Leukocytes Immunophenotype and Phagocytosis Activity in Pregnant and Nonpregnant Dromedary She Camels

Abstract

To Cite This Article: Hussen J, Shawaf T, Al-Mubarak AIA, Al Humam NA, Almathen F and Schuberth HJ, 2020. Leukocytes immunophenotype and phagocytosis activity in pregnant and nonpregnant dromedary she camels. Pak Vet J, 40(2): 239-243. http://dx.doi.org/10.29261/pakvetj/2019.117 INTRODUCTION Embryonic loss is one of the major factors responsible for high economic losses in food animals. The maintenance of pregnancy is associated with modulations in different immune mechanisms, which ensure protection against pathogens and at the same time prevent immunological destruction of the conceptus (Aluvihare et al., 2004; Somerset et al., 2004). The pregnancy-associated changes occur not only in the local environment of the uterus but extend also to the peripheral immune system (Ott and Gifford, 2010; Kamat et al., 2016). For different species like human (Spadaro et al., 2019), cows (Leung et al., 2000; Oliveira et al., 2012), mares (Bazzano et al., 2014; Piccione et al., 2015) and sows (Zhang et al., 2017), local and systemic immunomodulatory effects of pregnancy have been widely studied. According to studies in human and rodents, both lymphoid and myeloid immune cells including NK cells,  T cells, B cells, -T lymphocytes, neutrophils, monocytes, macrophages and dendritic cells play significant role in maintaining pregnancy (Lash et al., 2010; Nagamatsu and Schust, 2010; Groebner et al., 2011). In dairy cows, the presence of a conceptus resulted in increased numbers of peripheral blood myeloid cells with enhanced expression of chemotactic factors, which attract these cells to the uterus (Kamat et al., 2016). Little is known about the impact of pregnancy on the immune system of dromedary she camels. The aim of the current study was, therefore, the comparative analysis of immunophenotype of blood leukocytes and phagocytosis activity of neutrophils in pregnant and nonpregnant dromedary she camels. The results of the current study would lead to a better understanding of immunology of pregnancy and the identification of the immunologic factors associated with higher pregnancy rates in she camels. RESEARCH ARTICLE Pak Vet J, 2020, 40(2): 239-243. 240 MATERIALS AND METHODS Animals and blood sampling: In this study, 18 pregnant and 21 non-pregnant dromedary she camels (Camelus dromedarius), aged 10-14 years and maintained at the Camel Research Center, King Faisal University, Al-Ahsa, Saudi Arabia were used. The pregnant she camels were at their mid gestation (between 5 and 10 month based on sonographic examination and insemination history). Blood samples (5 ml blood from each she camel) were collected from she camels during the period between January and May 2019 by jugular venepuncture in EDTA containing vacutainer tubes (BD, Germany). All experimental procedures and management conditions used in this study were approved by the Ethics Committee at King Faisal University, Saudi Arabia (Permission number DSR 1811001). Microscopic counting of leukocytes: Whole blood was diluted 1: 4 in PBS and was then mixed with Türk’s solution (final dilution 1:20; Merck Millipore) and 10 μl of the mixture was poured onto the hemocytometer (Neubauer cell counter) for counting under the microscope. Leukocytes (in blue color were counted in four big squares of the cell counter and total leukocyte count was calculated (Camacho-Fernandez et al., 2018). Monoclonal antibodies: Nine commercially available monoclonal antibodies (mAbs) were used in this study, as shown in Table 1. Separation of blood leukocytes: Separation of whole blood leukocytes was done after hypotonic lysis of erythrocytes (Hussen et al., 2017). Briefly, blood was suspended in distilled water for 20 sec and double concentrated PBS was added to restore tonicity. This was repeated until complete erythrolysis indicated by the formation of clear white pellet of leukocytes. Separated cells were finally suspended in membrane immunofluorescence (MIF) buffer (PBS containing bovine serum albumin (5 g/L) and NaN3 (0.1 g/L)) at 5x10 cells/ml. The mean viability of separated cells was evaluated flow cytometrically by dye exclusion (propidium iodide; 2 μg/ml, Calbiochem, Germany) and was consistently >95%. Immunofluorescence and flow cytometry: Separated leukocytes (5x10 cells / ml) in PBS containing bovine serum albumin (5 g/L) and NaN3 (0.1 g/L) were labeled in 96 well round-bottom microtiter plates (1x10 / well; 20 min; 4°C) with monoclonal antibodies specific for CD4, WC1, MHCII and CD14 in three combinations, including CD4/WC1/CD14, CD14/MHCII and CD14/CD11a/CD18 (Eger et al., 2015; Hussen et al., 2018). After incubation with primary unlabeled antibodies, cells were washed twice and incubated with mouse secondary antibodies IgG1, IgM and IgG2a (BD) labelled with different fluorochromes. After washing the cells, directly labeled monoclonal antibodies were added to CD14, CD11a, CD11b and CD18. Finally, cells were washed and analyzed by flow cytometry (FACSCalibur, Becton Dickinson Biosciences). For each measurement 100,000 events were acquired and data were analyzed with FlowJo (FLOWJO LLC) (Fig. 1). Phagocytosis assay: Heat killed staphylococcus aureus (S. aureus) bacteria (Pansorbin, Calbiochem, Merck, Nottingham, UK) were labeled with fluorescein isothiocyanate (FITC, Sigma-Aldrich, St. Louis, Missouri, USA). FITC-conjugated and heat killed S. aureus bacteria were suspended in Roswell Park Memorial Institute (RPMI) medium and adjusted to 2x10 bacteria/ml. Separated camel leukocytes were plated in 96 well plates (1x10/well) and incubated at 37°C under 5% CO2 with labeled bacteria (50 bacteria/cell) for 40 minutes. After washing, cells were analyzed by flow cytometry (FACS Calibur, Becton Dickinson Biosciences, San Jose, California, USA). Phagocytosis-positive cells were defined as the percentage of green fluorescing cells among total cells. Phagocytosis capacity (as an indicator for the number of bacteria ingested by each cell) was defined as the mean green fluorescence intensity of gated phagocytosis-positive neutrophils. Table 1: List of antibodies Antigen Antibody clone Labelling Source Isotype CD4 GC50A1 Unlabeled WSU Mouse IgM WC1 BAQ128A Unlabeled WSU Mouse IgG1 CD14 TÜK4 PerCP Biorad mIgG2a MHCII TH81A5 WSU mIgG2a CD11a G43-25B PE BD mIgG2a CD18 6.7 FITC BD mIgG1 mIgG2a Polyclonal PE Invitrogen gIgG mIgG1 Polyclonal FITC Invitrogen gIgG mIgM Polyclonal APC Invitrogen gIgG Ig: Immunoglobulin; m: mouse; MHC-II: Major Histocompatibility Complex class II, g: goat, WSU: Washington State University. Fig. 1: Gating strategy for the identification of the main leukocyte populations in peripheral blood of she camel. In a SSC/FSC dot plot, camel granulocytes and mononuclear cells (PBMC) were gated according to their forward and side scatter characteristics. After setting a gate on granulocytes, eosinophils and neutrophils were identified according to their different auto-fluorescence intensities in the FL1 and FL2 fluorescence channels. In the PBMC gate, monocytes and lymphocytes were identified based on their different CD14 staining. S S C FSC F L 1 FL2 C D 1 4 SSC Eosinophils Neutrophils Monocytes Lymphocytes Figure 1 S S C FSC F L 1 FL2 C D 1 4 SSC Eosinophils Neutrophils Monocytes Lymphocytes Figure 1 S S C FSC F L 1 FL2 C D 1 4 SSC Eosinophils Neutrophils Monocytes Lymphocytes