Beta1 integrins differentially control extravasation of inflammatory cell subsets into the CNS during autoimmunity - PubMed (original) (raw)

Beta1 integrins differentially control extravasation of inflammatory cell subsets into the CNS during autoimmunity

Martina Bauer et al. Proc Natl Acad Sci U S A. 2009.

Abstract

Inhibiting the alpha(4) subunit of the integrin heterodimers alpha(4)beta(1) and alpha(4)beta(7) with the monoclonal antibody natalizumab is an effective treatment for multiple sclerosis (MS). However, the pharmacological action of natalizumab is not understood conclusively. Previous studies suggested that natalizumab inhibits activation, proliferation, or extravasation of inflammatory cells. To specify which mechanisms, cell types, and alpha(4) heterodimers are affected by the antibody treatment, we studied MS-like experimental autoimmune encephalomyelitis (EAE) in mice lacking the beta(1)-integrin gene either in all hematopoietic cells or selectively in T lymphocytes. Our results show that T cells critically rely on beta(1) integrins to accumulate in the central nervous system (CNS) during EAE, whereas CNS infiltration of beta(1)-deficient myeloid cells remains unaffected, suggesting that T cells are the main target of anti-alpha(4)-antibody blockade. We demonstrate that beta(1)-integrin expression on encephalitogenic T cells is critical for EAE development, and we therefore exclude alpha(4)beta(7) as a target integrin of the antibody treatment. T cells lacking beta(1) integrin are unable to firmly adhere to CNS endothelium in vivo, whereas their priming and expansion remain unaffected. Collectively, these results suggest that the primary action of natalizumab is interference with T cell extravasation via inhibition of alpha(4)beta(1) integrins.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

The clinical course of EAE is not altered in β1−/− BMCs. (A) Knockout efficiency for the indicated total and magnetically activated, cell-sorted populations was determined by Southern blotting. The number of samples for each population is given in each bar. Bars represent medians and interquartile ranges. (B) The relative weight normalized to day 0 and the clinical disease score of control and β1−/− BMCs with active EAE are shown. Data points indicate means of 13 mice from 3 independent experiments. Around day 16, mice were killed for histological and flow cytometric analyses. (C) Immunostaining of the spinal cord white matter of control and β1−/− BMCs with ongoing active EAE (clinical score 3) and healthy control mice. Infiltrating leukocytes were stained with Ly5.2, CD4, or Mac-1 antibodies (red), blood vessels were stained with a pan-laminin antibody (green), and nuclei were stained with DAPI (blue). (Scale bar, 100 μm.)

Fig. 2.

Fig. 2.

T lymphocytes depend on β1 integrins to enter the CNS. (A) Leukocytes and microglia cells were isolated from the brain and spinal cord of control or β1−/− BMCs with ongoing active EAE. Shown is the median number of isolated cells per CNS (average clinical score = 3, n = 5). (B and C) The isolated leukocytes were analyzed by flow cytometry. In B, the relative numbers of CD4+ T cells (CD4+), CD8+ T cells (CD8+), macrophages (Mac), and granulocytes (Gr) are shown (controls n = 8, β1−/− n = 9). (C) The β1 expression of the 4 leukocyte subsets was analyzed. Microglia cells are mainly host cell-derived and were excluded based on their cell surface expression of Ly-5.1. Each bar represents at least 5 mice. Bars in all panels represent medians and interquartile ranges.

Fig. 3.

Fig. 3.

β1 Integrin on T cells is important for EAE development. (A) Splenocytes from control (ctrl), β1fl/fl/MxCre+, or β1fl/fl/CD4Cre+ mice were stimulated unspecifically in vitro. Isotype control staining (iso) and β1-integrin expression of CD4+ T cells were analyzed by FACS. Shown are the medians and interquartile ranges of at least 5 animals. (B) The median day of disease onset of control and β1fl/fl/CD4Cre+ mice with active EAE is shown. (C) The clinical disease score of control and β1fl/fl/CD4Cre+ mice with active EAE is shown. Data points indicate means of 7 mice from 3 independent experiments. (D) Leukocytes and microglia cells were isolated by density gradient centrifugation from the CNS of control (n = 2) and β1fl/fl/CD4Cre+ (n = 3) mice with ongoing active EAE at day 31. The β1 expression of CD4+ and CD8+ T cells that were isolated from the CNS was quantified. Bars represent medians and interquartile ranges. (E) Shown are representative histograms of those T lymphocytes or of unspecifically stimulated splenocytes isolated from the same mice. Blue and red histograms represent control and β1−/− T cells, respectively. Isotype control stainings are shown in shaded gray histograms. (F) The clinical disease score of WT mice injected with encephalitogenic control or β1−/− MOG35–55-specific T cells is shown. Data points indicate means (controls n = 11, β1−/− n = 8; 4 independent experiments). The number of animals with EAE symptoms was compared by the Fisher exact test for each day (days 14–16: P < 0.05, days 17 and 18: P < 0.01, days 19 and 20: P < 0.005).

Fig. 4.

Fig. 4.

β1 Deficiency does not influence T cell proliferation and polarization. (A) Proliferation of control (ctrl) or β1−/− T cells was analyzed by flow cytometry. The division and proliferation index and the percentage of dividing cells were calculated from the generation sizes (n = 6). (B) CFSE stainings from 1 representative control and β1−/− sample are shown. Light gray-shaded histograms represent control animals that received DCs without OVA323–339 peptide. (C) Shown are the percentages of cytokine-expressing T cells. The intracellular cytokine expression of control and β1−/− T lymphocytes was analyzed by FACS. Bars represent medians and interquartile ranges (controls n = 4, β1−/− n = 5). ns, not significant.

Fig. 5.

Fig. 5.

In vivo firm adhesion of β1−/− T lymphocytes to the spinal cord microvasculature is dramatically reduced. (A) Integrin expression of proliferating control (blue) and β1−/− T lymphocytes (red) was analyzed by FACS. Isotype control stainings are shown in shaded gray histograms. Histograms are representative of 3 independent experiments. (B) Adhesion of T cell blasts to the endothelioma cell lines bEnd5 (WT) and bEndI1.1 (ICAM-1−/−) at room temperature. Graphs show medians and interquartile ranges of adherent T cells per field of view (fov) (n = 4). (C) Firm adhesion of control and β1−/− T cell blasts to the spinal cord microvascular wall was analyzed by IVM in WT mice with ongoing active EAE. Firm adhesion was analyzed 10 min, 30 min, and 1 h after infusion of T cells. Bars represent medians and interquartile ranges (n = 6). (D) Initial contact events of T cell blasts with endothelial cells were analyzed by IVM. From 6 experiments with control and β1−/− T cells, 46 and 30 vessels were analyzed, respectively. Shown is the percentage of rolling or captured T cells among the total number of T cells passing through a given venule during a 1-min observation period. Each dot represents one vessel, and the red line indicates the median. ns, not significant.

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