Data supporting the understanding of modulatory function of opioid analgesics in mouse macrophage activity

The data presented herein expand the current understanding of the modulatory function of opioid drugs in mouse macrophage activity described in our relevant research article (Filipczak-Bryniarska et al., 2017) [1], in which we characterize the influence of morphine, buprenorphine and oxycodone on humoral and cell-mediated immune response in mice. Among other things, we have shown the effects of treatment with assayed analgesics on macrophage ability to induce antigen-specific B-cell response to sheep red blood cells as well as to generate reactive oxygen intermediates and nitric oxide. The current data demonstrate the effects of morphine, buprenorphine or oxycodone administration on phagocytosis of sheep red blood cells and zymosan by mouse macrophages, supplementing the data on immune modulatory capacities of assayed drugs, recently reported by us (Filipczak-Bryniarska et al., 2017; Kozlowski et al., 2017) [1,2].


a b s t r a c t
The data presented herein expand the current understanding of the modulatory function of opioid drugs in mouse macrophage activity described in our relevant research article (Filipczak-Bryniarska et al., 2017) [1], in which we characterize the influence of morphine, buprenorphine and oxycodone on humoral and cellmediated immune response in mice. Among other things, we have shown the effects of treatment with assayed analgesics on macrophage ability to induce antigen-specific B-cell response to sheep red blood cells as well as to generate reactive oxygen intermediates and nitric oxide. The current data demonstrate the effects of morphine, buprenorphine or oxycodone administration on phagocytosis of sheep red blood cells and zymosan by mouse macrophages, supplementing the data on immune modulatory capacities of assayed drugs, recently reported by us (  Table   Subject area  Immunology  More specific subject area   Immunopharmacology Type of data Figure  How  The data presented here demonstrate the effects of morphine, buprenorphine or oxycodone administration on phagocytosis of either FITC-coupled sheep red blood cells or zymosan-green by mouse macrophages.
While zymosan-green reagent is commercially available, we have elaborated another, original phagocytosis assay with the use of FITC-coupled sheep red blood cells, prepared by us ex tempore.
The opioid analgesics were administered in vivo prior to macrophage harvest, which enables to evaluate their influence on immune cells taking into account the pharmacodynamics, tissue distribution and metabolism of tested drugs in living organism.

Data
The percentage of macrophages from mice treated with different opioid drugs emitting green fluorescence after incubation with fluorescein isothiocyanate (FITC)-coupled sheep red blood cells (Fig. 1, upper left graph) or with zymosan-green reagent (Fig. 1, upper right graph), and the geometric mean of emitted green fluorescence by these macrophages incubated with FITC-coupled sheep red blood cells (Fig. 1, lower left graph) or with zymosan-green reagent (Fig. 1, lower right graph). In addition, macrophage populations were divided into cells expressing high (FITC high , grey bars) or low (FITC low , black bars) green fluorescence emission. The raw data are included in supplementary table.

Mice
Ten week old male mice (22 7 2 g) of the inbred CBA strain were from the breeding unit of the Department of Immunology, Faculty of Medicine, Jagiellonian University Medical College, Krakow, Fig. 1. Opioid analgesics influence phagocytosis of fluorescein isothiocyanate (FITC)-coupled sheep red blood cells (SRBC) and zymosan-green by mouse macrophages. Oil-induced, peritoneal macrophages harvested from mice treated for a week with morphine, buprenorphine or oxycodone were incubated for 20 min in 37°C water-bath with SRBC-FITC or zymosan-green reagent, and then green fluorescence emission by these cells was measured cytometrically. Upper left graph shows the percentage of macrophages of mice treated with respective opioid drug that emit green fluorescence after incubation with SRBC-FITC, while upper right graph shows the percentage of macrophages emitting green fluorescence after incubation with zymosan-green reagent. Lower left graph demonstrates the geometric mean of emitted green fluorescence by control and opioid-treated macrophages incubated with SRBC-FITC and lower right graph depicts the geometric mean of emitted green fluorescence by control and opioid-treated macrophages incubated with zymosan-green reagent. According to the intensity of green fluorescence signal, macrophage populations were divided into cells expressing high (FITC high , grey bars) or low (FITC low , black bars) fluorescence emission.
Poland and were fed autoclaved food and water ad libitum. After random assignment to control or treatment groups, animals were subjected to the experiment conducted according to the guidelines of the 1st Local Ethics Committee in Krakow (approval No 123/2013).

Preparation of FITC-coupled sheep red blood cells (SRBC)
Powdered FITC (Sigma, St. Louis, MO, USA) was dissolved in Tris-HCl buffer (pH 9.0) to reach the concentration of 50 mg/ml. Then it was diluted 10 times with DPBS, adjusted to pH 8.0, and 10 ml of the final FITC solution was mixed with 1 ml of 100% suspension of SRBC (Graso Biotech, Starogard Gdanski, Poland), washed previously with DPBS, and incubated for 2 h at room temperature, in darkness, on hematological roller. Afterwards, SRBC-FITC were washed thrice with DPBS, filtered through nylon mesh and 1% suspension in DPBS was prepared for the phagocytosis assay.

Phagocytosis assay and cytometric analysis
Macrophages harvested from either drug-treated or untreated (control) mice were incubated with SRBC-FITC (in ratio 1:10) for 20 min in 37°C water-bath, which was followed with osmotic shock for lysis and removal of non-phagocytosed SRBC-FITC. Otherwise, macrophages were incubated with zymosan-green reagent (pHrodo TM Green Zymosan A BioParticles Conjugate; Life Technologies, Carlsbad, CA, USA), previously prepared according to manufacturer procedures, in a dose of 0.25 mg of zymosan per 5 × 10 6 macrophages for 20 min in 37°C water-bath. Then cells were analyzed by flow cytometry on FACS Calibur (BD Bioscience, San Jose, CA, USA), and acquired data were analyzed firstly with BD CellQuest Pro software, and then with GraphPad Prism and Excel software.