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Leucocyte adhesion deficiency (LAD) is a hereditary disorder caused by mutations in the CD18 (beta2 integrin) gene. Four missense mutations have been identified in three patients. CD18(A270V) supports, at a diminished level, CD11b/CD18 (Mac-1, alphaMbeta2 integrin) and CD11c/CD18 (p150,95, alphaXbeta2 integrin) expression and function but not CD11a/CD18 (LFA-1, alphaLbeta2 integrin) expression. Conversely, CD18(A341P) supports a limited level of expression and function of CD11a/CD18, but not of the other two CD11/CD18 antigens. CD18(C590R) and CD18(R593C) show a decreasing capacity to associate with the CD11a, CD11c and CD11b subunits. Transfectants expressing the CD11a/CD18 with the C590R and R593C mutations are more adhesive than transfectants expressing wild-type LFA-1, and express the reporter epitope of the monoclonal antibody 24 constitutively. Thus, the four mutations affect CD18 differently in its capacities to support CD11/CD18 expression and adhesion. These results not only provide a biochemical account for the clinical diversity of patients with leucocyte adhesion deficiency, but also offer novel insights into the structural basis of interaction between the alpha and beta subunits, which is an integral component in our understanding of integrin-mediated adhesion and its regulation.

In the primary sequence of the integrin beta subunit, the N-terminal region (NTR) and mid-region are separated by the I-like domain. To determine the spatial relationship and functional properties of the integrin beta(2) NTR and mid-region, we constructed beta(2)/beta(7) chimeras in which the NTR, I-like domain, and the mid-region of the beta(2) subunit were replaced by those of beta(7). Changing either the beta(2) NTR or mid-region, but not the I-like domain to that of beta(7) did not affect LFA-1 (alpha(L)beta(2)) formation and surface expression. Thus, the specificity of alpha(L)beta(2) pairing is conferred by the I-like domain but not the NTR or mid-region. Using these chimeras, the epitopes of six anti-beta(2) mAbs (H52, 7E4, AZN-L18, AZN-L27, KIM202, and MEM-148) were mapped. All except H52 require both the NTR and mid-region for epitope expression. Since these mAbs have distinct properties in terms of epitope expression and effect on LFA-1 binding to ICAM-1, we conclude that the beta(2) NTR and mid-region interact extensively. Although the I-like domain is located between the NTR and mid-region, its removal does not affect the folding of the beta(2) NTR/mid-region complex because this complex alone can be expressed as a soluble protein and precipitated by the appropriate mAbs. Finally, the mAbs H52 and 7E4, abrogated KIM185- but not Mg/EGTAinduced LFA-1/ICAM-1 binding and the epitope of MEM-148 is expressed on Mg/EGTA-activated but not resting LFA-1. These results suggest that the NTR/mid-region complex is involved in the regulation of LFA-1 function.

The cysteine-rich region (CRR) of the integrin beta subunits is organised into four repeating elements. By expression of a panel of truncated beta 2 subunits, and CRR segments fused to the C-terminal end of a CD4 soluble fragment, the segment required for the expression of two monoclonal antibody conformational epitopes was determined. This segment, E482-Q574, contains 16 cysteines representing two repeating units. We have thus defined the CRR unit motif of 'xC---C---C---CxCxxCxC---Cx', where 'x' represents a single residue, and '---' represents a stretch of four to 14 residues.

LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) are members of the beta2 integrins involved in leukocyte function during immune and inflammatory responses. We aimed to determine a minimized beta2 subunit that forms functional LFA-1 and Mac-1. Using a series of truncated beta2 variants, we showed that the subregion Q23-D300 of the beta2 subunit is sufficient to combine with the alphaL and alphaM subunits intracellularly. However, only the beta2 variants terminating after Q444 promote cell surface expression of LFA-1 and Mac-1. Thus, the major cysteine-rich region and the three highly conserved cysteine residues at positions 445, 447, and 449 of the beta2 subunit are not required for LFA-1 and Mac-1 surface expression. The surface-expressed LFA-1 variants are constitutively active with respect to ICAM-1 adhesion and these variants express the activation reporter epitope of the mAb 24. In contrast, surface-expressed Mac-1, both the wild type and variants, require 0. 5 mM MnCl2 for adhesion to denatured BSA. These results suggest that the role of the beta2 subunit in LFA-1- and Mac-1-mediated adhesion may be different.

Leucocyte adhesion deficiency type 1 (LAD-1) is characterized by the incapacity of leucocytes to carry out their adhesion functions via their CD11/CD18 antigens, which are also referred to as the leucocyte integrins. The patients generally suffer from poor wound healing and recurrent bacterial and fungal infections. In severe cases, the infections are often systemic and life-threatening. A LAD patient (AW) of moderate phenotype has been identified but, unlike most other cases, the level of CD11/CD18 antigens on her leucocytes are uncharacteristically high for a LAD patient. Molecular analysis revealed that she is a compound heterozygote for CD18 mutations. She has inherited a D231H mutation from her father and a G284S mutation from her mother. By transfection studies, it was established that the G284S mutation does not support CD11/CD18 antigen expression on the cell surface. In contrast, the D231H mutation does not affect CD18 forming integrin heterodimers with the CD11 antigens on the cell surface. However, the expressed integrins with the D231H mutation are not adhesive to ligands.

In the leukocyte adhesion deficiency (LAD)-1 syndrome, there is diminished expression of beta2(CD18) integrins. This is caused by lesions in the beta2-subunit gene and gives rise to recurrent bacterial infections, impaired pus formation, and poor wound healing. We describe a patient with clinical features compatible with a moderately severe phenotype of LAD-1 but who expresses the beta2 integrins lymphocyte function- associated molecule (LFA)-1 and Mac-1 at 40%-60% of normal levels. This level of expression should be adequate for normal integrin function, but both the patient's Mac-1 on neutrophils and LFA-1 on T cells failed to bind ligands such as fibrinogen and intercellular adhesion molecule (ICAM)-1, respectively, or to display a beta2-integrin activation epitope after adhesion-inducing stimuli. Unexpectedly, divalent cation treatment induced the patient's T cells to bind to ICAM-2 and ICAM-3. Sequencing of the patient's two CD18 alleles revealed the mutations S138P and G273R. Both mutations are in the beta2-subunit conserved domain, with S138P a putative divalent cation coordinating residue in the metal ion-dependent adhesion site (MIDAS) motif. After K562 cell transfection with alpha subunits, the mutated S138P beta subunit was coexpressed but did not support function, whereas the G273R mutant was not expressed. In summary, the patient described here exhibits failure of the beta2 integrins to function despite adequate levels of cell-surface expression.

The cysteine-rich region (CRR) of the beta2 integrin subunit was replaced by that of beta1 to give the chimera beta2NV1. Beta2NV1 can combine with alphaL to form a variant leukocyte-function-associated antigen (LFA)-1 on COS cell surface, suggesting that the specificity of the beta2 interaction with alphaL does not lie in the CRR. Unlike those expressing wild-type LFA-1, COS cells expressing alphaL beta2NV1 are constitutively active in intercellular adhesion molecule (ICAM)-1 adhesion. These results suggest that activation of LFA-1 involves the release of an intramolecular constraint, which is maintained, in part, by the authentic beta2 CRR.