The CD5 ectodomain interacts with conserved fungal cell wall components and protects from zymosan-induced septic shock-like syndrome

Abstract

The CD5 lymphocyte surface receptor is a group B member of the ancient and highly conserved scavenger receptor cysteine-rich superfamily. CD5 is expressed on mature T and B1a cells, where it is known to modulate lymphocyte activation and/or differentiation processes. Recently, the interaction of a few group B SRCR members (CD6, Spalpha, and DMBT1) with conserved microbial structures has been reported. Protein binding assays presented herein indicate that the CD5 ectodomain binds to and aggregates fungal cells (Schizosaccharomyces pombe, Candida albicans, and Cryptococcus neoformans) but not to Gram-negative (Escherichia coli) or Gram-positive (Staphylococcus aureus) bacteria. Accordingly, the CD5 ectodomain binds to zymosan but not to purified bacterial cell wall constituents (LPS, lipotheicoic acid, or peptidoglycan), and such binding is specifically competed by beta-glucan but not by mannan. The K(d) of the rshCD5/(1-->3)-beta-d-glucan phosphate interaction is 3.7 +/- 0.2 nM as calculated from tryptophan fluorescence data analysis of free and bound rshCD5. Moreover, zymosan binds to membrane-bound CD5, and this induces both MAPK activation and cytokine release. In vivo validation of the fungal binding properties of the CD5 ectodomain is deduced from its protective effect in a mouse model of zymosan-induced septic shock-like syndrome. In conclusion, the present results indicate that the CD5 lymphocyte receptor may sense the presence of conserved fungal components [namely, (1-->3)-beta-d-glucans] and support the therapeutic potential of soluble CD5 forms in fungal sepsis.

Fig. 1.

Interaction of the CD5 ectodomain with whole fungal cells. (A) Biotin-labeled rshCD5 (rshCD5-b) or rshCD6 (rshCD6-b) (15 μg) was incubated with 10⁶ S. pombe, C. albicans, or C. neoformans cells. Cell-bound protein was solubilized and run on SDS-PAGE. Detection of total and bound biotin-labeled proteins was performed by Western blot analysis by using HRP-streptavidin (SAv). (B) Binding of different amounts (1–20 μg) of rshCD5-b to C. albicans was analyzed as in A. The binding of 20 μg of rshCD5-b in presence of 5 mM EDTA is also shown. (C) Binding of a fixed amount (15 μg) of rshCD5-b or rshCD6-b to 10⁸ E. coli or S. aureus bacteria is shown. (D) Culture supernatants from HEK 293-EBNA transfectants expressing individual ectodomains (DI, DII, or DIII) of rshCD5 were incubated with 10⁶ C. albicans or C. neoformans overnight at 4 °C. Unbound protein was washed off and precipitated with 10% (weight/vol) trichloroacetic acid. Unbound precipitated (NB) and cell-bound (B) proteins were analyzed by Western blot with a rabbit polyclonal anti-CD5 antiserum plus HRP-labeled sheep anti-rabbit Ig antiserum. (E) Fungal cell aggregation was assayed by incubating FITC-labeled C. albicans (10⁶) overnight at 4 °C with 5–10 μg of BSA, rshCD5, or rshCD6 in the presence or absence of 20 μg of zymosan (ZYM), β-d-glucan (β-d-GLU), or mannan (MAN) as a competitor. Cells were then transferred onto glass slides and visualized by fluorescence microscopy. (Magnification: ×400.) Tot prot., total protein.

Fig. 2.

The CD5 ectodomain binds to zymosan through recognition of β-d-glucans. (A) ELISA plates coated with BSA, zymosan (ZYM), LPS, PGN, or LTA were incubated with increasing amounts (0.01–2 μg) of rshCD5-b. Bound protein was detected with HRP-streptavidin (SAv). (B) Binding of rshCD6-b to BSA-, ZYM-, LPS-, PGN-, or LTA-coated ELISA plates was analyzed as in A. (C) Binding of rshCD5 to ZYM is competed by β-d-glucans. A fixed amount (2 μg) of rshCD5-b (Left) and rshCD6-b (Right) was incubated with ZYM-coated ELISA plates in the presence or absence of increasing amounts (0.01–20 μg) of unlabeled competitors (β-d-glucans, zymosan, mannan, or BSA). Bound protein was detected with HRP-SAv. (D) Binding of rshCD5 to whole fungal cells is competed by β-d-glucans. A fixed amount (15 μg) of rshCD5-b was incubated with 10⁸ C. albicans or C. neoformans cell suspensions in the presence of increasing amounts (1–20 μg) of unlabeled competitors: Zymosan (S. cerevisiae), β-d-glucan (barley), β-1,3-glucan (E. gracilis), glucan (S. cerevisiae), and mannan (S. cerevisiae). Bound rshCD5-b was detected by Western blot analysis using HRP-SAv.

Fig. 3.

Zymosan (ZYM) binds to membrane-bound CD5 and induces downstream signaling events. (A) Increasing amounts (1–30 μg) of FITC-labeled ZYM were incubated with 2G5 cells either untransfected (Left) or transfected (Right) to express the WT membrane CD5 receptor (2G5-CD5.WT). Fluorescence intensity of stained cells was analyzed by flow cytometry. (B) 2G5-CD5.WT transfectants were stained with a fixed amount (15 μg) of FITC-labeled ZYM in the presence or absence of increasing amounts (1–30 μg) of ZYM, β-d-glucan (β-GLU), and mannan (MAN). Fluorescence intensity was analyzed by flow cytometry. (C) 2G5 cells (2 × 10⁶) either untransfected or stably expressing WT (2G5-CD5.WT) or cytoplasmic tail-truncated (2G5-CD5-K384^STOP) CD5 surface molecules were pulsed for 0, 5, 15, and 30 min with 40 μg/mL ZYM at 37 °C. Subsequently, cell solubilizates were analyzed by Western blot with anti-pERK1/2, anti-pMEK, and anti-cdk4 antisera plus HRP-labeled sheep anti-rabbit or anti-mouse Ig antisera. (D) HEK 293 cells either stably expressing TLR2 or not were transfected in transient for expression of WT (CD5.WT) or cytoplasmic tail-truncated (CD5.K384^STOP) membrane CD5 forms. Cells were then pulsed for 24 h with 20 μg/mL ZYM in the presence or absence of rshCD5 (25 μg) and assayed for IL-8 secretion by ELISA.

Fig. 4.

rshCD5 protects from septic shock-like syndrome induced by zymosan in mice. rshCD5 pretreatment protects from zymosan-induced sepsis. The toxicity score, peritoneal total leukocyte count (10³ cells/mm³), IL-6 and IL-1β serum levels, and liver MPO activity (mU/mg protein) of CD1 mice pretreated with BSA or rshCD5 (25 μg, i.p.) 1 h before infusion of zymosan (500 mg/kg, i.p.) were assessed at 18 h. The survival of mice from the same groups was monitored for 12 days.