Neisseria gonorrhoeae kills carcinoembryonic antigen-related cellular adhesion molecule 1 (CD66a)-expressing human B cells and inhibits antibody production

Abstract

Neisseria gonorrhoeae cells (gonococci [GC]), the etiological agents for gonorrhea, can cause repeated infections. During and after gonococcal infection, local and systemic antigonococcal antibody levels are low. These clinical data indicate the possibility that GC may suppress immune responses during infection. Carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1 or CD66a), a receptor for GC opacity (Opa) proteins, was shown to mediate inhibitory signals. In the present study, human B cells were activated by interleukin-2 to express CEACAM1 and then stimulated to secrete antibodies and simultaneously coincubated with Opa- and OpaI GC of strain MS11. Our results show that this OpaI GC has the ability to inhibit antibody production. The interaction of GC and CEACAM1 with human peripheral B cells also results in induction of cell death. The same findings were observed in DT40 B cells. This CEACAM1-promoted cell death pathway does not involve the inhibitory signals or the tyrosine phosphatases SHP-1 and SHP-2 but depends on Bruton's tyrosine kinase in DT40 cells. Our results suggest that Neisseria gonorrhoeae possesses the ability to suppress antibody production by killing CEACAM1-expressing B cells.

FIG. 1.

The increasing expression of CEACAM1 in human B cells involves IL-2 and results in the enhancement of adherence to Opa⁺ bacteria. Human PBMC were treated with IL-2 for 3 days. The expression level of CEACAM on B cells was determined with flow cytometry by double staining of B cells with allophycocyanin-anti-CD19 (F3) and FITC-anti-CEACAM (F1) antibodies. (A and B) PBMC with and without IL-2 treatment. The top right section contains CEACAM1-expressing B cells. The adherence and invasion of GC and E. coli with purified B cells were determined by direct counting of the number of bacteria per cell (C) and by recovery of CFU after treatment with gentamicin (D), respectively. DT40-CEACAM1 and DT40-CEACAM3 cells serve as control cell lines.

FIG. 2.

Cell proliferation assay and OpaI-induced inhibition of antibody production by CEACAM1-expressing B cells. (A) The B cells collected each day were treated with MTT. Color absorbance (optical density at 540 nm) is directly proportional to the number of surviving cells and was measured by ELISA plate reader. It should be noted that the control cell line, DT40, goes into apoptosis immediately after its growth reaches the peak at day 4. (B) B cells with or without treatment with IL-2 were sorted out from PBMC, then treated with anti-CD40 and -Mμ antibodies (the stimulators), and simultaneously challenged with Opa⁻ or OpaI GC for 72 h. The supernatants were collected, and antibody level was measured by ELISA. The bars (B) represent the means from six experiments, in which each experiment was performed in duplicate or triplicate.

FIG. 3.

ITIM does not participate in CEACAM1-mediated the cell death and invasion. Both cDNAs of CEACAM1-long (L) and CEACAM1-short (S) forms were stably transfected into DT40 B cells (A) and HeLa cells (B). The expression levels of CEACAM1 in DT40 B-cell and HeLa cell transfectants were determined by flow cytometry using antibody YTH71.3, which recognizes CEACAM1, CEACAM6 (CD66c), and CEACAM3 (CD66d). Untransfected DT40 B or HeLa cells were used as a negative control (filled-in curve). DT40 or HeLa transfectants were challenged with Opa⁻ and OpaI GC or E. coli and assayed for cell death (A) and phagocytic ability (B), respectively. The OpaI-mediated cell death of DT40-CEACAM1-L and -S cells was also tested in the presence of caspase-3 inhibitor (open bar, panel A). DT40 B cells and their transfectants that bound annexin V-FITC or took up PI were counted as dead cells. The percentage of dead cells is indicated on the y axis. CFU recovered after gentamicin treatment in HeLa cells were quantified as phagocytosed bacteria (B).

FIG. 4.

OpaI GC and E. coli kill CEACAM1-expressing human B cells. CEACAM1-expressing B cells were treated with Opa⁻ GC, OpaI GC, Opa⁻ E. coli, or OpaI E. coli or not treated for 2.5 h as described in the legend to Fig. 2. Human B cells that bound annexin V-FITC or took up PI were regarded as dead cells. The percentage of dead cells is indicated on the right.

FIG. 5.

BTK is involved in the signal pathway of CEACAM1-mediated cell death. CEACAM1-L and CEACAM1-L-Y459F were stably transfected and expressed in mutants with DT40 mutations: ΔSHIP, ΔSHP-1, ΔSHP-2, ΔSyk, and ΔBTK. The expression levels of mutants in stable DT40 B-cell transfectants were determined by flow cytometry using anti-CEACAM antibody (B). DT40-CEACAM3 and DT40-CEACAM3-Y196F were used as the positive and negative controls, respectively (11). Untransfected DT40 B cells were used as another negative control (filled-in curve). These transfectants were challenged with OpaI GC. The percentage of dead cells is indicated on the y axis (A).