CD45 exclusion- and cross-linking-based receptor signaling together broaden FcεRI reactivity

Abstract

For many years, the high-affinity receptor for immunoglobulin E (IgE) FcεRI, which is expressed by mast cells and basophils, has been widely held to be the exemplar of cross-linking (that is, aggregation dependent) signaling receptors. We found, however, that FcεRI signaling could occur in the presence or absence of receptor cross-linking. Using both cell and cell-free systems, we showed that FcεRI signaling was stimulated by surface-associated monovalent ligands through the passive, size-dependent exclusion of the receptor-type tyrosine phosphatase CD45 from plasma membrane regions of FcεRI-ligand engagement. Similarly to the T cell receptor, FcεRI signaling could also be initiated in a ligand-independent manner. These data suggest that a simple mechanism of CD45 exclusion-based receptor triggering could function together with cross-linking-based FcεRI signaling, broadening mast cell and basophil reactivity by enabling these cells to respond to both multivalent and surface-presented monovalent antigens. These findings also strengthen the case that a size-dependent, phosphatase exclusion-based receptor triggering mechanism might serve generally to facilitate signaling by noncatalytic immune receptors.

Fig. 1. Mast cells sense the presence of surfaces.

(A) FcεRIγ phosphorylation detected on Western blots of whole-cell lysates of RBL-2H3 cells grown for 24 hours in suspension (Susp) or adherent (Adh) culture (left) or after contacting uncoated plastic for 0 to 15 min (right). The blots were incubated with anti-pY47 FcεRIγ (top) or anti-FcεRIγ (bottom) antibodies. (B) Anti-pY47 FcεRIγ signal relative to anti-FcεRIγ signal normalized against that for cell stimulate with anti-TNP IgE and TNP_BSA. Statistical significance was calculated versus non-contacting cells (0 min). (C and D) Interference reflection microscopy (IRM) images of WT, PP2-treated WT, FcεRI-ve, and FcεRI+ve RBL-2H3 cells 30 min after contacting uncoated glass in growth medium (C). The size of the contact areas of cells under the indicated conditions were quantified (D). Each spot represents a single cell. (E and F) Differential interference contrast images of WT, FcεRI-ve, and FcεRI+ve RBL-2H3 cells cultured for 24 hours on uncoated glass revealing dendritic morphology (E). The percentage of the indicated cells with a dendritic morphology were quantified (F). (G) Percentage of RBL-2H3 cells that grew spontaneously in suspension under normal culturing conditions. (H) Percentage of cells that detach at different levels of mechanical agitation. For (G) and (H), WT cells are in red; FcεRI-ve cells are in green; FcεRI+ve cells are in blue.. (I) The contact area of RBL-2H3 cells 30 min after making contact with PLL-coated glass. Each spot represents a single cell. (J) The percentages of WT, FcεRI-ve, and FcεRI+ve RBL-2H3 cells that exhibited Ca²⁺ flux responses within 10 min of making contact with uncoated (glass) or PLL-coated glass. For all bar graphs, data are means ± SD of three independent experiments. Blots and images are representative of three independent experiments. Scale bars, 5 µm. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

Fig. 2. FcεRI responds to immobilized ligands and is sensitive to constitutive kinase and phosphatase activities.

(A) Percentages of RBL-2H3 cells that exhibited Ca²⁺ flux responses within 10 min of making contact with glass or SLBs functionalized with His-Fcε or TNP_BSA-IgE versus uncoated surfaces or in response to soluble His-Fcε. Statistically significant differences refer to differences from zero. (B) Normalized anti-pY47 FcεRIγ signals relative to anti-FcεRIγ signals in whole-cell lysates from RBL-2H3 cells that made contact with the indicated surfaces. Statistically significant differences are in comparison to cells at time zero.. Data are from four independent experiments. (C) Western blotting analysis of FcεRIγ phosphorylation in RBL-2H3 cells after acute inhibition of kinase or phosphatase activity with PP2 or pervanadate (Na₃VO₄), respectively. (D) Quantitation of the effects measured in (C) in RBL-2H3 cells (left) and primary human basophils (right). Significances represent differences from unstimulated cells. Replicate primary basophil measurements were performed with blood from different donors. For all bar graphs, data are means ± SD of three independent experiments, unless stated otherwise. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

Fig. 3. CD45 and FcεRI segregate following engagement of surface-associated IgE.

(A to E) Left: Confocal fluorescence images of Syk, FcεRI, and CD45 at the basal surfaces of RBL-2H3 cells that had made contact with (A) TNP_BSA-IgE-coated glass, (B) a His-Fcε-functionalized SLB, (C) Ni-glass, (D) uncoated glass, or (E) PLL-coated glass. Timings indicate the period of contact with the surface. Right: Intensity line profiles correspond to the arrows in the merged views. (F) Exclusion of CD45 relative to FcεRI was observed for primary human basophils that were loaded with anti-Der f 1 IgE and then interacted with Der f 1–coated glass. Images are representative of observations made in at least three independent experiments. Replicates with primary basophils were performed with blood from different donors. Scale bars, 5 µm.

Fig. 4. CD45 exclusion is passive and independent of integrin activity.

(A) Confocal fluorescence images of Syk, FcεRI, and CD45 at the basal surface of an RBL-2H3 cells treated with PP2 after making contact with TNP_BSA-IgE-coated glass (a line profile is shown in fig. S5). (B) Effect of PP2, as used in (A), on FcεRI triggering as measured by Ca²⁺ flux intensity. Images are 10-min maximum intensity projections for Ca²⁺ intensity scaled as a heat map; blue, low intensity; red, high intensity. (C) Confocal fluorescence images of Syk, FcεRI, and CD45 at the basal surface of RBL-2H3–derived GPMVs after making contact with TNP_BSA-IgE-coated glass (upper) or a His-Fcε–functionalized SLB (lower). (D) Confocal fluorescence images of Syk, FcεRI, and CD45 at the basal surface of RBL-2H3 cells after making contact for the indicated times with TNP_BSA-IgE-coated glass in the presence of EDTA to chelate divalent cations. (E) Confocal fluorescence images of Syk, FcεRI, and CD45 at the basal surface of CD29-ve RBL-2H3 cells after making contact for the indicated times with TNP_BSA-IgE-coated glass. (F) Ratio of CD45 fluorescence intensity outside (O) versus inside (I) areas of enriched FcεRI intensity, for WT, CD29-ve, and EDTA-treated RBL-2H3 cells. Data are means ± SD of three independent experiments, with > 5 cells assessed per experiment. (G) Percentages of WT, CD29-ve, and EDTA-treated RBL-2H3 cells that produced Ca²⁺ flux responses within 10 min of making contact with glass coated with TNP_BSA-IgE or TNP_BSA-IgE and rat laminin peptide. Data are means ± SD of four independent experiments. Images are representative of observations made in at least three independent experiments. Scale bars, 5 µm. **P < 0.01; ns, not significant.

Fig. 5. CD45 exclusion is sensitive to the size of the surface-associated ligand.

(A) Schematic representation of CD43Fcε chimeras. (B and C) Confocal fluorescence images of Syk, FcεRI, and CD45 at the basal surface of an RBL-2H3 cell after making contact with an SLB (B) or Ni-glass (C) presenting extended CD43₁Fcε and CD43₃Fcε chimeras. (D) Percentages of RBL-2H3 cells producing Ca²⁺ fluxes within 10 min of making contact with an SLB or Ni-coated glass functionalized with extended CD43₁Fcε and CD43₃Fcε chimeras versus the same surfaces presenting His-Fcε. Data are means ± SD of four independent experiments. (E) Confocal fluorescence image of CD45 (that is, anti-CD45 Fab) at the basal surface of primary human basophils after making contact with anti-IgE–coated glass. (F) Normalized anti-pY47 FcεRIγ signals relative to anti-FcεRIγ signals obtained from Western blots of whole-cell lysates from primary human basophils after making contact anti-IgE conjugated directly or indirectly (through donkey anti-mouse; DaM) to glass. Data are means ± SD of three independent experiments. Resting denotes cells kept in suspension. (G) Percentage maximal degranulation of RBL-2H3 cells 30 min after making contact with an SLB or Ni-coated glass functionalized with extended CD43₁Fcε and CD43₃Fcε chimeras versus the same surfaces presenting His-Fcε. Data are means ± SD of four independent experiments. Images are representative of observations made in at least three independent experiments. Replicates with primary basophils were performed with blood from different donors. Scale bars, 5 µm. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

Fig. 6. FcεRI triggering relies on the large extracellular domain of CD45.

(A) Schematic of WT and chimeric CD45 constructs, which are colored purple to reflect their color in the fluorescence images. (B and C) Left: FcεRI and CD43CD45 (B) or CD86CD45 (C) fluorescence at the basal surface of RBL-2H3 cells cultured for 24 hours on glass. Right: Line profiles of fluorescence intensity corresponding to the arrows in the images. (D) Local concentration, that is, the average number of molecules in the observation volume (measured by fluorescence correlation spectroscopy), for CD45 and CD43CD45 at the basal versus apical surfaces of RBL-2H3 cells compared to those for CD86CD45. Data are means ± SD of three independent experiments, with >3 cells analyzed per experiment. (E) Confocal fluorescence images of Syk, FcεRI, and CD43CD45 at the basal surface of an RBL-2H3 cell after making contact for the indicated times with TNP_BSA-IgE-coated glass. (F) Fluorescence intensity for CD45, CD86CD45, and CD43CD45 outside (O) versus inside (I) areas of enriched FcεRI intensity. Data are means ± SD of three independent experiments, with >5 cells assessed per experiment. (G) Confocal fluorescence images of Syk, FcεRI, and CD86CD45 at the basal surface of an RBL-2H3 cell after making contact for the indicated times with TNP_BSA-IgE-coated glass. (H) Percentages of RBL-2H3 cells expressing CD45, CD86CD45, or CD43CD45, that produced Ca²⁺ fluxes within 10 min of making contact with an SLB or Ni-coated glass functionalized with His-Fcε, TNP_BSA-IgE-coated glass, or after being incubated with soluble anti-TNP and TNP_BSA. Data are means ± SD of four independent experiments. (I) Normalized anti-pY47 FcεRIγ signals relative to anti-FcεRIγ signals for whole-cell lysates prepared from RBL-2H3 cells expressing CD45, CD86CD45, and CD43CD45 after making contact for the indicated times with Ni-coated glass functionalized with His-Fcε (top) or with TNP_BSA-IgE-coated glass (bottom). Data are means ± SD of three independent experiments. All images are representative of observations made in at least three independent experiments. Scale bars, 5 µm. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

Fig. 7. FcεRI signaling can be triggered by unilamellar vesicles.

(A) Confocal fluorescence images of Syk, FcεRI, and CD45 at the equatorial plane of RBL-2H3 cells that interacted with GUVs (top) or laudan-stained LUVs (bottom) loaded with Fcε. G, GUV; L, LUV. Scale bars, are 5 µm. Images are representative of observations made in three independent experiments. (B) Schematic representation of the non-triggered state of the resting basophil surface (top) and after the triggering of FcεRI by polyvalent antigen (pAg; middle) or surface-associated monovalent antigen (mAg; bottom).