Leukocyte adhesion deficiency-I variant syndrome (LAD-Iv, LAD-III): molecular characterization of the defect in an index family

Abstract

Leukocyte adhesion deficiencies are rare clinical syndromes of impaired host defense that provide novel insights into regulation of immune and inflammatory responses. Leukocyte adhesion deficiency (LAD)-I variant (LAD-Iv), also called LAD-III, is a unique disorder in which inside-out signaling of β₁, β₂, and β₃ integrins on leukocytes and platelets is disrupted, leading to impaired cellular adhesion, recurrent infections, and bleeding. We originally reported the second patient with this disorder to be identified and characterized the adhesive deficiencies and functional phenotype of this subject's leukocytes. Here, we show that the molecular defect in this index subject is a new mutation in FERMT3 (KINDLIN-3) which encodes KINDLIN-3, a cytoskeletal protein that interacts with the cytoplasmic tails of β₁, β₂, and β₃ integrins and is required for inside-out and outside-in signaling of these heterodimers. We also report clinical features and previously unrecognized defects in cells from a new patient, a sibling of the original subject that we described who carries the same FERMT3 mutation. Mutations in FERMT3 have now been shown to be the basis for LAD-Iv/LAD-III in each of the four original patients or families that established this syndrome, including the family that we describe.

Figure 1

Leukocytes from Patient 2 demonstrated the LAD-Iv/LAD-III phenotype of impaired inside-out signaling of β₁ and β₂ integrins. A. PMNs from Patient 2 did not adhere to β₂ integrin ligands when activated. PMNs from Patient 2 or a healthy age-matched infant were incubated for 10 min at 37°C in wells coated with bovine serum albumin (BSA), gelatin, or fibrinogen (FB) in the absence of an agonist or with n-formyl-methionyl-phenylalanine (fMLP) (10⁻⁷M). Adhesion was quantitated as described in Methods. This figure illustrates results from a single experiment (performed in triplicate). The same pattern was demonstrated in two other experiments in which PMNs from Patient 2 and control subjects were activated with fMLP or PMA (10⁻⁷M) (data not shown). B. Activated PMNs from Patient 2 did not adhere to fibronectin. PMNs from Patient 2 or a control subject were incubated on wells coated with fibronectin in the presence or absence of PMA or fMLP as in panel A. In parallel, PMNs in some wells were treated with the β₁ integrin activating antibody TS 2/16 (“act mAb”; 10μg/ml). This figure indicates the pattern of adhesion in a single experiment (performed in triplicate) and is representative of two additional experiments examining primary PMNs from Patient 2. C. Adhesion of EBV-transformed lymphoblasts from Patient 2 to immobilized ligands was induced by extracellular Mn⁺⁺ or mAb TS 2/16 (“activating mAb”; see legend to panel B). Lymphoblasts from Patient 2 or a control subject were incubated for 15 min at 37°C in wells coated with fibronectin with buffer alone, or with PMA (10⁻⁷M) (performed in triplicate). In parallel wells lymphoblasts were treated with activating mAb, Mn⁺⁺ (1μM), or Mn⁺⁺ together with a blocking anti- β₁ mAb (mAb p4C10, 10 μg/ml). A similar pattern was seen in a second experiment. In additional experiments, extracellular Mn⁺⁺ induced adhesion of lymphoblasts from Patient 2 to immobilized ICAM-1, a ligand for β₂ integrins; this was inhibited by a blocking anti- β₂ mAb (mAb 60.3; 10μg/ml) (N=2, data not shown).

Figure 2

Monocytes from Patient 2 did not adhere or differentiate in culture. Mononuclear cells from a healthy volunteer (A) or a Patient 2 (B) were incubated in culture wells for 45 min, nonadherent cells were removed, and the adherent monocytes were washed, covered with fresh medium, and incubated at 37°C in 5% CO₂ for an additional 48 hr. Representative fields were then photographed. These figures illustrate the cellular morphology in one experiment examining monocytes from this subject.

Figure 3

KINDLIN-3 is absent in EBV-transformed lymphoblasts from Patient 1. Lymphoblasts from Patient 1 and two control subjects were lysed, subjected to electrophoresis, and immunoblotted for KINDLIN-3 or actin as described in Methods.