Endotoxin upregulates CCR7 and its ligands in the lymphatic-free mouse iris

Purpose: Using time lapse intravital microscopy and histology, we previously reported that we could not detect migration of antigen-presenting cells from the iris to the regional lymph node. Dendritic cells (DC) in other peripheral tissues migrate to lymph nodes in response to chemokines, CCL19 (ELC) and CCL21b (SLC), that activate the CCR7 receptor. We hypothesized that DCs in an inflamed iris might show a different chemokine receptor and ligand profile, thus explaining the DC's inability to migrate. Methods: Eyes of 35 BALB/c mice were injected intravitreally with 2 μl of 250 ng E. coli lipopolysaccharide (LPS) or phosphate buffered saline (PBS). Five mice served as naïve controls. After 3 and 6 h, the iris-ciliary bodies were dissected and pooled in groups of five. Total RNA was isolated, and reverse-transcriptase polymerase chain reaction (RT-PCR) for chemokine receptor and ligand mRNA was performed. In addition, one eye from each of the three animals was taken 6 h after LPS injection for immunohistology (IHC). Results: The naïve iris, the iris after PBS injection, and the iris after LPS injection contained CCR5 mRNA at approximately equal levels and did not have detectable CCR6 mRNA. No CCR7 mRNA expression was found in the naïve iris, but it was weakly expressed in PBS-injected eyes and was approximately 3.4 fold upregulated after LPS injection. This was confirmed by IHC with no staining for CCR7 in the control iris but positive staining in the inflamed eyes. Transcripts for the CCR7 ligands, CCL19 and CCL21b, were found after LPS or PBS injection but not in naïve iris-ciliary bodies. Conclusions: The clear upregulation of CCR7 and its ligands in the inflamed iris suggests that another mechanism prevents iris DCs from migrating. Other possibilities include the absence of co-factors, inhibitory substances, the lack of lymphatics inside the eye, or inadequate biological activity of these chemotactic factors and ligands.