Isolated B cells (106/ml) were stimulated with lipopolysaccharide (LPS) (2 g/ml) in the presence of the RP high-performance liquid chromatography (RP-HPLC) fractions (1 l/well), and proliferation was assessed after 48 hr of culture by [3H]thymidine incorporation (upper panel). not suppress T-cell proliferation. Delayed addition of BIP-enriched fractions, up to 7 hr after LPS addition, inhibited the proliferation of isolated B cells. BIP-enriched fractions dramatically inhibited both OspA- and OspC-induced proliferation of isolated B cells. These results strongly suggest that BIP may facilitate Rabbit Polyclonal to SYTL4 transmission by preventing B-cell activation, and also highlights the potential of BIP as a therapeutic agent in B-cell maladies. spp.4spp., the causative agent of Lyme disease, express several distinct structures with stimulatory Cinoxacin activity for mammalian B cells, including the outer surface proteins (Osp) A and C.5 Immunosuppressive molecules in tick saliva may aid in the immune evasion of by preventing saliva induced a dramatic inhibition of host B cells by preventing interleukin (IL)-10 and tumour necrosis factor- (TNF-) production, CD69 expression and proliferation after stimulation with lipopolysaccharide (LPS).13 This B-cell inhibitory activity acts directly on B cells, without inducing B-cell necrosis or apoptosis.13 B lymphocytes play a crucial role in antimicrobial immunity. Immediately after pathogen entry, circulating natural antibodies contribute to effectively eliminate most of the circulating antigens by rapid exotoxin neutralization and enhancing opsonization. T-independent (TI)-1 antigens, such as bacterial LPS, are potent B-cell mitogens, capable of non-specific, polyclonal activation of B cells. TI-2 antigens, consisting of highly repetitive structures on the surface of pathogens, then activate antigen-specific B lymphocytes, which initiates a rapid T-independent response.14 AntigenCC3d complement complexes bound to dendritic cells allow unique immediate isotype switching.15 These antibodies can be secreted at a rate sufficient to keep up with the rapid multiplication of invading infectious micro-organisms.16 Subsequent to this early phase, cognate T-cellCB-cell interactions allow affinity maturation, more complete isotype switching and memory B-cell development. TCB-cell interaction is dependent on the presentation of antigen by the major histocompatibility complex (MHC) class II molecules of B cells. By inhibiting T-cell-independent and T-cell-dependent B-cell activation, the B-cell inhibitory activity we have identified is therefore likely to play a major role Cinoxacin in enhancing tick-vectored pathogen transmission. Our present work was undertaken to further characterize the factor responsible for this recently described B-cell inhibitory activity in saliva. We report that a protein, of 18 000 molecular weight (MW), termed the B-cell inhibitory protein (BIP), was responsible for this activity. BIP-enriched fraction activity was maximal when BIP was added between 0 and 7 hr after LPS addition. BIP-enriched fractions dramatically inhibit the B-lymphocyte proliferation induced by the lipoproteins OspA and OspC, suggesting that BIP may be crucial to transmission enhancement. Materials and methods SGE stability to thermal, chemical and protease treatment Tick salivary glands extracts (SGE) were obtained from partially fed adult female ticks collected from freshly killed deer, as described previously.13 Briefly, tick salivary glands were homogenized in Cinoxacin ice-cold sterile phosphate-buffered saline (PBS) by sonication and then freezeCthawed three times. SGE were clarified by centrifugation at 10 000 for 10 min at 4 and then stored at ?20. For the study of BIP temperature stability, SGE were thawed just before the test or incubated at 4, room temperature or 37 for 16 hr, at 56 for 1 hr, or at 98 for 3 min and then frozen at ?20. For trypsin digestion of SGE, trypsin-agarose beads (01 U, 50 l; Sigma, Poole, UK) were washed three times in RPMI-1640 (Life Technologies, Paisley, UK), pH 8, and incubated with 200 l of RPMI-1640 plus 100 l of SGE (2 mg/ml), BSA (1 mg/ml) or PBS for 6 hr at 37 with continuous shaking. The trypsin-agarose beads were then removed from the samples by centrifugation at 2600 for 5 min and the supernatants were harvested and frozen at ?20. The protein digestion was confirmed by sodium dodecyl sulphateCpolyacrylamide gel electrophoresis (SDSCPAGE), as described below. SGE and a control PBS sample were also subjected to 01% trifluoroacetic acid (TFA) and 90% acetonitrile (ACN) for 1 hr at room temperature before freezing at ?20. Gel filtration liquid chromatography Glutathione-binding glutathione-S-transferases (GSTs) were first removed from SGE by applying SGE in PBS to a 1-ml GSTrapFF column (Amersham Biosciences, Bucks., UK) according to the manufacturer’s instructions. GST-depleted SGE (200 g) was then concentrated to 200 l by centrifugation on a 10 000-MW cut-off filter unit (Sigma) and finally Cinoxacin loaded onto a Superdex-200 HR 10/30 FPLC gel filtration column (Amersham Biosciences). Elution was performed in 10 mm Tris-HCl, pH 74, containing 150 mm NaCl,.