Mouse basophils reside in extracellular matrix-enriched bone marrow niches which control their motility

Abstract

Basophils co-express FcεRIα and CD49b, the α-2 chain of integrin-type receptor VLA-2 (α2β1), which recognizes type-1 collagen as a major natural ligand. The physiological relevance of this integrin for interactions with extracellular bone marrow matrix remains unknown. Herein, we examined the expression of several receptors of this family by bone marrow-derived basophils sorted either ex-vivo or after culture with IL-3. Having established that both populations display CD49d, CD49e and CD49f (α-4, α-5 and α-6 integrins subunits, respectively), we addressed receptor functions by measuring migration, adhesion, proliferation and survival after interacting with matched natural ligands. Type I collagen, laminin and fibronectin promoted basophil migration/adhesion, the former being the most effective. None of these ligands affected basophil viability and expansion. Interactions between basophils and extracellular matrix are likely to play a role in situ, as supported by confocal 3D cell imaging of femoral bone marrow sections, which revealed basophils exclusively in type-1 collagen-enriched niches that contained likewise laminin and fibronectin. This is the first evidence for a structure/function relationship between basophils and extracellular matrix proteins inside the mouse bone marrow.

Figure 1. Bone marrow-derived basophils express integrin and migrate in response to their respective extracellular matrix ligands.

The procedure used for direct isolation of resident medullary basophils yields a population that contains 30–50% CD49b⁺FcεRIα⁺ basophils . Basophil-enriched populations were also generated from total bone marrow cells cultured for 8 days with IL-3, as described before , . Panel a depicts cytofluorometric profiles of constitutively expressed integrin-type ECM receptors for type-1 collagen, fibronectin and laminin, on basophils isolated ex vivo or from IL-3-driven cultures. Panel b shows that freshly isolated (ex vivo) or bone marrow-derived basophils (in vitro) migrate through collagen, fibronectin and laminin, and that the migratory response is highest in response to type-1 collagen, as compared to laminin and fibronectin. Panel c reveals that in all cases, membrane expression of corresponding receptors is not changed. Panel d shows that neutralizing monoclonal antibodies to CD49b, CD49f and CD49e completely abrogated specific migration through type-1 collagen, laminin and fibronectin, respectively. Experiments in this figure were performed with 2–3-month-old mice, with at least 5 animals being evaluated per group. In all panels, values correspond to specific migration after subtracting background adhesion to wells coated with BSA. Results are expressed as means ± SE. * p<0.05; ** p<0.01; *** p<0.001.

Figure 2. Bone marrow-derived basophils adhere onto purified extracellular matrix through integrin-type receptors.

The panel shows that basophils obtained from IL-3 stimulated bone marrow cultures are able to adhere onto selected ECM proteins, namely type-1 collagen, laminin and fibronectin. Freshly isolated basophils behave similarly. Adhesion is abrogated by monoclonal antibodies that recognize matching integrin-type receptors. Experiments were performed with 2–3-month-old mice, with at least 5 animals being evaluated per group, and the adhesion assay was performed in 24-well culture plates coated with ECM proteins or BSA. Total numbers of adhered basophils correspond to specific adhesion after subtracting the background values from plates coated with BSA. Results are expressed as mean ± SE*** p<0.001.

Figure 3. Extracellular matrix ligands do not modulate expansion or survival of basophils derived from bone marrow cultures.

Percentages of basophils (phenotypically defined by CD49b-FcεRIα co-expression) expanded from bone marrow cells cultured with IL-3 remain unchanged whatever the ECM ligand added during culture: type I collagen (T-I col), fibronectin (FN) or laminin (LM) (panel a). Lack of ECM ligand effect on survival of mature basophils as assessed by means of XTT staining of viable cells, quantified by absorbance (panels b and c). Results represented in panel b were obtained by positive selection based on CD49b expression and confirmed with one experiment using depletion of CD117⁺ cells (panel c). Cells were incubated during 24 hours in culture medium alone or supplemented with IL-3 and no significant differences were induced by the addition of integrin ligands. Data are expressed as means ± SE from three distinct experiments except for Fig. 3c that represents one experiment.

Figure 4. Basophils reside in extracellular matrix-enriched bone marrow microenvironments and adhere onto ECM ligands.

Panel a represents a confocal image in which cell nuclei are labeled in blue by DAPI, type I collagen is seen in red, whereas the two basophil markers CD49b and FcεR1 are in green and magenta, respectively. Basophils were defined, using of Pearson's coefficient to identify CD49b and FcεR1 co-localization, and are shown in white in panel b, whereas the corresponding volume profile can be seen in panel c, where one basophil is seen in close contact with type I collagen fibrils. Panel d shows another microscopic field, in which a cluster of basophils can be identified (in white), in close apposition to fibronectin, thus showing that the basophil niche also contains fibronectin (labeled in red). The volume reconstruction of this same field can be seen in panel e. Panel f shows volume profiles in which it becomes clear that basophils (seen in white (defined by CD49b plus FcεR1 co-localization) are anchored in the fibronectin network (in red). Co-localization of two markers was defined after calculation of Pearson's coefficient with imageJ (v1.45) and JaCoP plugin, whereas volume profiles were settled using the Imaris software, after analyzing stacked confocal images. In all pictures, corresponding magnifications are shown in the white bars.