Association of human B cells with <i>S</i>. <i>epidermidis</i> is mediated primarily by complement.

<p><b>A)</b> FITC-labeled <i>S</i>. <i>epidermidis</i> (strain 1457) was opsonized with NHS, heat-treated NHS (HT Tx), or DPBS (unopsonized control) and binding of B cells to <i>S</i>. <i>epidermidis</i> was assessed by flow cytometry. <b>B)</b> FITC-labeled <i>S</i>. <i>epidermidis</i> was opsonized with NHS, NHS treated with cobra venom factor (+CVF), NHS treated with FUT-175 (+FUT-175), HT Tx, or DPBS and binding of B cells to <i>S</i>. <i>epidermidis</i> was assessed by flow cytometry (60 min time point). Data in panels <b>A</b> and <b>B</b> are the mean ± SEM of 5 separate experiments using different blood donors. <b>C)</b> FITC-labeled <i>S</i>. <i>epidermidis</i> was combined with 5 μg anti-human CD35 (+αCR1), CD21 (+αCR2), CD11b (+αCR3), CD11c (+αCR4), or isotype IgG₂ control (isotype) mAbs and binding to B cells was determined using flow cytometry (30 min time point). <b>D)</b> FITC-labeled <i>S</i>. <i>epidermidis</i> was combined with 4 μg of soluble recombinant CD35 (+rCR1), CD21 (+rCR2), CD11b (+rCR3), CD11c (+rCR4), or DPBS and binding to B cells was determined using flow cytometry (60 min time point). Data in panels C and D are the mean ± SEM of at least 3 separate experiments using different blood donors. Experiments in panels A-D were performed using 1 × 10⁶ purified human peripheral blood mononuclear cells (PBMCs) and 1 × 10⁶ CFUs <i>S</i>. <i>epidermidis</i>. B cells were identified as the CD19⁺ leukocyte population of PBMCs. **<i>P</i><0.01, ***<i>P</i><0.001, and ****<i>P</i><0.0001 versus DPBS controls for panels A and D, NHS for panel B, or isotype IgG₂ for panel C. Statistics were determined by using a repeated-measures one-way ANOVA and Dunnett’s post-test.</p

Immunofluorescence microscopy analysis of purified B cells and monocytes associated with <i>S</i>. <i>aureus</i> or <i>S</i>. <i>epidermidis</i>.

<p><b>A</b>) Representative images (100×) of B cells or monocytes purified from heparinized human blood 80 min after addition of 1 × 10⁶ CFU/mL <i>S</i>. <i>aureus</i> or <i>S</i>. <i>epidermidis</i>. Extracellular bacteria are red and internalized bacteria are green (appear turquoise in the context of blue nuclei). <b>B</b>) Quantitation of the association and internalization of <i>S</i>. <i>aureus</i> or <i>S</i>. <i>epidermidis</i> by B cells or monocytes for assays described in panel A. Data in panel B are the mean ± SEM of at least 5 separate experiments. *<i>P</i><0.05 for comparison of <i>S</i>. <i>aureus</i> with <i>S</i>. <i>epidermidis</i> as determined by paired two-tailed Student’s t test.</p

Association of <i>S</i>. <i>aureus</i> or <i>S</i>. <i>epidermidis</i> with PMNs, monocytes, B cells, and T cells in human blood.

<p><b>A</b>) Representative flow cytometry dot plots of CD3⁺ lymphocytes (T cells), CD19⁺ lymphocytes (B cells), CD14⁺ monocytes, and CD66^High granulocytes (PMNs) 40 min after combining heparinized human blood with FITC-labeled <i>S</i>. <i>aureus</i> or <i>S</i>. <i>epidermidis</i>. <b>B-E</b>) Flow cytometry analyses of leukocyte subsets associated with FITC-labeled <i>S</i>. <i>aureus</i> or <i>S</i>. <i>epidermidis</i>. <b>F & G</b>) Relative distribution of CD66^High granulocytes, CD14⁺ monocytes, CD19⁺ lymphocytes, and CD3⁺ lymphocytes associated with <i>S</i>. <i>aureus</i> or <i>S</i>. <i>epidermidis</i> at 40 min. Data are from experiments presented in panels B-E. Data in all panels represent 3 experiments with different blood donors using 5 × 10⁵ CFU/mL bacteria. *<i>P</i><0.05 and **<i>P</i><0.01 versus <i>S</i>. <i>aureus</i> as determined with a paired two-tailed Student’s t test.</p

Relative inability of <i>S</i>. <i>aureus</i> to bind B cells is conserved among diverse strains and influenced by bacterial viability.

<p><b>A)</b> Association of B cells with FITC-labeled <i>S</i>. <i>aureus</i> strains (USA100, USA200, and USA300) or <i>S</i>. <i>epidermidis</i> strains (1457, 12228, and RP62A) in heparinized human blood was determined by flow cytometry (60 min time point). ~2.5–5 × 10⁵ CFU/mL of bacteria were used in these assays. <b>B)</b> Association of B cells with untreated or UV-irradiated USA300 or <i>S</i>. <i>epidermidis</i> strain 1457 (1 × 10⁷ CFU/mL for each) in heparinized human blood was determined by flow cytometry (40 min time point). Data in panels A and B are the mean ± SEM of 5 separate experiments using different blood donors. For panel A, ^#, ^, *<i>P</i>≤0.002 versus 1457, 12228, or RP62A as determined by repeated-measures one-way ANOVA and Tukey’s post-test. For panel B, **<i>P</i><0.01 as determined by paired two-tailed Student’s t test. NS, not significant.</p

Comparison of the binding of <i>S</i>. <i>aureus</i>, <i>S</i>. <i>epidermidis</i> and zymosan with human B cells.

<p><b>A)</b> Flow cytometry analysis of B cells associated with <i>S</i>. <i>aureus</i> strain USA300 or <i>S</i>. <i>epidermidis</i> strain 1457 in heparinized human blood 40 min after the addition of 5 × 10⁵, 1 × 10⁶, or 1 × 10⁷ CFU/mL of staphylococci. <b>B)</b> Percentage of B cells with bound bacteria or zymosan 40 min after combining heparinized human blood with 8 × 10⁶ CFU/mL of FITC-labeled USA300 or <i>S</i>. <i>epidermidis</i> strain 1457, or the indicated numbers of FITC-labeled zymosan particles. Data in panels A and B are the mean ± SEM of at least 4 separate experiments using different blood donors. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, and ****<i>P</i><0.0001 versus USA300 as determined by paired two-tailed Student’s t test for data in panel A or by repeated-measures one-way ANOVA and Dunnett’s post-test for data in panel B.</p

Bovine CCL28 Mediates Chemotaxis via CCR10 and Demonstrates Direct Antimicrobial Activity against Mastitis Causing Bacteria

<div><p>In addition to the well characterized function of chemokines in mediating the homing and accumulation of leukocytes to tissues, some chemokines also exhibit potent antimicrobial activity. Little is known of the potential role of chemokines in bovine mammary gland health and disease. The chemokine CCL28 has previously been shown to play a key role in the homing and accumulation of IgA antibody secreting cells to the lactating murine mammary gland. CCL28 has also been shown to act as an antimicrobial peptide with activity demonstrated against a wide range of pathogens including bacteria, fungi and protozoans. Here we describe the cloning and function of bovine CCL28 and document the concentration of this chemokine in bovine milk. Bovine CCL28 was shown to mediate cellular chemotaxis via the CCR10 chemokine receptor and exhibited antimicrobial activity against a variety of bovine mastitis causing organisms. The concentration of bovine CCL28 in milk was found to be highly correlated with the lactation cycle. Highest concentrations of CCL28 were observed soon after parturition, with levels decreasing over time. These results suggest a potential role for CCL28 in the prevention/resolution of bovine mastitis.</p></div

Bovine CCL28 mediates migration of CCR10 transfected cells Supernatant fluids from Cos-7 cells transfected with mCCL28, bCCL28, or empty vector controls were placed in the bottom well of a Transwell migration chamber.

<p>Cells expressing mCCR10 were placed in the upper chamber and allowed to migrate for 1.5hrs. Both murine and bovine CCL28 mediated migration of CCR10 transfectants significantly better than empty vector controls *p<0.05, as determined by Mann Whitney <i>U</i> test. Results are from four separate experiments. Average migration of cells migrating to mCCL28 was 18.4% (SE 3.71), migration to bCCL28 9.4% (SE 2.12), and migration to empty vector 0.1% (SE 0.01). Error bars represent standard error of the mean. NS = Not Significant.</p

Primer names and sequences.

<p>Primer names and sequences.</p

Hla-dependent human T cell and B cell plasma membrane permeablity occurs following 60 minutes exposure to USA300 supernatant.

<p>A) Representative flow cytometry dot plots of human PBMCs stained with propidium iodide following intoxication with filtered 5 hour supernatants from USA300 or USA300Δ<i>hla</i> diluted to a final concentration of 1∶6 for 30 minutes for CD14⁺ PBMCs or 180 minutes for CD3⁺ and CD19⁺ PBMCs. B) Representative histograms of data in part A with CD3⁺, CD14⁺, and CD19⁺ PBMCs exposed to supernatant from USA300 indicated by a dashed line, supernatant from USA300Δ<i>hla</i> indicated with grey shading, and TSB media alone indicated with black shading. Compiled results from at least 4 separate experiments shown in part A using different blood donors for C) CD3⁺, D) CD14⁺ and E) CD19⁺ PBMCs at indicated times following intoxication with *P<0.05, **P<0.01, and ***P<0.001 relative to USA300 as determined by paired one-tailed t-test.</p

Hla induces programmed cell death in human monocytes, T cells and B cells during USA300 infection.

<p>Compiled flow cytometry analysis of human PMNs or PBMCs infected at a MOI of 10 with USA300, USA300Δ<i>hla</i>, USA300Δ<i>hla</i> Comp, USA300Δ<i>saeR/S</i>, or a DPBS control then examined using an Annexin V binding assay at 3 hours (A, C, E, and G) or ApoBrdU Tunel assay at 6 hours (B, D, F, and H). Figures represent 4 separate experiments using different blood donors with *P<0.05, **P<0.01, and ***P<0.001 relative to USA300 as determined by paired one-tailed t-test for A, C, E and G or one-way repeated measures ANOVA with Tukey’s post-test for B, D, F, and H.</p