Characterization of the B-cell inhibitory protein factor in Ixodes ricinus tick saliva: a potential role in enhanced Borrelia burgdoferi transmission

Abstract

We recently described the inhibition of host B lymphocytes by Ixodes ricinus tick saliva. In this study, we characterized the factor responsible for this activity and examined the modulation of lipopolysaccharide (LPS)- and Borrelia burgdorferi outer surface protein (Osp)-induced proliferation of naive murine B lymphocytes by an enriched fraction of this factor. The B-lymphocyte inhibitory activity was destroyed by trypsin treatment, indicating that a proteinaceous factor was responsible for this activity. The removal of glutathione-S-transferase (GST) from tick salivary glands extracts (SGE) showed that this B-cell inhibitory protein (BIP) was not a GST. Gel filtration liquid chromatography indicated that BIP has a native molecular weight of approximately 18,000. An enrichment protocol, using a combination of anion-exchange and reverse-phase liquid chromatography, was established. BIP-enriched fractions did 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 B. burgdorferi transmission by preventing B-cell activation, and also highlights the potential of BIP as a therapeutic agent in B-cell maladies.

Figure 1

A stable tick protein inhibits lipopolysaccharide (LPS)-induced B-lymphocyte proliferation. B cells (10⁶/ml), isolated from BALB/c splenocytes by negative selection, were cultured in medium alone (Med.), with concanavalin A (Con A) (2 µg/ml) or with LPS (2 µg/ml) in the presence or absence of tick salivary glands extracts (SGE) (2 µg/ml). (a) SGE were thawed and used immediately in the assay (−20°), or incubated for 16 hr at 4°, room temperature (RT) or 37°, or for 1 hr at 56° or 3 min at 98° prior to the assay. c.p.m., counts per minute; n/a, not applicable. (b) SGE were untreated or digested by trypsin for 6 hr at 37° prior to the assay. Phosphate-buffered saline (PBS) instead of SGE was subjected to trypsin digestion as a control. B-cell proliferation was assessed after 48 hr of culture by [³H]thymidine incorporation. Results are expressed as the mean of triplicate samples ± standard deviation from one experiment that is representative of three carried out.

Figure 2

The B-cell inhibitory protein (BIP) is not a glutathione-binding glutathione-S-transferase (GST). GSTs were removed from tick salivary glands extracts (SGE) by affinity chromatography. B cells (10⁶/ml) were cultured with lipopolysaccharide (LPS) (2 µg/ml) alone, LPS + SGE (column 1, 60 ng/ml; column 2, 220 ng/ml; column 3, 670 ng/ml; column 4, 2000 ng/ml), LPS + GST-depleted SGE (SGE-GST) (column 1, 60 ng/ml; column 2, 220 ng/ml; column 3, 670 ng/ml; column 4, 2000 ng/ml), or LPS + isolated GST (column 1, 26 ng/ml; column 2, 78 ng/ml, column 3, 234 ng/ml, column 4, 700 ng/ml). B-cell proliferation was assessed after 48 hr of culture by [³H]thymidine incorporation. B cells were also incubated with medium alone or with 2 µg/ml concanavalin A (Con A), yielding proliferation values of 266 ± 31 and 156 ± 35 counts per minute (c.p.m.), respectively. Results are expressed as the mean value of triplicate samples ± standard deviation from one experiment that is representative of three carried out.

Figure 3

The B-cell inhibitory protein (BIP) is ≈ 18 000 molecular weight (MW). Chromatogram of 200 µg of glutathione-S-transferase (GST)-depleted tick salivary glands extracts (SGE) monitored at 280 nm from a Superdex-200 HR 10/30 gel filtration column. Fractions containing BIP are highlighted (black line). Inset: log MW of gel-filtration standard proteins (bovine γ-globulin, 158 000; chicken ovalbumin, 44 000; equine myoglobin, 17 000; and vitamin B12, 1350) versus elution volume/void volume ratio (V_e/V_o) is shown. The V_o was 7·68 ml. Results shown are from one experiment that is representative of three conducted.

Figure 4

Partial purification of the B-cell inhibitory protein (BIP) by anion-exchange chromatography. Glutathione-S-transferase (GST)-depleted tick salivary glands extracts (SGE) were fractionated by stepwise anion-exchange chromatography, eluted with 3 ml of 0, 0·06, 0·12, 0·25, 0·5 and 1 m NaCl. Isolated B cells (10⁶/ml) were stimulated with lipopolysaccharide (LPS) (2 µg/ml) in the presence of SGE fractions (1 µl/well), and proliferation was assessed after 48 hr of culture by [³H]thymidine incorporation. B cells were also incubated with medium alone (100% inhibition) and in the presence of LPS without any SGE material (0% inhibition). The percentage inhibition of B-cell proliferation is presented (white circles). The protein concentration was assessed by the Bradford assay in each fraction (black squares). Arrows indicate the three fractions pooled and carried onto the reverse phase high-performance liquid chromatography (RP-HPLC) enrichment step. Results shown are from one experiment that is representative of four conducted.

Figure 5

Partial purification of the B-cell inhibitory protein (BIP) by reverse-phase chromatography. (a) Pooled active fractions from the anion-exchange step (see Fig. 4) were injected onto a C18 reverse phase (RP) column and the elution monitored at 280 nm (lower panel). Isolated B cells (10⁶/ml) were stimulated with lipopolysaccharide (LPS) (2 µg/ml) in the presence of the RP high-performance liquid chromatography (RP-HPLC) fractions (1 µl/well), and proliferation was assessed after 48 hr of culture by [³H]thymidine incorporation (upper panel). B cells were also incubated with medium alone (100% inhibition) and in the presence of LPS without any tick salivary glands extracts (SGE) material (0% inhibition). ACN, acetonitrile. (b) Increasing volumes (0–5 µl) of the two fractions exhibiting the highest BIP activity were tested for inhibition of B-cell proliferation. The protein concentration was assessed in each fraction by the NanoOrange assay. Data are presented as the mean of triplicate samples ± standard deviation. Results shown are from one experiment that is representative of three conducted. c.p.m., counts per minute.

Figure 6

Delayed addition of the B-cell inhibitory protein (BIP)-enriched fraction inhibits lipopolysaccharide (LPS)-induced B-cell proliferation. The BIP-enriched fraction (5 µl/well) (grey bars) and a control, non-active fraction (5 µl/well) (black bars) were added at 0, 3, 7, 22 and 30 hr after LPS (2 µg/ml) stimulation of isolated B cells (10⁶/ml). Proliferation was assessed after 48 hr of culture by [³H]thymidine incorporation. The controls of medium alone and concanavalin A (Con A) (2 µg/ml) were 2600 ± 1260 counts per minute (c.p.m.) and 3950 ± 450 c.p.m., respectively. Results are expressed as the mean of triplicate samples ± standard deviation from one experiment that is representative of four conducted.

Figure 7

The B-cell inhibitory protein (BIP)-enriched fractions inhibit Borrelia burgdorferi outer surface protein (OspA and OspC)-induced proliferation of isolated B lymphocytes. (a) Effect of the BIP-enriched fraction (5 µl/well) on the proliferation of isolated B cells (10⁶/ml) cultured with lipopolysaccharide (LPS) (2 µg/ml), OspC (5 µg/ml) or OspA (1 µg/ml). The controls of medium alone and concanavalin A (Con A) (2 µg/ml) were 1550 ± 89 counts per minute (c.p.m.) and 917 ± 87 c.p.m., respectively. (b) Dose–response curve of the effect of the BIP-enriched fraction on B-cell (10⁶/ml) proliferation induced by OspC (1 µg/ml). B-cell proliferation was assessed after 48 hr of culture by [³H]thymidine incorporation. Results are expressed as the mean of triplicate samples ± standard deviation from one experiment that is representative of three (a) and two (b) conducted.