CD6 binds to pathogen-associated molecular patterns and protects from LPS-induced septic shock

Abstract

CD6 is a lymphocyte receptor that belongs to the scavenger receptor cysteine-rich superfamily. Because some members of the scavenger receptor cysteine-rich superfamily act as pattern recognition receptors for microbial components, we studied whether CD6 shares this function. We produced a recombinant form of the ectodomain of CD6 (rsCD6), which was indistinguishable (in apparent molecular mass, antibody reactivity, and cell binding properties) from a circulating form of CD6 affinity-purified from human serum. rsCD6 bound to and aggregated several Gram-positive and -negative bacterial strains through the recognition of lipoteichoic acid and LPS, respectively. The Kd of the LPS-rsCD6 interaction was 2.69 +/- 0.32 x 10(-8) M, which is similar to that reported for the LPS-CD14 interaction. Further experiments showed that membrane CD6 also retains the LPS-binding ability, and it results in activation of the MAPK signaling cascade. In vivo experiments demonstrated that i.p. administration of rsCD6 before lethal LPS challenge significantly improved mice survival, and this was concomitant with reduced serum levels of the proinflammatory cytokines TNF-alpha, IL6, and IL-1beta. In conclusion, our results illustrate the unprecedented bacterial binding properties of rsCD6 and support its therapeutic potential for the intervention of septic shock syndrome or other inflammatory diseases of infectious origin.

Fig. 1.

Binding of rsCD6 to Gram-positive and Gram-negative bacteria. (A) Western blot analysis of the affinity-purified biotin-labeled proteins. (B) Protein binding to E. coli and S. aureus. (C) Calcium influence on the binding of rsCD6 and rSpα to E. coli and S. aureus. (D) Competition binding assays of rsCD6 to E. coli and S. aureus in the presence of increasing concentrations of LPS or LTA. For bacterial binding studies, biotin-labeled proteins were incubated with a suspension of 5 × 10⁷ bacteria. Unbound protein was washed off, and then bacteria and bound protein were solubilized with SDS/PAGE loading buffer and electrophoresed. Detection of biotin-labeled proteins was performed by Western blot using HRP–streptavidin.

Fig. 2.

Characterization of affinity-purified circulating CD6 from human serum. (A) Coomassie blue staining of affinity-purified nsCD6 from human serum. (B) Western blot analysis of biotin-labeled purified nsCD6 and rsCD6 proteins and membrane CD6 (mCD6) immunoprecipitated from surface biotinylated HUT-78 cells with streptavidin–HRP. (C) Membranes containing the same proteins as in B, Western blotted with a rabbit polyclonal antiserum specific for the extracellular region of CD6. (D) Flow cytometry analysis of the reactivity of biotinylated rsCD5, rsCD6, nsCD6, or BSA (used as a negative control) with the K652 and Raji cells. Bound protein was detected with streptavidin–Tricolor.

Fig. 3.

Binding of rsCD6 to LPS. (A) ELISA showing direct binding of nsCD6 and rsCD6 to LPS. Several concentrations of biotinylated rsCD6, nsCD6, or BSA (as negative control) were added to the LPS-coated wells, and bound protein was detected with streptavidin–HRP. (B and C) Binding of rsCD6 or rsCD5 to Re-LPS was monitored by changes in FITC–Re-LPS fluorescent properties. (B) rsCD6, but not rsCD5, induces a significant increase in fluorescence anisotropy upon binding to FITC–Re-LPS, which increases with increasing rsCD6 concentration. (C) Net change in fluorescence emission intensity of FITC–Re-LPS at 520 nm upon addition of increasing amounts of rsCD6 or rsCD5. The apparent K_d for FITC–Re-LPS/sCD6 complexes, calculated from the saturation curve fitted to a rectangular hyperbola, was 2.69 ± 0.32 × 10⁻⁸ M.

Fig. 4.

LPS from E. coli binds to cell-surface CD6 and activates ERK1/2. (A) Flow cytometry analysis showing direct binding of increasing amounts of LPS–FITC to parental and CD6.wt-transfected 2G5 cells. (B) To ease comparison, mean fluorescence intensities of A were plotted against the amount of LPS–FITC added to each cell line. (C) Competition studies of LPS–FITC binding to the 2G5-CD6.wt transfectants. Cells were incubated with 10 μg of LPS–FITC in the presence of increasing amounts of rsCD6 or rsCD5. (D and E) Analysis of ERK1/2 phosphorylation after LPS or PHA stimulation of parental and CD6.wt-transfected 2G5 cells (D) and COS-7 cells transiently expressing wild-type (CD6.wt) or cytoplasmic tail-truncated CD6 (CD6.P527^stop) molecules (E). In both cases, serum-starved cells were stimulated for 40 min with 100 μg/ml LPS or 100 ng/ml PHA at 37°C. Cell lysates were resolved by SDS/PAGE, transferred to nitrocellulose, and subjected to immunoblotting with anti-phospho ERK1/2 (p-ERK1/2) antiserum. Further reprobing with anti-ERK1/2 antiserum was used as loading control.

Fig. 5.

rsCD6 induces bacterial aggregation. (A) FITC-labeled E. coli and S. aureus bacterial suspensions were incubated overnight at room temperature with rsCD6 or rsCD5 (2 μM) in the presence of 5 mM Ca²⁺. Equimolar concentrations of rSpα and HSA were used as positive and negative control, respectively. Aggregation was observed by direct examination on a fluorescence microscope. (B) Kinetics of Ca²⁺-dependent Re-LPS aggregation in the absence (filled circles) and presence of increasing concentrations of rsCD6, as described in Materials and Methods. The final concentrations of Re-LPS, Ca²⁺, and EDTA were 100 μg/ml, 2.5 mM, and 5 mM, respectively. One representative experiment of two performed is shown.

Fig. 6.

Effect of rsCD6 and rsCD5 on survival rate and cytokine serum levels after LPS-induced septic shock. (A) Survival graph. C57BL/6J mice (8 weeks old) were injected i.p. with a lethal dose of LPS (30 mg/kg) 1 h after i.p. administration of sterile saline solution (n = 26), rsCD5 (n = 10), or rsCD6 (n = 16) (25 μg each). The percentage of survival mice was analyzed, and the log-rank t test P values were calculated. (B–D) Circulating levels of cytokines in LPS-challenged mice. Plasma levels of TNF-α (B), IL-1β (C), and IL-6 (D) were quantified by ELISA at different times after LPS injection. Data are expressed as mean ± SEM. Statistical differences in the results were evaluated by the two-tailed Student t test. ∗, Statistically significant difference (P < 0.05).