Flow cytometric analysis of CD19+ B cells from PB of HDs tested for adhesion to the integrins of interest. that in peripheral blood, spleens, and tonsils from healthy donors the highest expression levels of the integrins LFA-1 and VLA-4 are also found on MBCs. Consistent with this, we found MBCs to have a higher capacity to adhere to ICAM-1 and VCAM-1 than na?ve B cells. In patients with the autoimmune disease rheumatoid arthritis, it is the MBCs that have the highest levels of LFA-1 and VLA-4; moreover, compared with healthy donors, na?ve B and MBCs of patients NNC 55-0396 receiving anti-TNF medication have enhanced levels of the active form of LFA-1. Commensurate levels of the active L subunit can be induced on B cells from healthy donors by exposure to the integrin ligands. Thus, our findings establish the selective use of the integrins LFA-1 and VLA-4 in the localization Rabbit Polyclonal to CSGLCAT and adhesion of MBCs in both mice and humans. 0.05; ** 0.01; *** 0.001; **** 0.0001. Results Sustained Treatment With Anti-integrin Antibodies Depletes MBCs in the Spleen The integrins of interest in this study are LFA-1 and VLA-4, and their ligands ICAM-1 and VCAM-1 (Physique 1A). Starting with a populace of mature B cells identified as CD19+CD93?CD43?GL7? lymphocytes, MBCs were defined as CD80+CD73+/?PDL2+/? based on the differential expression of the CD80, CD73, and PDL2 surface markers (15), (Supplementary Physique 1; Physique 1B). These are to be explained in more detail elsewhere (Aranburu et. al. in preparation); here it suffices to note that this MBCs in SLC?/? mice contain mainly IgM-expressing cells (Physique 1C). Open in a separate window Physique 1 MBCs present in the spleen of SLC?/? mice are dependent on integrins for their retention (A). The integrins (subunits) of interest and their ligands (BCD). Circulation cytometric analysis of spleen from SLC?/? mice (B) NNC 55-0396 Gating strategy for MBCs (C) Percentage of IgM-expressing cells in MBCs (D) Percentages of MBCs isolated from spleens of SLC?/? mice treated for 2 weeks with anti-LFA-1 and anti-4. = 6 (treated), = 5 (isotype control); error bars show mean +/CSD; data are representative of two impartial experiments. An unpaired two-tailed Student 0.01). To determine whether the adhesion of mouse MBCs in the spleen depends on integrins, we treated SLC?/? mice with antibodies against LFA-1 and VLA-4. After a 2-week period, the presence of MBCs was significantly reduced (Physique 1D), showing that MBCs rely on the conversation with ICAM-1 and VCAM-1 for their retention in the spleen. Acute Treatment With anti-VLA-4 Antibodies Induces the Release of MBCs Into PB To investigate whether the observed integrin-mediated loss of MBCs from your spleen resulted in their accumulation in the blood circulation, we started by looking at the number of leukocytes in the PB of SLC?/? mice soon (5 h) after the injection of the blocking antibodies. Compared to the injection of control antibodies, leukocyte number more than doubled after the injection of antibodies against both LFA-1 and VLA-4 together, but did not alter significantly when each antibody was used alone (Physique 2A). This is to be contrasted with the situation for the MBCs, where the anti-VLA-4 acted selectively, NNC 55-0396 increasing their release into the blood (Supplementary Physique 2; Physique 2B). On the other hand, the number of MZ B cells was selectively increased by anti-LFA-1 treatment, and blocking with both antibodies increased numbers of MBCs as well as MZ B cells at least 3-fold (Figures 2B,C). Comparison of the proportions of MBCs and MZ B cells as well as their ratios in the blood.