Effects of Salivary Gland Extract from Rhipicephalus sanguineus on Immunoglobulin Class Productivity of Canine Peripheral Blood Lymphocytes.

Abstract

ABSTRACT. Effects of salivary gland extract (SGE) from Rhipicephalus sanguineus on immunoglobulin class productivity of canine peripheral blood lymphocytes (PBL) in vitro were studied. The detectable limit of the ELISA for canine total immunoglobulin, IgM and A was at least 1, 1 and 15 ng/ml, respectively, and it seems to be useful for the evaluation of non-specific immunoglobulin class productivity in vitro. SGE from R. sanguineus suppressed pokeweed mitogen-or lipopolysaccharide-induced total immunoglobulin and IgA productivity of canine PBL although IgM productivity was not suppressed. These results suggested that the suppression was caused partly by the direct effect of SGE on B lymphocytes.KEY WORDS: canine, immunoglobulin class productivity, Rhipicephalus sanguineus.J. Vet. Med. Sci. 63(3): 325-328, 2001 It is known that tick infestation itself has immune suppressive effects on host animals, which aids the transmission of tick-borne diseases [9,10]. Rhipicephalus sanguineus is the three-host dog tick, and is known to be a vector for various diseases of dogs. Suppressive effects of the infestation of R. sanguineus on dogs, such as antibody production, neutrophil function and lymphocyte blastogenic response have been found in our previous works [2][3][4]. The effects of tick saliva on the inhibition of neutrophil function and lymphocyte blastogenic response have been proven by in vitro experiments; however, immunoglobulin productivity has not been proven yet. In this study, we examined the effects of salivary gland extract (SGE) of adult R. sanguineus on in vitro immunoglobulin class productivity of canine peripheral blood lymphocytes (PBL) induced by pokeweed mitogen (PWM) or lipopolysaccharide (LPS) stimulation.SGE was obtained from females of partially-engorged R. sanguineus kept in our laboratory [3]. The ticks were allowed to feed for 4 days on the ears of rabbits (Japanese white, Kyudo, Japan) and collected. Salivary glands were dissected from each tick and washed with phosphate buffered saline (PBS, pH 7.2) and homogenized with a ground glass homogenizer in 1.0 ml of PBS containing 0.1 mM phenylmethylsulfonyl fluoride (PMSF, Nakalai Tasque, Japan) over an ice bath. This crude extract was centrifuged at 7740 × g for 5 min. Supernatant was filtered through a 0.22 µm filter and the protein concentration was determined with a DC Protein Assay Kit (Bio-Rad, U.S.A.). The obtained SGE was stored at -20°C until use.Peripheral blood was collected in heparinized tubes (20 IU/ml) from six tick-naive, adult male Beagles bred at Yamaguchi University. A density gradient method with Lymphoprep (1.077 g/ml, Nycomed Pharma AS, Oslo, Norway) was used to separate mononuclear cells containing lymphocytes from peripheral blood [5]. Contaminated red blood cells were lysed with ammonium chloride Tris buffer (0.017 M Tris, pH 7.2 containing 0.75% NH 4 Cl). The mononuclear cells were washed twice with sterile PBS, and finally resuspended at 1 × 10 6 cells/ml in Dulbecco's Modified Eagle Medium (GIBCO BRL, U.S.A.) containin...

Effects of Salivary Gland Extract from Rhipicephalus sanguineus on IgG Subclass Production and Cytokine mRNA Expression in Mononuclear Cells of Canine Peripheral Blood.

“…The ConA-induced production of IgG2 was suppressed significantly, but that of IgG1 was not. In our previous study, IgA productivity was suppressed in canine PBMCs, but not in IgM [15]. The expression of IFN-γ mRNA was significantly suppressed by SGE.…”

“…In our previous study, the suppressive effects by salivary gland extract (SGE) from R. sanguineus were observed on the production of total immunoglobulin and IgA by the mononuclear cells in canine peripheral blood (PBMCs), but they did not suppress the production of IgM [15]. The class and subclass of immunoglobulin produced are influenced by cytokines, for example, IFN-γ, IL-4 and IL-5 induce the production of IgG2, IgG1 and IgE, and IgA, respectively [1,4,5,11,22].…”

“…The salivary glands were dissected from partiallyengorged female R. sanguineus kept in our laboratory [8], and SGE was prepared as described previously [15].…”

“…PBMCs were isolated as described previously [10]. The culture of PBMCs for the production of IgG subclass and the mRNA expression of cytokines were performed as described by Matsumoto et al [15] and Pinelli et al [19], respectively with some modifications, that is, for the production of IgG and mRNA expression of cytokines, PBMCs were cultured at a density of 5 × 10 5 cells/ml for 6 days and 2 × 10 6 cells/ml for 18 hr, respectively, with 1 µg/ml of ConA (SIGMA, U.S.A.) and SGE at a final concentrations: 100, 25, 6.25, 1.56 and 0 µg/ml at 37°C in a humidified chamber of 5 % CO 2 .…”

“…After cultured in 6 days, the supernatant of the PBMCs culture was collected to determine IgG1 and IgG2 concentrations by ELISA, as described previously [15] with some modifications. To quantify IgG1 and IgG2, goat anti-dog IgG (1 µg/ml, BETHYL, U.S.A.) and sheep anti dog IgG (1:1,000, ICN, U.S.A.) in 0.05 M carbonate buffer (pH 9.6), respectively were immobilized on the surface of ELISA plates (Greiner, Germany).…”