Abstract

The combination of indirect immunofluorescent labeling and flow cytometry has proven to be a sensitive method for labeling of the κ-opioid receptor on mouse thymocytes. In the present study, this labeling procedure was applied, along with phenotypic analysis, to mature immune cell populations to determine whether κ-opioid receptor expression is present after immune cell maturation. Unfixed primary splenocytes from 6- to 8-week-old C57BL/6ByJ male mice were incubated with the fluorescein-containing, κ-selective ligand fluorescein-conjugated 2-(3,4-dichlorophenyl)-N-methyl-N-[1-(3-aminophenyl)-2-(1-pyrrolidinyl)ethyl]acetamide (FITC-AA). Amplification of FITC-AA binding to the κ-opioid receptor was attained by adding a biotin-conjugated antifluorescein antibody, followed by extravidin-R-phycoerythrin. It has been shown previously that greater than 60% of immature thymocytes (CD4⁺/CD8⁺) demonstrated specific κ-opioid receptor labeling. However, the present report shows that less than 25% of either T-helper or T-cytotoxic splenic lymphocytes expressed the κ-opioid receptor. Likewise, only 16% of all splenic B lymphocytes were labeled for the κ-opioid receptor. These findings demonstrate a decrease in κ-opioid receptor expression on maturation of mouse lymphocytes. Interestingly, resident peritoneal macrophages showed a greater magnitude of specific receptor labeling, compared with either thymocytes or splenocytes, and approximately 50% of the resting Mφ expressed the κ-opioid receptor. However, elicitation of Mφ with thioglycollate resulted in the complete loss of the expression of this receptor. Taken together, these findings demonstrate the diversity in the expression of the κ-opioid receptor on immune cells at varying stages of differentiation, with preferential expression demonstrated by resident, peritoneal macrophages.