C-C motif chemokine ligand (CCL) production in equine peripheral blood mononuclear cells identified by newly generated monoclonal antibodies

Chemokines are soluble molecules directing immune cell trafficking and homing, mediating inflammation, and initiating immune responses to infection. In horses, the analysis of chemokines has been limited by the lack of specific antibodies. We generated mAbs specific for the equine C-C motif chemokine ligands (CCL) CCL2 (MCP-1), CCL3 (MIP-1 alpha), CCL5 (RANTES) and CCL11 (eotaxin) using hybridoma technology. Antibody specificity was confirmed by intracellular staining of Chinese Hamster Ovary cells transfected with expression vectors encoding for CCL2, CCL3, CCL5, or CCL11. Transfectants were stained with the anti-CCL mAbs. Flow cytometric analysis confirmed the specificity of the different mAbs for the respective chemokine. In addition, equine PBMC were stained after isolation, culture in medium, or stimulation with LPS, or PMA and ionomycin. CCL2 was detected in few cluster of differentiation (CD)14(+) monocytes in PBMC stimulated with PMA and ionomycin for 2 h. CCL3 was produced by CD14(+) monocytes after 4-6 h culture in medium. After stimulation with PMA and ionomycin for 12-24 h, CCL3 was also expressed in lymphocytes, mainly in CD4(+) T cells. Stimulation with LPS reduced the percentage of CCL3(+) monocytes in PBMC. CCL5 was detected in PBMC ex vivo in CD4(+) and CD8(+) T cells. Culture of PBMC for longer than 6 h or stimulation with PMA and ionomycin reduced the percentage of CCL5(+) cells. CCL11 was produced by CD4(+) T cells in PBMC after stimulation with PMA and ionomycin for 4-24 h. After LPS stimulation of PBMC, CCL2, CCL5, and CCL11 production were comparable to culture in medium alone. ELISAs for each of the four chemokines were developed using pairs of anti-equine CCL mAbs. Supernatants from PMA and ionomycin stimulated PBMC contained detectable amounts of CCL2, CCL3 and CCL5, while CCL11 secretion could be stimulated from equine tracheal epithelial cells in response to IL-4. The newly generated mAbs for equine CCL chemokines facilitate the quantitative analysis of intracellular chemokine production by flow cytometry and soluble chemokines by ELISA. The CCL mAbs are valuable tools to improve the evaluation of innate immune responses in horses.