RPA could reliably quantify levels of IL-1 p, IL-4, IL-6, IL-10, IL-12 p40, IL-18, GM-CSF, IFNyand TGF0 mRNA. IL-8 and TNFa - specific riboprobes require optimisation for use with the developed protocol. The amount of RNA required for analysis using the RPA exceeded what was isolated from the DC subsets. However, the RPA makes it possible to evaluate levels ofnine cytokine mRNA transcripts in single RNA samples isolated from sheep. This will help support studies of ovine immunology and immunopathology, where cytokine responses are the readout.