Detection of opioid receptors on murine lymphocytes by indirect immunofluorescence: Mature normal and tumor bearing mice lymphocytes

Abstract

Opioid peptides modulate immune responses via ligation to classical opioid receptors (μ, δ and κ), expressed on immune cells, or in an indirect fashion via the central nervous system. The combination of immunofluorescent technique and flow cytometry has proven to be sensitive methods for detection of opioid receptors on leukocytes. In the current study a fluorescein isothiocyanate-conjugated naltrexone (FITC-NTX) derivative in the absence or presence of naltrexone, as a competitor, was used to detect opioid receptors on thymocytes and then on splenocytes of normal and tumor bearing Balb/c mice. Tumor bearing mice were made by intraperitoneal injection of fibrosarcoma cell line. In a two weeks interval, tumor grew and then mice splenocytes were harvested. Cells were incubated with FITC-NTX alone (direct fluorescence), or FITC-NTX followed by biotin-conjugated anti-fluorescein IgG and extravidin- R-phycoerythrin (indirect immunofluorescence). Using flow cytometry we found that, with direct fluorescence staining there is only nonspecific cell staining. In contrast, indirect staining of cells demonstrated labeling of opioid receptors. Thymocytes displayed 37.5±7% specific labeling by current staining procedure. However, this specific staining was 17.2±4% and 7.5±2% in splenocytes of normal and tumor bearing mice, respectively. Taken together, these results showed that, direct fluorescence staining failed to stain opioid receptors expressed on lymphocytes. These receptors can only be detected by a biotin–streptavidin amplification procedure. We also found that the level of opioid receptors on mature lymphocytes is less than that of immature ones and are even lesser in the tumor bearing mice lymphocytes.