Alpha4beta7 integrin/MAdCAM-1 adhesion pathway is crucial for B cell migration into pancreatic lymph nodes in nonobese diabetic mice

Abstract

Although B cells are crucial antigen-presenting cells in the initiation of T cell autoimmunity to islet beta cell autoantigens in type 1 diabetes (T1D), adhesion molecules that control migration of B cells into pancreatic lymph nodes (PanLN) in the nonobese diabetic (NOD) mouse model of human T1D have not been defined. In this study, we found that B cells from PanLN of 3-4-week-old female NOD mice expressed high levels of alpha(4) integrin and LFA-1 and intermediate levels of beta(7) integrin; half of B cells were L-selectin(high). In short-term in vivo lymphocyte migration assays, B cells migrated from the bloodstream into PanLN more efficiently than into peripheral LNs. Moreover, antibodies to mucosal addressin cell adhesion molecule 1 (MAdCAM-1) and alpha(4)beta(7) integrin inhibited >90% of B cell migration into PanLN. In contrast, antibodies to peripheral node addressin, L-selectin or LFA-1 partially inhibited B cell migration into PanLN. Furthermore, one intraperitoneal injection of anti-MAdCAM-1 antibody into 3-week-old NOD mice significantly inhibited entry of B cells into PanLN for at least 2 weeks. Taken together, these results indicate that the alpha(4)beta(7) integrin/MAdCAM-1 adhesion pathway plays a predominant role in migration of B cells into PanLN in NOD mice. Thus, specific blockage of alpha(4)beta(7) integrin/MAdCAM-1 adhesion pathway-mediated B cell migration may be a potential treatment for T1D.

Figure 1. Expression of adhesion molecules on PanLN B cells

Lymphocytes were isolated from PanLN, PLN, MLN and PP of 3-4-wk-old female NOD mice, stained with a FITC-anti-B220 mAb combined with a PE-conjugated mAb to α₄ integrin, β₇ integrin, L-selectin or LFA-1, or a PE-conjugated negative control mAb, and evaluated by FACS analysis. The histograms in each panel show the staining with the anti-lymphocyte adhesion molecule mAb (unshaded) and the isotype control mAb (shaded) on B220⁺ B cells. The numbers in each panel indicate the percentage of B cells that express each adhesion molecule (top) and the mean fluorescence intensity of the B cell staining (bottom). Data are one representative of three experiments. In each experiment, lymphocytes were pooled from 3-4 mice.

Figure 2. B cells migrate into PanLN more efficiently than into PLN

Fifty million TRITC-labeled lymphocytes from 3-4-wk-old female NOD mice were transferred i.v. into age-matched female NOD mice. Two h after cell transfer, donor B cells in PanLN, PLN, MLN and PP of host mice were identified by immunofluorescence staining with a FITC-anti-B220 mAb followed by FACS analysis. Representative FACS plots show donor B cells (TRITC⁺B220⁺ cells in box) in LNs and PP of host mice. Numbers in FACS plots are mean ± standard derivation of donor B cells as a percentage of total lymphocytes. One-way ANOVA test, *p<0.05 and **p<0.01 compared with PanLN, n=4 mice in each group.

Figure 3. MAbs to MAdCAM-1, α₄β₇ integrin, PNAd, L-selectin and LFA-1 inhibit migration of B cells into PanLN

Short-term in vivo lymphocyte migration assays were used to determine the role of each adhesion molecule in migration of B cells into PanLN in 3-4-wk-old female NOD mice. (A, C): Fifty million TRITC-labeled lymphocytes from 3-4-wk-old female NOD mice were transferred i.v. into age-matched female NOD mice treated with a mAb to MAdCAM-1 (A) or PNAd (C), or with a negative control mAb (A, C). (B, D, E): Fifty million TRITC-labeled lymphocytes from 3-4-wk-old female NOD mice were pretreated with a mAb to α₄β₇ integrin (B), L-selectin (D) or LFA-1 (E), or with a negative control mAb (B, D, E) and were transferred i.v. into age-matched female NOD mice. In all experiments, host mice were sacrificed 2 h after cell transfer. Donor B cells in PanLN, PLN, MLN and PP of host mice were identified by suspension staining with a FITC-anti-B220 mAb and FACS analysis. Migration of donor B cells (TRITC⁺B220⁺ cells) into the anti-adhesion molecule mAb-treated group is expressed as the percentage of the migration into the negative control mAb-treated group, in which migration is set at 100% (horizontal dotted line). One-way ANOVA test, *p<0.05 and **p<0.01 compared with control mAb-treated group, n=3-4 mice in each group.

Figure 4. Blockage of MAdCAM-1 prevents homeostatic migration of B cells into PanLN

(A, B) Three-week-old female NOD mice were injected i.p. with an anti-MAdCAM-1 mAb or a negative control mAb (30 μg/g body weight). (A): Mice were sacrificed at 4, 5 or 8 wk of age. The absolute numbers of B cells in PanLN, PLN and MLN were determined from hemacytometer counts and FACS analysis. Absolute number of B cells in each LN of the anti-MAdCAM-1 mAb-treated group is given as the percentage of that in the negative control-mAb-treated group, in which the absolute number of B cells is set at 100% (horizontal dotted line). One-way ANOVA test, **p<0.01 anti-MAdCAM-1 mAb group compared to the control mAb group. (B): Mice at 4, 5 or 8 wk of age were injected i.v. with 5×10⁷ TRITC-labeled lymphocytes from 3-4-wk-old female NOD mice. Two h after cell transfer, donor B cells in LNs of host mice were evaluated using FACS analysis. Migration of donor B cells (TRITC⁺B220⁺ cells) into the anti-MAdCAM-1 mAb-treated group is expressed as the percentage of the migration into the negative control mAb-treated group, in which migration is set at 100% (horizontal dotted line). One-way ANOVA test, **p<0.01 anti-MAdCAM-1 mAb group compared with the negative control mAb group (horizontal line, migration=100%), n=3-4 mice in each group.