A novel form of integrin dysfunction involving β1, β2, and β3 integrins (original) (raw)
Patient FM was born at term by elective Caesarean section to nonconsanguineous Maltese parents. She had two female siblings, of whom one is 7 years old and well, whereas the other died within hours of birth with widespread bruising and bleeding. Despite her atraumatic delivery, patient FM was noted to have extensive bruising and petechiae within hours of birth. She had an antenatal intraventricular hemorrhage and later required insertion of a ventriculo-peritoneal shunt for posthemorrhagic hydrocephalus. A platelet count and routine clotting screen performed at this point were in the normal range. The umbilical cord separated normally at 1 week of age. At three months of age she was referred for investigation of prolonged bleeding following minor trauma.
The platelet count, thrombin time, activated partial thromboplastin time, prothrombin time, and fibrinogen levels were all within the normal range. However, platelet aggregation was absent in response to ADP, collagen, and arachidonic acid. In contrast, platelets from both parents aggregated normally in response to these agonists. Flow cytometry revealed that the patient’s platelets had normal expression of GPIb and αIIbβ3. These results suggested a diagnosis of type 2 GT with dysfunctional, rather than absent, platelet αIIbβ3. She has been managed with tranexamic acid and platelet transfusions as required.
From 5 months of age the patient developed recurrent bacterial infections and at 11 months she developed leg ulcers and was commenced on prophylactic antibiotics. She was found to have leukocytosis (38.4 × 109/l; normal range 5 × 109–15 × 109/l), suggestive of a leukocyte adhesion defect, but had normal expression of the leukocyte integrin αL, αM, αX, and β2 subunits and the selectin ligand sialyl Lewis x. She had normal humoral immune responses to tetanus toxoid and Haemophilus influenza, neutrophil phagocytosis of Staphylococcus aureus, and oxidative burst. Her lymphocyte count, however, was high (15.7 × 109/l; normal range of 1.5 × 109–4 × 109/l). Her T cell mitogenic activity to phytohemagglutinin was reduced by 50% compared with healthy age-matched controls. The patient is now 3 years old and has undergone a successful bone marrow transplant. There is no family history of either LAD-1– or GT-type disorders.
Integrin expression on the patient’s platelets, neutrophils, and T cells. Because the patient displayed symptoms indicative of both leukocyte and platelet integrin dysfunction, the cell surface expression of the major integrins on platelets, neutrophils and T cells was analyzed by flow cytometry. The overlapping profiles in Figure 1 show that the expression of the αIIb and β3 subunits on platelets, the αL, αM, αX, and β2 subunits on neutrophils, and the αL, β2, α4, α5, and β1 subunits on T cells was similar for the patient and a control donor.
Comparison of integrin expression on platelets, T cells, and neutrophils from patient and control. Expression of (a) αIIb and β3 subunits on platelets (n = 2). (b) αL, αM, αX, and β2 subunits on neutrophils. (c) αL, β2, α4, α5, and β1 subunits on T cells from control (black line) and patient (gray region). Background binding is indicated (dotted line) and is identical for control and patient. Representative histograms (n = 3) are shown.
To determine whether the patient’s integrins were abnormally processed or posttranslationally modified, cell lysates were subjected to SDS-PAGE and Western blotting for the relevant integrin subunits. No differences were detected in the electrophoretic characteristics of the αIIb or β3 integrin subunits of platelet lysates (Figure 2a) or the αL, β2, α4, or β1 subunits of T cell lysates (Figure 2b) prepared from the patient and a control donor. Therefore, the patient’s integrins resembled normal controls in both expression and biochemical characteristics.
Electrophoretic characteristics of integrin subunits from patient and control platelets and T cells. (a) Platelet lysates from a control donor (C) and the patient (P) blotted for αIIb and β3 subunits. (b) T cell lysates from a control donor (C) and the patient (P) blotted for αL, β2, α4, and β1 subunits. Representative blots (n = 2) are shown.
Functional analysis of αIIbβ3 on the patient’s platelets. Although the patient had normal cell surface expression of the three classes of integrins tested, it was possible that her symptoms were due to the inability of these integrins to function normally. The function of the platelet integrin αIIbβ3 was assessed by aggregometry. Although control platelets responded to 5 μM ADP as expected, the patient’s platelets did not aggregate (Figure 3a). We next assessed the ability of two standard platelet agonists, ADP and TRAP, which signal through two distinct platelet receptors, to cause platelets to bind soluble fibrinogen (Figure 3b). Both stimuli induced binding of control platelets to fibrinogen, and this was inhibited by the αIIbβ3 antagonist, eptifibatide; however, the patient’s platelets failed to bind soluble fibrinogen under any circumstances of inside-out stimulation. These agonists induced upregulation of α-granule contents, such as P-selectin, indicating platelet activation was normal (data not shown). Another way to activate integrins is to use mAb’s, such as LIBS-6, which stimulate αIIbβ3 by direct activation of the ectodomain (termed outside-in signaling) (13). LIBS-6 induces expression of the αIIbβ3 activation epitope recognized by the mAb PAC-1 (14). Here LIBS-6 induces the PAC-1 epitope on both patient and control platelets (Figure 3c).
Comparison of integrin αIIbβ3 function in patient and control platelets. (a) Platelet aggregation in response to 5 μM ADP (single experiment performed in duplicate). (b) Binding of FITC-conjugated antifibrinogen to platelets in the presence (black line) or absence (gray region) of 20 μg/ml eptifibatide. Platelets were either unstimulated or stimulated for 20 minutes with 10 μM ADP or 1 μM TRAP. Data are representative of four separate experiments. (c) Binding of mAb PAC-1 to platelets stimulated with 10 μg/ml β3 integrin mAb LIBS-6 in the presence (black line) and absence (gray region) of eptifibatide. Data are representative of two separate experiments.
Functional analysis of Mac-1 on the patient’s neutrophils. The function of the β2 integrin Mac-1 was examined by inducing neutrophil binding to immobilized fibrinogen. A variety of activating stimuli were used, which tested both inside-out and outside-in signaling to integrins. All treatments caused control neutrophils to bind to fibrinogen in a β2 integrin-dependent manner (Figure 4). In contrast, the patient’s neutrophils failed to bind in response to either FMLP or the phorbol ester PdBu (inside-out signaling), but did bind to fibrinogen following exposure to the β2 activating mAb KIM 185. Therefore, the ability of Mac-1 on the patient’s neutrophils to bind fibrinogen was impaired in response to typical stimulants of inside-out signal transduction.
Comparison of Mac-1–mediated adhesion of patient and control neutrophils. The binding of control (light gray bars) and patient (black bars) neutrophils to fibrinogen-coated plates when stimulated with 100 nM FMLP, 50 nM PdBu, or 10 μg/ml KIM 185 for 30 minutes. The presence of β2 mAb IB4 at 10 μg/ml inhibits adhesion of control (white bars) and patient (dark gray bars) cells. Data (mean of triplicates ± SD) from one representative experiment (n = 2) are shown.
However, the patient’s neutrophils were able to mobilize intracellular Ca2+ in response to FMLP and the Ca2+ mobilizing agent thapsigargin, and FMLP induced similar levels of L-selectin shedding and Mac-1 upregulation in patient and control neutrophils (data not shown). Therefore, the patient’s neutrophils are responsive to FMLP in non-integrin–dependent ways.
Functional analysis of LFA-1 on the patient’s T cells. The function of the β2 integrin LFA-1 was examined on T cells by inducing adhesion to immobilized ligands. PdBu, the Ca2+ mobilizer ionomycin, or CD3 mAb UCHT-1, which cross links the T cell receptor/CD3 complex, were used to test LFA-1 activation by inside-out signaling, whereas the β2-activating mAb KIM 185, Mg2+/EGTA, or Mn2+ were used to directly activate LFA-1. All the stimuli induced LFA-1–mediated binding of control T cells to both ICAM-1 and ICAM-3 (Figure 5, a and b). However, only KIM 185, Mg2+/EGTA, and Mn2+ induced adhesion of the patient’s T cells. None of the stimuli that act through intracellular signaling pathways induced LFA-1–mediated adhesion of the patient’s T cells to either ligand (Figure 5, a and b).
Adhesion of patient and control T cells to LFA-1 ligands ICAM-1 and ICAM-3, to α4β1 ligand VCAM-1, and to α4β1/α5β1 ligand fibronectin. The binding of control (light gray bars) and patient (black bars) T cells to plates coated with (a) ICAM-1, (b) ICAM-3, (c) VCAM-1, and (d) fibronectin when stimulated with 50 nM PdBu, 1 μM ionomycin, 10 μg/ml UCHT-1, 10 μg/ml KIM 185 or TS2/16, 5 mM MgCl2/1 mM EGTA, or 0.5 mM MnCl2. The presence of αL mAb 38 at 10 μg/ml in a and b, α4 mAb HP2/1 at 10 μg/ml in c, and α4 mAb HP2/1 plus α5 mAb SAM-1 both at 10 μg/ml in d inhibits adhesion of control (white bars) and patient (dark gray bars) cells. Data (mean of triplicates ± SD) from one representative experiment (n = 3) are shown. Unstim, unstimulated.
Functional analysis of α4β1 and α5β1 on the patient’s T cells. T cells express β1 as well as β2 integrins, with α4β1 and α5β1 being involved in many immune processes in association with the β2 integrins. When adhesion to the α4β1 ligand VCAM-1 (Figure 5c) or the α4β1/α5β1 ligand fibronectin (Figure 5d) was assessed, all the stimuli tested induced adhesion of control T cells, whereas the patient’s T cells only adhered when stimulated by Mg2+/EGTA, Mn2+, or the β1-activating mAb TS2/16. Thus β1 and β2 integrins on the patient’s T cells were able to bind their ligands when stimulated directly through the ectodomain, but failed to bind when inside-out stimuli were used. These results suggested a possible lesion in an intracellular signaling pathway.
Functional analysis of β1 and β2 integrins on the patient’s B cells. To assess whether the defect in inside-out stimulation of integrin-mediated adhesion also affected B cells, we used EBV-transformed B lymphoblastoid cells derived from the patient’s blood and a control donor’s blood. Both these cell lines were able to adhere to ICAM-1 (Figure 6a) or fibronectin (Figure 6b) when stimulated with Mn2+, but the patient’s cells failed to adhere when stimulated with PdBu.
Adhesion of patient and control B lymphoblastoid cells to fibronectin and ICAM-1. The binding of control (gray bars) and patient (black bars) EBV-transformed B cells to plates coated with (a) ICAM-1 and (b) fibronectin when stimulated with 50 nM PdBu or 0.5 mM MnCl2 for 30 minutes. Data (mean of triplicates ± SD) from one representative experiment (n = 3) are shown.
The state of integrin affinity and avidity on the patient’s T cells. We next investigated the activation state of the integrins on the T cells. The β2 integrin LFA-1, when in higher affinity form, is recognized by mAb 24 (15) and binds soluble ICAM-1 with increased affinity (11). Exposure to Mn2+ of control and patient T cells induced equivalent levels of both mAb 24 and soluble ICAM-1 binding (Figure 7a), indicating that the capacity for LFA-1 to adopt a higher-affinity form was intact when stimulated from outside the cell. Similarly, β1 integrins from patient and control T cells could be induced to express the β1 activation epitope, recognized by mAb HUTS 21 (16), and to bind soluble VCAM-1 (α4β1 only) (Figure 7a). Therefore, the lack of β1 and β2 integrin function on the patient’s T cells could not be explained by an inability to assume a higher affinity conformation.
Comparison of the affinity and avidity state of integrins. (a) Control (black line) and patient (gray region) T cells incubated with mAbs 24 (β2 integrin activation reporter) or HUTS 21 (β1 integrin activation reporter) at 25 μg/ml, or ICAM-1Fc (300 μg/ml) or VCAM-1Fc (2 μg/ml) for 20 minutes at 37°C in the presence of 0.5 mM MnCl2; control (dotted line) and patient (dashed line) T cells incubated with mAbs or soluble ligand as above for 20 minutes at 4°C in the presence of 1 mM EDTA. Data are from one representative experiment (n = 3). (b) T cells were either unstimulated or treated with 5 mM Mg2+/1 mM EGTA, 50 nM PdBu, or 5 μM thapsigargin then labeled with LFA-1 mAb G25.2 and analyzed by confocal microscopy. A false color scheme is employed, which depicts the intensity of the fluorescent signal from blue (low) to yellow (high) (2). One optical section is shown at midheight of the cells. Data are representative of four experiments. The total fluorescent signal was quantified and averaged over four experiments as follows: no treatment, control 61.5 ± 5.0, patient 98.8 ± 4.9; Mg2+/EGTA, control 59.5 ± 3.5, patient 105.5 ± 6.8; PdBu, control 78.8 ± 2.9, patient 101.0 ± 1.8; thapsigargin, control 81.0 ± 3.4, patient 96.5 ± 6.6.
When β2 integrins on leukocytes are triggered through intracellular pathways, they become laterally mobile and cluster (2, 17). T cell adhesion to ICAM-1 is dependent on this clustered form of LFA-1 (2, 3). When viewed by confocal microscopy, control T cells exhibited increased LFA-1 clustering following exposure to PdBu or the Ca2+ mobilizer thapsigargin, but not when exposed to Mg2+/EGTA (Figure 7b), as reported previously (2). In contrast, LFA-1 was already in a clustered state on the patient’s T cells and additional stimulation with PdBu and thapsigargin caused no further increase (Figure 7b). Preliminary evidence indicated parallel findings for β1 integrins on control and patient T cells using both α4 and β1 mAbs (n = 2; data not shown). Other abundant cell surface membrane proteins such as CD2, CD4, CD8, CD55, and MHC class I were not clustered on the patient’s cells (data not shown). These findings suggest a disruption of signaling pathways causing dysregulation of integrin clustering or lateral mobility on the patient’s T cells.
Analysis of the cytoskeleton. For leukocytes and platelets, the link between integrins and the cytoskeleton is critical for their function, and the cytoskeleton is also involved in the process of integrin clustering. To address whether associations with the cytoskeleton are defective in the patient’s T cells, lysates were blotted for some of the most commonly reported integrin-associated cytoskeletal proteins. Filamin, talin, α-actinin, vinculin, ezrin, paxillin (not shown), and actin (Figure 8a) were all present at equivalent levels in the control and patient’s T cells and migrated as expected on SDS-PAGE. The findings indicate that none of these cytoskeletal proteins in the patient’s cells had been cleaved or subjected to altered posttranslational modification.
Western blotting of cytoskeletal proteins and morphology of migrating T cells from patient and control. (a) Control (C) and patient (P) T cell lysates were titrated and blotted for the indicated cytoskeletal proteins and also for the αL subunit of LFA-1. Representative blots are shown (n = 3). (b) Video microscopy pictures of control (C) and patient (P) T cells migrating on ICAM-1–coated coverslips following stimulation with 5 mM MgCl2/1mM EGTA. (c) Cell tracks of randomly migrating T cells treated as in b.
When T cells are stimulated through LFA-1 they polarize and migrate on immobilized ICAM-1 (18), suggesting that activated LFA-1 can signal remodeling of the cytoskeleton. Therefore, to determine whether a component of the patient’s T cell cytoskeletal network was dysfunctional, Mg2+/EGTA-treated T cells were adhered to ICAM-1 and their ability to polarize and migrate assessed. Both the patient and control T cells polarized (Figure 8b) and migrated (Figure 8c) on ICAM-1 in a comparable manner. The average speed of control T cells was calculated to be 12.7 ± 6.3 μm/min and of patient T cells was 12.4 ± 5.9 μm/min. These data provide evidence that both the cytoskeleton and the adapter proteins linking the cytoskeleton and integrins function normally in the patient’s cells.
Expression of GTPases, PKCs, and other adhesion-related molecules. Inside-out signaling pathways leading to integrin activation are poorly characterized. However, in an attempt to discover the nature of the signaling lesion giving rise to the lack of integrin function in the patient, we decided to assess the expression of various signaling molecules that have been associated with adhesion of leukocytes. The adaptor protein SLAP-130 (19, 20), the guanine nucleotide exchange factor Vav-1 (21), and the GTPase Rap-1 (22) have been shown recently to have a role in LFA-1 clustering and adhesion. The GTPases RhoA, Rac-1, and Cdc42 are involved in integrin-mediated cell migration (23) and mutations in Rac-2 give rise to neutrophil defects similar to the patient’s abnormalities (24). PKCs have been implicated in several aspects of leukocyte adhesion (18, 25), and the patient’s leukocytes do not adhere in response to activation of PKC with phorbol ester. Finally, the integrin-linked kinase (ILK) is involved in adhesion of several classes of integrin (26). The expression and electrophoretic characteristics of 12 of the 13 proteins tested were identical in patient and control T cells (Figure 9). Expression of PKC-α, however, was elevated 2.5-fold in the patient’s T cells.
Western blotting of Rho family GTPases, PKC isoforms, and other adhesion-related molecules. Control (C) and patient (P) T cell lysates were blotted for the indicated proteins as for Figure 8. Representative blots are shown (n = 3).