We report a patient with a novel form of inherited integrin dysfunction in which the β1 and β2 integrins are expressed on the leukocyte cell surface at normal levels, but cannot be stimulated to bind ligand by intracellular signaling pathways. The defect also affects the integrin αIIbβ3, because the patient’s platelets cannot be induced to bind soluble fibrinogen or to aggregate by platelet agonists. However, all three classes of integrins can be activated directly either with divalent cations Mg2+/Mn2+ or activating mAbs.
Characteristically LAD-1 patients lacking β2 integrins have elevated numbers of circulating neutrophils and fail to clear bacterial infections because these cells have restricted ability to traffic into infected tissue. The patient described here also has an abnormally elevated number of circulating lymphocytes, a feature that is not evident in classical LAD-1. This is probably due to the lack of function of both the β1 and β2 families of integrins, resulting in impaired responses of both myeloid cells and lymphocytes to inflammatory signals. In experiments with knockout mice, lack of β2 integrin LFA-1 can be partially compensated for by the β1 integrins, but blocking function of both classes of integrins further increases the numbers of circulating neutrophils and lymphocytes and substantially impairs inflammatory responses (27
To date, four patients with nonclassical forms of LAD-1 have been described. The leukocytes in two cases expressed approximately 60% of the normal levels of β2 integrins, but had no β2 integrin function (8
). These LAD-1 variant cases are related to classical LAD-1, having a mutation in one allele that prevents expression and a mutation in the other allele that allows expression but not ligand binding. Similar GT patients have been described who have αIIbβ3 expression but no function (29
There are two reported LAD-1 variant cases in which no mutations in the integrin β subunit genes have been detected. The first case had the clinical indicators of LAD-1 and the β2 integrins were expressed but nonfunctional (30
). After some years, defects in β3 integrin function also became apparent; but, in contrast to our patient, β1 integrins were not affected. For the second patient, there were problems with β2 and β1 integrin function, and a bleeding problem then developed that was not explored experimentally (31
). A speculation is that the genetic lesions in these two patients and patient FM are individual, but may be related and potentially highlight a specific common pathway dedicated to integrin activation.
Because the activities of at least three integrin families are affected in patient FM and the integrins can function when activated directly from outside the cell, it is unlikely that the dysfunction is due to mutations in the integrin subunits themselves. It is more probable that the faulty gene encodes a protein that is critical for integrin function. Moreover, the expression of the affected gene product is predicted to be confined to hematopoietic cells or have redundant function in other cell types. The fact that murine β1 integrin knockouts are embryonic lethal (32
) implies that the patient would not have survived if β1 integrin functioning was universally affected. The results demonstrating that other leukocyte and platelet functions are relatively normal provide evidence that the lesion is confined to a component of a key pathway dedicated to integrin activation.
Further insight into the nature of the patient’s lesion has come from confocal microscopy, which revealed that LFA-1 and the β1 integrins on the patient’s T cells are constitutively clustered and that the state of clustering does not change when the cells are activated. Integrin clustering is believed to be dynamic, but much is unknown about the sequence of events regulating this process. It has been proposed that integrins on resting cells are tethered to the cytoskeleton in an unclustered form and that activation of the cell removes the cytoskeletal constraint allowing the integrin to move and form clusters in the membrane, mediate firm ligand binding, and potentially reassociate with the cytoskeleton (2
). The observation that the patient has constitutively clustered integrins, yet these integrins do not function, implies that dynamic clustering is required for inside-out stimulated adhesion and that, in the patient’s cells, integrin mobility may be restricted. Although exposure to low concentrations of cytochalasin D promotes adhesion of naive T cells by removing the cytoskeletal tethering of the integrins (3
), this procedure did not alter the adhesion capabilities of the patient’s T cells (data not shown). It is interesting that outside-in signaling through LFA-1 was sufficient for T cell adhesion, polarization, and migration, suggesting that the state of clustering is irrelevant for these adhesion-dependent activities, or, alternatively, that it can be altered by signaling directly through LFA-1.
Although the platelet integrin αIIbβ3 is activated primarily through signaling that leads to a change in affinity (reviewed in ref. 5
), avidity regulation also plays a role in the activation of this integrin (4
). In the patient’s platelets, inside-out agonists, such as ADP and thrombin, failed to induce binding of soluble fibrinogen, suggesting that this pathway is not functioning. The relationship between affinity and avidity regulation remains to be resolved, but the evidence gathered from the unusual patient described here suggests that, at least in platelets, both integrin activation pathways may be defective or, alternatively, that they are interdependent.
As far as LFA-1 clustering is concerned, the adapter protein SLAP-130 (also known as Fyb and ADAP) and the Rac-1 guanine nucleotide exchange factor,Vav-1, recently have been reported to be involved (19
). Deletion of either protein in murine T cells prevents LFA-1 clustering and adhesion to ICAM-1 and the β1 integrin ligand fibronectin in response to CD3 engagement. These cells, however, do adhere when stimulated with phorbol ester, which is not the case for the patient’s T cells. Caution is necessary when extrapolating results obtained with mouse cells to human studies, but the above findings, as well as the fact that the expression of SLAP-130 and Vav-1 in the patient’s T cells is normal, suggests that it is unlikely that these proteins are defective in this patient. Additionally, SLAP-130 is not expressed in B cells, whereas adhesion of the patient’s EBV-transformed B lymphoblastoid cells is defective. Expression of an active form of the GTPase Rap-1 in thymocytes also leads to constitutive clustering of LFA-1 (22
), and Rap-1 is reported to lie in the signaling pathway leading to LFA-1, Mac-1, and αIIbβ3 activation (reviewed in ref. 33
). Rap-1, however, is not hematopoietic cell specific, is expressed at normal levels in the patient, and, as with SLAP-130 and Vav-1, the effects on clustering and adhesion differ from the patient’s problem, which consists of constitutively clustered nonfunctioning integrins.
Other Rho family GTPases, RhoA, Rac, and Cdc42, have been directly implicated in adhesion and are involved in changes in the F-actin cytoskeleton that are important for cell migration (23
). Of interest is another patient with clinical features of LAD-1, where the lesion was found to be a mutation in the gene encoding the hematopoietic cell-specific small GTPase Rac-2, preventing GTP binding (24
). However, the patient described here has elevated numbers of lymphocytes and an intact superoxide burst, distinguishing her from the Rac-2 defective patient for whom the described dysfunctions are restricted to neutrophils. We found no difference in the expression or electrophoretic characteristics of Rac-1, Rac-2, RhoA, or Cdc42, or with T cell migration, suggesting that our patient’s problem does not lie with these proteins.
PKC isozymes have been implicated in adhesion processes (34
). In our study, phorbol ester was unable to activate β1 or β2 integrin–mediated adhesion, suggesting that the lesion could be either in a PKC subtype or downstream of such a kinase. Consistent with this phenotype, phorbol ester–sensitive Jurkat cells mutant in ERK-1 have been generated, in which β1 and β2 integrin function is lacking (25
). The patient’s cells, however, have normal ERK and p38 MAP kinase expression (data not shown). PKC-β1 has been suggested to have a role in LFA-1–mediated adhesion stimulated from outside (18
), a pathway that is functional in the patient’s cells.
We found T cells to express normal amounts of PKC-β, -δ, -θ, and -ζ, but 2.5-fold increased levels of PKC-α, and it is of interest to speculate about the meaning of this increase. The use of the Ca2+
mobilizer, thapsigargin, is one of the inside-out signaling protocols that failed to induce adhesion of the patient’s cells. As this form of adhesion is not sensitive to the broadly based PKC inhibitor Ro 31-8220 (2
), it seems that the patient’s adhesion lesion is evident in at least one model of β1 and β2-induced adhesion that is not PKC dependent. It is therefore unlikely that the increased PKC-α level is the cause of the lesion, but is probably a consequence of it. PKC-α has been implicated in various adhesion phenomena. It is physically associated with β1 integrins and involved in membrane trafficking by controlling integrin internalization (35
). Overexpression of a number of PKC isozymes, including PKC-α, cause adhesion of Jurkat T cells (36
). Taken together, these results suggest that the overexpression of PKC-α observed in the patient’s T cells may well be a downstream effect of the clustered state of the integrins. In any event, this observation provides a valuable clue to the aberrant molecular events in the patient’s leukocytes and will require further investigation.
Various other factors associated with integrin activity seemed unlikely to explain the patient’s lesion. A number of cytoskeletal proteins, reported to associate with integrins (reviewed in ref. 37
), were present at normal levels and with normal electrophoretic characteristics in the patient’s T cells. Moreover, T cell polarization and migration following triggering of LFA-1 were normal, suggesting a functioning cytoskeleton. Therefore, the dysfunction is probably not due to lack of expression of one of the cytoskeletal proteins involved in migration. Other factors that associate more directly with integrins and the cytoskeleton, such as cytohesin-1, which binds to LFA-1, or β3-endonexin, which binds αIIbβ3, are integrin-type specific, diminishing the likelihood of their involvement in this integrin dysfunction (see ref. 37
). In this context, the kinase ILK is also of interest because it links several types of integrins to the cytoskeleton (26
). ILK, however, is expressed at normal levels in the patient and is, moreover, best characterized for its role in β1 integrin-matrix interactions that are key for epithelial cells, not hematopoietic cells, where the problems lie in this patient. We have proposed previously that inside-out signaling in T cells leads to clustering of LFA-1, which is dependent upon cytoskeletal rearrangement and activity of the cysteine protease, calpain (2
). Calpain is also activated following FMLP stimulation of neutrophils (38
). However, in platelets, calpain activation is reported to lie downstream of αIIbβ3 ligation, because calpain inhibitors have no effect on platelet aggregation but do affect fibrin clot retraction (39
). Because the patient in this study has no platelet aggregation, it is unlikely that the defect affects calpain.
PI3K is reported to have a role in chemokine induction of LFA-1 affinity increase in murine T cells (40
). In contrast, we have failed to find a role for PI3K in LFA-1–mediated adhesion of human T cells stimulated by TCR-CD3 or phorbol esters, as assessed by the lack of effect of inhibitor LY294002 (B. Leitinger, unpublished data). Therefore, it is unlikely that the patient’s adhesion defect lies in the PI3K pathway. In addition, the p110δ subunit of PI3K is predominantly expressed in leukocytes, and recent characterization of mice expressing an inactive form of p110δ show CD3-stimulated α4β1/α5β1- and LFA-1–mediated adhesion to be normal (41
In summary, a defect in the activation of three classes of integrin on leukocytes and platelets has been described. A key observation is that the β2 and β1 leukocyte integrins are constitutively clustered, and it is speculated that the lack of regulation of this clustering leads to a defect in the ability of the integrins to function correctly and, in turn, to the LAD-1- and GT-like symptoms of this unique patient. It is hoped that study of this patient will yield further key insights into the common features that lead to integrin activity on leukocytes.