CX3CR1 is expressed by human B lymphocytes and mediates [corrected] CX3CL1 driven chemotaxis of tonsil centrocytes

Abstract

Background: Fractalkine/CX(3)CL1, a surface chemokine, binds to CX(3)CR1 expressed by different lymphocyte subsets. Since CX(3)CL1 has been detected in the germinal centres of secondary lymphoid tissue, in this study we have investigated CX(3)CR1 expression and function in human naïve, germinal centre and memory B cells isolated from tonsil or peripheral blood.

Methodology/principal findings: We demonstrate unambiguously that highly purified human B cells from tonsil and peripheral blood expressed CX(3)CR1 at mRNA and protein levels as assessed by quantitative PCR, flow cytometry and competition binding assays. In particular, naïve, germinal centre and memory B cells expressed CX(3)CR1 but only germinal centre B cells were attracted by soluble CX(3)CL1 in a transwell assay. CX(3)CL1 signalling in germinal centre B cells involved PI3K, Erk1/2, p38, and Src phosphorylation, as assessed by Western blot experiments. CX(3)CR1(+) germinal centre B cells were devoid of centroblasts and enriched for centrocytes that migrated to soluble CX(3)CL1. ELISA assay showed that soluble CX(3)CL1 was secreted constitutively by follicular dendritic cells and T follicular helper cells, two cell populations homing in the germinal centre light zone as centrocytes. At variance with that observed in humans, soluble CX(3)CL1 did not attract spleen B cells from wild type mice. OVA immunized CX(3)CR1(-/-) or CX(3)CL1(-/-) mice showed significantly decreased specific IgG production compared to wild type mice.

Conclusion/significance: We propose a model whereby human follicular dendritic cells and T follicular helper cells release in the light zone of germinal centre soluble CX(3)CL1 that attracts centrocytes. The functional implications of these results warrant further investigation.

Figure 1. Expression of CX₃CR1 in tonsil and blood B cells.

(A) Flow cytometric analysis of CX₃CR1 expression in tonsil and blood B cells. Representative histograms from each B cell fractions, and THP-1 and Raji cell lines, tested as positive and negative controls, respectively, are shown. (B) Quantization of CX₃CR1 by real time PCR in peripheral blood and tonsil B cells. Data are normalized to the expression of POLR2A. Values are expressed as arbitrary units calculated as fold of CX₃CR1 expression relative to the THP-1 cell line, arbitrarily set at 1. Raji cell line was tested as negative control. Data are median, minimum and maximum values from two different experiments performed in quadruplicate. (C) Displacement experiments of ¹²⁵I-CX₃CL1 in tonsil and blood B cells. Left panel. Cells were incubated for 2 h at 4°C with 1 nM ¹²⁵I-CX₃CL1 in the absence or presence of 1, 10 and 100 nM cold CX₃CL1. Percentage of binding inhibition by unlabeled CX₃CL1, calculated as ratio between cell-bound cpm in the presence of unlabeled ligand and cell-bound cpm in the absence of unlabeled ligand multiplied by 100, was used as a measure for competition between ¹²⁵I-labeled and unlabelled CX₃CL1. THP-1 and Raji cell lines were tested as positive and negative controls, respectively. Right panel. The experiments shown in the left panel were repeated using 100 nM cold CXCL8 as negative control.

Figure 2. Expression and chemotactic activity of CX₃CR1 in tonsil B cell subsets.

(A) Apoptosis evaluation in purified tonsil GC B cells. The proportion of early apoptotic GC B cells was detected by Annexin V staining at time 0 and after 1, 2, 3, and 4h culture. Results are expressed as median, minimum and maximum values from five different GC B cell suspensions. (B) Flow cytometric analysis of CX₃CR1 expression on freshly purified tonsil GC, naïve, and memory B cells. Results are expressed in box plot as median percent positive cells, minimum and maximum values, and quartiles, from ten different experiments. (C) Chemotaxis of GC, naïve, and memory B lymphocytes to rCX₃CL1. Results are median numbers of migrated cells, maximum and minimum values, from five different experiments for each B cell subset. * P = 0.043 for both 300 and 600 ng/ml rCX₃CL1. ▪ = Chemotaxis of non-GC B cells to 300 ng/ml rCXCL12 tested as control. (D) Freshly isolated GC B cells were pre-incubated with or without PTX and subjected to chemotaxis to 300 ng/ml CX₃CL1 or medium (nil). Results are median numbers of migrated cells, minimum and maximum values from three different experiments.

Figure 3. CX₃CR1 driven signal transduction.

(A–B) Western blot analysis of phosphorylated (p) and non-p PI3K, p38, AKT, MEK/Erk1,2 and kinase assay of Hck activity (Src family) in GC and non-GC B cells. One representative experiment out of four performed is shown. (C) Chemotaxis of GC B cells to 300 ng/ml rCX₃CL1 following pre-incubation with or without the PI3K inhibitor LY294002 (20 µM), the MEK inhibitor PD98059 (1 µM), the AKT inhibitor (1 µM), the p38 inhibitor SB203580 (1 µM)or the Src inhibitor (10 µM). All cells in the bottom chamber were collected and counted. Results are median numbers of migrated cells, minimum and maximum values from five different experiments. **P = 0.009 for PI3K and p38 inhibitors; *P = 0.03 for MEK and Src inhibitors.

Figure 4. Immunophenotypic and molecular analyses of CX₃CR1⁺ and CX₃CR1⁻ GC B cells.

(A) CX₃CR1⁺ and CX₃CR1⁻ GC B cells were analyzed after staining with mAbs. Results are percent positive cells, minimum to maximum ranges from ten independent experiments. (B) V_H5(D)J_H-μ rearrangement sequences were evaluated in CX₃CR1⁺ and CX₃CR1⁻ GC B cells. 104 molecular clones from five different CX₃CR1⁺ GC B cell fractions were compared to 63 molecular clones from three different CX₃CR1⁻ GC B lymphocyte fractions. 34 molecular clones from three different CD10^-CD27⁻ naive B cell fractions were tested as controls. Results are ratios between number of mutations and number of molecular clones.

Figure 5. Immunophenotypic and functional dissection of CX₃CR1⁺ GC B cells.

(A) Double staining of CX₃CR1⁺ GC B cells with CD27 and CD23 mAbs. Each B cell fraction was further characterized with mAbs (CD27⁺ cells, right lower panel; CD23⁺ cells, left lower panel). Results are median percent positive cells, maximum and minimum values from ten different experiments. (B) CX₃CR1⁺ GC B cells were subjected to chemotaxis to 300 ng/ml rCX₃CL1. Migrated cells were double stained for CD27 and CD23. One representative experiment out of seven is shown. (C) Purified GC B cells were subjected to CX₃CL1-driven chemotaxis and migrated cells were collected and stained with a panel of mAbs. Results are median percent positive cells, maximum and minimum values from four different experiments.

Figure 6. CX₃CL1 expression in tonsil germinal center populations.

(A) Formalin-fixed tonsil tissue section was stained with anti-CX₃CL1 mAb (brown). GC, germinal center; FM, follicular mantle, SE, subepithelial area. One representative experiment out of three is shown. (B) GC B cells, non-GC B cells and T_FH cells were tested for CX₃CL1 production in 24h culture supernatants by ELISA. GC B cells were cultured with or without rCD40L, non-GC B cells with or without anti-Ig antibody plus rCD40L, and T_FH cells with or without PHA plus IL-2. Results are median pg/ml CX₃CL1, minimum and maximum values from four different experiments.* P = 0.028. (C) Double staining of formalin-fixed tonsil tissue section for CX₃CL1 (red) and CD21 (brown). Individual double positive cells are shown in the small right panel. (D) CX₃CL1 production by human FDC cell lines HK and EF cultured 24 h without or with TNF (10 µg/ml) or IFN-γ (10 µg/ml). Results are median pg/ml CX₃CL1, minimum and maximum values from four different experiments. *P = 0.026.

Figure 7. Impaired OVA specific IgG production in CX₃CR1⁻/⁻ or CX₃CL1⁻/⁻mice.

(A) Splenocytes from WT mice double stained with anti-CX₃CR1 and B220 mAbs were analyzed by flow cytometry. Histograms from two different WT mice are shown. (B) Splenocytes from WT mice were tested for chemotaxis to murine rCX₃CL1 or rCXCL12 (control). Migrated B cells were enumerated by flow cytometry using anti-B220 mAb. Results are median number of migrated cells, minimum and maximum values from four different experiments. (C) CX₃CR1⁻/⁻, CX₃CL1⁻/⁻ or WT mice were immunized with OVA and tested at different dilutions for specific IgG production by ELISA. Results were expressed as median optical densities (OD), maximum and minimum values, from ten different experiments. Serum from two WT mice was titrated for OVA IgG antibodies (1: 200, 1:500, 1:1000, 1:10000, 1:50000, 1:100000 dilutions) and used in each tests as standard curve, whereas serum from mice before immunization was used as negative control.