ß2 Integrin Signaling Cascade in Neutrophils: More Than a Single Function.

Neutrophils are the most prevalent leukocytes in the human body. They have a pivotal role in the innate immune response against invading bacterial and fungal pathogens, while recent emerging evidence also demonstrates their role in cancer progression and anti-tumor responses. The efficient execution of many neutrophil effector responses requires the presence of ß2 integrins, in particular CD11a/CD18 or CD11b/CD18 heterodimers. Although extensively studied at the molecular level, the exact signaling cascades downstream of ß2 integrins still remain to be fully elucidated. In this review, we focus mainly on inside-out and outside-in signaling of these two ß2 integrin members expressed on neutrophils and describe differences between various neutrophil stimuli with respect to integrin activation, integrin ligand binding, and the pertinent differences between mouse and human studies. Last, we discuss how integrin signaling studies could be used to explore the therapeutic potential of targeting ß2 integrins and the intracellular signaling cascade in neutrophils in several, among other, inflammatory conditions in which neutrophil activity should be dampened to mitigate disease.

Transmembrane and Juxtamembrane Structure of αL Integrin in Bicelles.

The accepted model for the interaction of α and ß integrins in the transmembrane (TM) domain is based on the pair αIIbß3. This involves the so-called outer and inner membrane association clasps (OMC and IMC, respectively). In the α chain, the OMC involves a GxxxG-like motif, whereas in the IMC a conserved juxtamembrane GFFKR motif experiences a backbone reversal that partially fills the void generated by TM separation towards the cytoplasmic half. However, the GFFKR motif of several α integrin cytoplasmic tails in non-bicelle environments has been shown to adopt an α-helical structure that is not membrane-embedded and which was shown to bind a variety of cytoplasmic proteins. Thus it is not known if a membrane-embedded backbone reversal is a conserved structural feature in α integrins. We have studied the system αLß2 because of its importance in leukocytes, where integrin deactivation is particularly important. Herein we show that the backbone reversal feature is not only present in αIIb but also in αL-TM when reconstituted in bicelles. Additionally, titration with ß2 TM showed eight residues clustering along one side of αL-TM, forming a plausible interacting face with ß2. The latter orientation is consistent with a previously predicted reported polar interaction between αL Ser-1071 and ß2 Thr-686.

α7 helix region of αI domain is crucial for integrin binding to endoplasmic reticulum chaperone gp96: a potential therapeutic target for cancer metastasis.

Integrins play important roles in regulating a diverse array of cellular functions crucial to the initiation, progression, and metastasis of tumors. Previous studies have shown that a majority of integrins are folded by the endoplasmic reticulum chaperone gp96. Here, we demonstrate that the dimerization of integrin αL and ß2 is highly dependent on gp96. The αI domain (AID), a ligand binding domain shared by seven integrin α-subunits, is a critical region for integrin binding to gp96. Deletion of AID significantly reduced the interaction between integrin αL and gp96. Overexpression of AID intracellularly decreased surface expression of gp96 clients (integrins and Toll-like receptors) and cancer cell invasion. The α7 helix region is crucial for AID binding to gp96. A cell-permeable α7 helix peptide competitively inhibited the interaction between gp96 and integrins and blocked cell invasion. Thus, targeting the binding site of α7 helix of AID on gp96 is potentially a new strategy for treatment of cancer metastasis.

Probing the cell membrane by magnetic particle actuation and Euler angle tracking.

The mechanical properties of the cell membrane and the subjacent actin cortex are determinants of a variety of processes in immunity and cell division. The lipid bilayer itself and its connection to the actin cortex are anisotropic. An accurate description of the mechanical structure of the cell membrane and the involved dynamics therefore necessitates a measurement technique that can capture the inherent anisotropy of the system. Here, we combine magnetic particle actuation with rotational and translational particle tracking to simultaneously measure the mechanical stiffness of monocytic cells in three rotational and two translational directions. When using particles that bind via integrins to the cell membrane and the subjacent cortex, we measured an isotropic stiffness and a characteristic power-law dependence of the shear modulus on the applied frequency. When using particles functionalized with immunoglobulin G, we measured an anisotropic stiffness with a 10-fold-reduced value in one dimension. We suggest that the observed reduced stiffness in the plane of the cell membrane is caused by a local detachment of the lipid bilayer from the subjacent cytoskeletal cortex. We expect that our technique will enable new insights into the mechanical properties of the cell membrane that will help us to better understand membrane processes such as phagocytosis and blebbing.

An unusual allosteric mobility of the C-terminal helix of a high-affinity alphaL integrin I domain variant bound to ICAM-5.

Integrins are cell surface receptors that transduce signals bidirectionally across the plasma membrane. The key event of integrin signaling is the allosteric regulation between its ligand-binding site and the C-terminal helix (alpha7) of integrin's inserted (I) domain. A significant axial movement of the alpha7 helix is associated with the open, active conformation of integrins. We describe the crystal structure of an engineered high-affinity I domain from the integrin alpha(L)beta(2) (LFA-1) alpha subunit in complex with the N-terminal two domains of ICAM-5, an adhesion molecule expressed in telencephalic neurons. The finding that the alpha7 helix swings out and inserts into a neighboring I domain in an upside-down orientation in the crystals implies an intrinsically unusual mobility of this helix. This remarkable feature allows the alpha7 helix to trigger integrin's large-scale conformational changes with little energy penalty. It serves as a mechanistic example of how a weakly bound adhesion molecule works in signaling.

Intercellular adhesion molecule-3 binding of integrin alphaL beta2 requires both extension and opening of the integrin headpiece.

The leukocyte-restricted integrin alpha(L)beta(2) is required in immune processes such as leukocyte adhesion, migration, and immune synapse formation. Activation of alpha(L)beta(2) by conformational changes promotes alpha(L)beta(2) binding to its ligands, ICAMs. It was reported that different affinity states of alpha(L)beta(2) are required for binding ICAM-1 and ICAM-3. Recently, the bent, extended with a closed headpiece, and extended with open headpiece conformations of alpha(L)beta(2), was reported. To address the overall conformational requirements of alpha(L)beta(2) that allow selective binding of these ICAMs, we examined the adhesion properties of these alpha(L)beta(2) conformers. alpha(L)beta(2) with different conformations were generated by mutations, and verified by using a panel of reporter mAbs that detect alpha(L)beta(2) extension, hybrid domain movement, or I-like domain activation. We report a marked difference between extended alpha(L)beta(2) with closed and open headpieces in their adhesive properties to ICAM-1 and ICAM-3. Our data show that the extension of alpha(L)beta(2) alone is sufficient to mediate ICAM-1 adhesion. By contrast, an extended alpha(L)beta(2) with an open headpiece is required for ICAM-3 adhesion.

Role of the beta-subunit arginine/lysine finger in integrin heterodimer formation and function.

Formation of the integrin alphabeta heterodimer is essential for cell surface expression and function. At the core of the alphabeta interface is a conserved Arg/Lys "finger" from the beta-subunit that inserts into a cup-like "cage" formed of two layers of aromatic residues in the alpha-subunit. We evaluated the role of this residue in heterodimer formation in an alphaA-lacking and an alphaA-containing integrin alphaVbeta3 and alphaMbeta2 (CD11b/CD18), respectively. Arg261 of beta3 was mutated to Ala or Glu; the corresponding Lys252 of beta2 was mutated to Ala, Arg, Glu, Asp, or Phe; and the effects on heterodimer formation in each integrin examined by ELISA and immunoprecipitation in HEK 293 cells cotransfected with plasmids encoding the alpha- and beta-subunits. The Arg261Glu (but not Arg261Ala) substitution significantly impaired cell surface expression and heterodimer formation of alphaVbeta3. Although Lys252Arg, and to a lesser extent Lys252Ala, were well tolerated, each of the remaining substitutions markedly reduced cell surface expression and heterodimer formation of CD11b/CD18. Lys252Arg and Lys252Ala integrin heterodimers displayed a significant increase in binding to the physiologic ligand iC3b. These data demonstrate an important role of the Arg/Lys finger in formation of a stable integrin heterodimer, and suggest that subtle changes at this residue affect the activation state of the integrin.

Aggregatibacter actinomycetemcomitans leukotoxin requires beta-sheets 1 and 2 of the human CD11a beta-propeller for cytotoxicity.

Aggregatibacter actinomycetemcomitans leukotoxin (Ltx) is a repeats-in-toxin (RTX) cytolysin that kills human leukocyte function-associated antigen-1 (LFA-1; alpha(L)/beta(2))-bearing cells. In order to determine whether the alpha(L) portion of the heterodimer is involved in Ltx recognition, we transfected human, mouse and bovine alpha(L) cDNAs into J-beta(2).7, an alpha(L)-deficient cell line, and looked for restoration of Ltx susceptibility. Cells expressing either bovine or human alpha(L) in conjunction with human beta(2) were efficiently killed by Ltx, an indication that bovine alpha(L) could substitute for its human counterpart in critical regions used by Ltx for attachment to LFA-1. On the other hand, cells expressing murine alpha(L) and human beta(2) were not susceptible to the lethal effects of Ltx indicating that the toxin recognition sites are not present in the corresponding mouse sequence. To further identify the region(s) of alpha(L) recognized by Ltx, we constructed and evaluated a panel of chimeric human/murine alpha(L) genes in J-beta(2).7 cells. Analysis of the alpha(L) mutant panel showed that the presence of human N-terminal 128 amino acids on a mouse CD11a background, a region that includes beta-sheets 1 and 2 of the beta-propeller of the human alpha(L) chain, was sufficient for Ltx cytolysis.

Antigen-specific suppression of experimental autoimmune encephalomyelitis by a novel bifunctional peptide inhibitor.

The objective of this study is to evaluate the activity of a novel peptide, i.e., bifunctional peptide inhibitor (BPI), which targets the immunological synapse and inhibits autoimmune responses in an antigen-specific manner. Proteolipid protein (PLP)-BPI was designed by conjugating two peptides, an encephalitogenic epitope of proteolipid protein (PLP(139-151)) and an intercellular adhesion molecule-1-binding peptide derived from alpha(L) integrin (CD11a(237-246)), via a spacer peptide. The therapeutic effect of PLP-BPI was studied in experimental autoimmune encephalomyelitis (EAE) in female SJL/J mice as a model for human multiple sclerosis. Mice that received i.v. injections of PLP-BPI showed significantly lower EAE disease scores and incidence than those treated with vehicle, PLP(139-151) peptide only, CD11a(237-246) peptide only, unlinked mixture of PLP(139-151), and CD11a(237-246) peptides, or other control peptides. Multiple injections of antigenic peptide can produce anaphylactic responses; interestingly, PLP-BPI-treated animals have significantly lower anaphylactic response than do the PLP(139-151)-treated group. Therefore, PLP-BPI can effectively inhibit the disease severity and incidence of EAE with a lower possibility of inducing fatal anaphylaxis. These results suggest that BPI-type molecules can be used to treat different autoimmune diseases in which antigenic epitopes have been identified.

Rolling adhesion of alphaL I domain mutants decorrelated from binding affinity.

Activated lymphocyte function-associated antigen-1 (LFA-1, alphaLbeta2 integrin) found on leukocytes facilitates firm adhesion to endothelial cell layers by binding to intercellular adhesion molecule-1 (ICAM-1), which is up-regulated on endothelial cells at sites of inflammation. Recent work has shown that LFA-1 in a pre-activation, low-affinity state may also be involved in the initial tethering and rolling phase of the adhesion cascade. The inserted (I) domain of LFA-1 contains the ligand-binding epitope of the molecule, and a conformational change in this region during activation increases ligand affinity. We have displayed wild-type I domain on the surface of yeast and validated expression using I domain specific antibodies and flow cytometry. Surface display of I domain supports yeast rolling on ICAM-1-coated surfaces under shear flow. Expression of a locked open, high-affinity I domain mutant supports firm adhesion of yeast, while yeast displaying intermediate-affinity I domain mutants exhibit a range of rolling phenotypes. We find that rolling behavior for these mutants fails to correlate with ligand binding affinity. These results indicate that unstressed binding affinity is not the only molecular property that determines adhesive behavior under shear flow.

Differential activation of LFA-1 and Mac-1 ligand binding domains.

Nine integrin alpha subunits contain an 'inserted' or I-domain, known to involve in ligand binding. Mutation of an invariant isoleucine residue in the I-domains of alphaL and alphaM has previously been reported to activate LFA-1 and Mac-1, respectively. In this article, we report notable differences in the regulation of adhesion of these two integrins. We find that mutation of the isoleucine residue in the proposed "socket for isoleucine" in full-length alphaL does not lead to an active LFA-1, although mutation of the equivalent residue in alphaM does convey constitutive activity to Mac-1. In addition, we observe the isolated I-domain of alphaL to be constitutively active. This challenges reports that state the alphaL I-domain exists in an inactive, closed conformation, and requires the presence of activating agents for ligand binding. These results shed further light on the many questions surrounding regulation of integrin activation.

Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins.

Although critical for development, immunity, wound healing, and metastasis, integrins represent one of the few classes of plasma membrane receptors for which the basic signaling mechanism remains a mystery. We investigated cytoplasmic conformational changes in the integrin LFA-1 (alphaLbeta2) in living cells by measuring fluorescence resonance energy transfer between cyan fluorescent protein-fused and yellow fluorescent protein-fused alphaL and beta2 cytoplasmic domains. In the resting state these domains were close to each other, but underwent significant spatial separation upon either intracellular activation of integrin adhesiveness (inside-out signaling) or ligand binding (outside-in signaling). Thus, bidirectional integrin signaling is accomplished by coupling extracellular conformational changes to an unclasping and separation of the alpha and beta cytoplasmic domains, a distinctive mechanism for transmitting information across the plasma membrane.

Characterization of ICAM-4 binding to the I domains of the CD11a/CD18 and CD11b/CD18 leukocyte integrins.

Intercellular adhesion molecule-4 (ICAM-4, LW blood group antigen), a member of the immunoglobulin superfamily expressed on red cells, has been reported to bind to CD11a/CD18 and CD11b/CD18 leukocyte integrins. The location of the ICAM-4 binding sites on CD11a/CD18 and CD11b/CD18 are not known. CD11/CD18 integrin I domains have been found to act as major binding sites for physiological ligands and a negatively charged glutamic acid in ICAMs is considered important for binding. ICAM-4 lacks such a residue, which is replaced by an arginine. However, we demonstrate here that ICAM-4 in red cells and transfected fibroblasts interacts specifically with the I domains of CD11a/CD18 and CD11b/CD18 integrins. The binding was inhibited by anti-I domain and anti-ICAM-4 antibodies and it was dependent on divalent cations. Interestingly, ICAM-4 negative red cells were still able to bind to the CD11b/CD18 I domain but the binding of these cells to the CD11a/CD18 I domain was clearly reduced. Using a solid phase assay, we were able to show that isolated I domains directly and specifically bind to purified recombinant ICAM-4 in a cation dependent manner. Competition experiments indicated that the binding sites in ICAM-4 for the CD11a and CD11b I domains are different. However, the ICAM-4 binding region in both I domains seems to overlap with the regions recognized by the ICAM-1 and ICAM-2. Thus we have established that the I domains contain an ICAM-4 binding region in CD11a/CD18 and CD11b/CD18 leukocyte integrins.