Structural and ICAM-1-docking properties of a cyclic peptide from the I-domain of LFA-1: an inhibitor of ICAM-1/LFA- 1-mediated T-cell adhesion

The purpose of this work was to study the conformation of cyclic peptide 1, cyclo(1,12)-Pen1-Ile2-Thr3-Asp4-Gly5-Glu6-Ala7- Thr8-Asp9-Ser10-Gly11-Cys12-OH, derived from the I-domain of the LFA-1 alpha-subunit. We found that cyclic peptide 1 can bind to the D1-domain of ICAM-1 and inhibit ICAM-1/LFA-1-mediated homotypic and heterotypic T-cell adhesion. To understand the bioactive conformation and binding requirements for cyclic peptide 1, its solution structure was studied using NMR, CD, and molecular dynamics simulations. Furthermore, possible binding properties between the cyclic peptide and the D1-domain of ICAM-1 were evaluated using docking experiments. This cyclic peptide has a stable betaII -turn at Asp4- Gly5-Glu6-Ala7 and a betaI-turn at Pen1-Ile2-Thr3-Asp4; a less stable betaV-turn is found at the C-terminal region. The beta-turn at Asp4- Gly5-Glu6-Ala7 was also found in the X-ray structure of the I-domain of LFA-1. Our CD studies showed that the peptide binds to calcium/magnesium and forms a 1:1 (peptide:calcium/magnesium) complex with low cation concentrations and multiple types of complexes with higher cation concentrations. Binding to divalent cations causes a conformational change in peptide 1; this is consistent with our previous study that binding of peptide 1 to ICAM-1 was influenced by divalent cations. Docking studies show the interaction between cyclic peptide 1 and the D1-domain of ICAM-1; it indicates that the Ile2-Thr3-Asp4-Gly4-Glu6-Ala7-Thr8 sequence interacts with the F and C strands of the D1-domain. Finally, these studies will help us design a new generation of selective peptides that may bind better to the D1-domain of ICAM-1.     

A Ca2+ binding cyclic peptide derived from the alpha-subunit of LFA-1: inhibitor of ICAM-1/LFA-1-mediated T-cell adhesion.

The objective of this work is to study the conformation of cyclic peptide (1), cyclo (1, 12) Pen1-Gly2-Val3-Asp4-Val5-Asp6-Gln7-+ ++Asp8-Gly9-Glu10-Thr11-Cys12, in the presence and absence of calcium. Cyclic peptide 1 is derived from the divalent cation binding sequence of the alpha-subunit of LFA-1. This peptide has been shown to inhibit ICAM-1-LFA-1 mediated T-cell adhesion. In order to understand the structural requirements for this biologically active peptide, its solution structure was studied by nuclear magnetic resonance (NMR), circular dichroism (CD) and molecular dynamics simulations. This cyclic peptide exhibits two types of possible conformations in solution. Structure I is a loop-turn-loop type of structure, which is suitable to bind cations such as EF hand proteins. Structure II is a more extended structure with beta-hairpin bend at Asp4-Val5-Asp6-Gln7. There is evidence that alterations in the conformation of LFA-1 upon binding to divalent cations cause LFA-1 to bind to ICAM-1. To understand this mechanism, the cation-binding properties of the peptide were studied by CD and NMR. CD studies indicated that the peptide binds to calcium and forms a 1 : 1 (peptide: calcium) complex at low calcium concentrations and multiple types of complexes at higher cation concentrations. NMR studies indicated that the conformation of the peptide is not significantly altered upon binding to calcium. The peptide can inhibit T-cell adhesion by directly binding to ICAM-1 or by disrupting the interaction of the alpha and beta-subunits of LFA-1 protein. This study will help us to understand the mechanism(s) of action of this peptide and will improve our ability to design a better inhibitor of T-cell adhesion.     

Inhibition of ICAM-1/LFA-1-mediated heterotypic T-cell adhesion to epithelial cells: design of ICAM-1 cyclic peptides

In this work, we have designed cyclic peptides (cIBL, cIBR, cIBC, CH4 and CH7) derived from the parent IB peptide (ICAM-1(1-21)) that are inhibitors of ICAM-1/LFA-1-mediated T-cell adhesion to Caco-2 cell monolayers. Cyclic peptide cIBR has the best activity of any of the peptides evaluated. The active ICAM-1 peptides have a common Pro-Arg-Gly sequence that may be important for binding to LFA-1.     

ICAM-3 interacts with LFA-1 and regulates the LFA-1/ICAM-1 cell adhesion pathway

The interaction of lymphocyte function-associated antigen-1 (LFA-1) with its ligands mediates multiple cell adhesion processes of capital importance during immune responses. We have obtained three anti-ICAM-3 mAbs which recognize two different epitopes (A and B) on the intercellular adhesion molecule-3 (ICAM-3) as demonstrated by sequential immunoprecipitation and cross-competitive mAb-binding experiments. Immunoaffinity purified ICAM-3-coated surfaces were able to support T lymphoblast attachment upon cell stimulation with both phorbol esters and cross-linked CD3, as well as by mAb engagement of the LFA-1 molecule with the activating anti-LFA-1 NKI-L16 mAb. T cell adhesion to purified ICAM-3 was completely inhibited by cell pretreatment with mAbs to the LFA-1 alpha (CD11a) or the LFA-beta (CD18) integrin chains. Anti-ICAM-3 mAbs specific for epitope A, but not those specific for epitope B, were able to trigger T lymphoblast homotypic aggregation. ICAM-3-mediated cell aggregation was dependent on the LFA-1/ICAM-1 pathway as demonstrated by blocking experiments with mAbs specific for the LFA-1 and ICAM-1 molecules. Furthermore, immunofluorescence studies on ICAM-3-induced cell aggregates revealed that both LFA-1 and ICAM-1 were mainly located at intercellular boundaries. ICAM-3 was located at cellular uropods, which in small aggregates appeared to be implicated in cell-cell contacts, whereas in large aggregates it appeared to be excluded from cell-cell contact areas. Experiments of T cell adhesion to a chimeric ICAM-1-Fc molecule revealed that the proaggregatory anti-ICAM-3 HP2/19 mAb was able to increase T lymphoblast attachment to ICAM-1, suggesting that T cell aggregation induced by this mAb could be mediated by increasing the avidity of LFA-1 for ICAM-1. Moreover, the HP2/19 mAb was costimulatory with anti-CD3 mAb for T lymphocyte proliferation, indicating that enhancement of T cell activation could be involved in ICAM-3-mediated adhesive phenomena. Altogether, our results indicate that ICAM-3 has a regulatory role on the LFA-1/ICAM-1 pathway of intercellular adhesion.     

A peptide derived from LFA-1 protein that modulates T-cell adhesion binds to soluble ICAM-1 protein

Leukocyte function associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) have been shown to be critical for adhesion process and immune response. Modulation or inhibition of the interaction between LFA-1/ICAM-1 interactions can result in therapeutic effects. Our group and others have shown that peptides derived from ICAM-1 or LFA-1 inhibit adhesion in a homotypic T-cell adhesion assay. It is likely that the peptides derived from ICAM-1 bind to LFA-1 and peptides derived from LFA-1 bind to ICAM-1 and inhibit the adhesion interaction. However, there are no concrete experimental evidence to show that peptides bind to either LFA-1 or ICAM-1 and inhibit the adhesion. Using NMR, CD and docking studies we have shown that an LFA-1 derived peptide binds to soluble ICAM-1. Docking studies using "autodock" resulted in LFA-1 peptide interacting with the ICAM-1 protein near Glu34. The proposed model based on our experimental data indicated that the LFA-1 peptide interacts with the protein via three intermolecular hydrogen bonds. Hydrophobic interactions also play a role in stabilizing the complex.     

Structural basis for LFA-1 inhibition upon lovastatin binding to the CD11a I-domain.

The lymphocyte function-associated antigen (LFA-1) belongs to the family of beta2-integrins and plays an important role in T-cell activation and leukocyte migration to sites of inflammation. We report here that lovastatin, a drug clinically used for lowering cholesterol levels, inhibits the interaction of human LFA-1 with its counter-receptor intercellular adhesion molecule-1. Using nuclear magnetic resonance spectroscopy and X-ray crystallography we show that the inhibitor binds to a highly conserved domain of the LFA-1 alpha-chain called the I-domain. The first three-dimensional structure of an integrin inhibitor bound to its receptor reveals atomic details for a hitherto unknown mode of LFA-1 inhibition. It also sheds light into possible mechanisms of LFA-1 mediated signalling and will support the design of novel anti-adhesive and immunosuppressive drugs.     

Functional involvement of the LFA-1/ICAM-1 adhesion system in the autologous mixed lymphocyte reaction

The integrin surface molecule termed lymphocyte functional antigen-1 (LFA-1), and its physiological ligand intercellular adhesion molecule-1 (ICAM-1), have been proven to play a relevant role in several immune reactions where cell-to-cell contact is required: these reactions include allogeneic mixed lymphocyte reaction (MLR) and direct cytotoxicity. In the present study, we show that monoclonal antibodies (mAbs) directed to LFA-1 as well as to ICAM-1 molecules are able to inhibit T cell proliferation in autologous MLR (AMLR). Such an in vitro reaction is generally considered a functional model of Ia-mediated immunocompetent cell cooperation, and is impaired in several pathological conditions. It is noteworthy that the LFA-1 molecule is largely represented on the T cell surface, whereas ICAM-1 is poorly expressed on resting T cells: autologous stimulation slightly increases ICAM-1 expression. Pretreatment studies indicate that the inhibitory effect of anti-ICAM-1 mAb on T cell proliferation in AMLR is exerted on responder T cells.     

Cutting edge: a small molecule antagonist of LFA-1-mediated cell adhesion.

LFA-1 (CD18,CD11a) is a cell-adhesion molecule that mediates critical immunological processes. In this paper we report the discovery and characterization of (R)-5-(4-bromobenzyl)-3-(3, 5-dichlorophenyl)-1,5-dimethylimidazolidine-2,4-dione (BIRT 377), an orally bioavailable small molecule that interacts specifically with LFA-1 via noncovalent binding to the CD11a chain and prevents LFA-1 from binding to its ligand, ICAM-1. BIRT 377 inhibits lymphocyte activity both in vitro and in vivo, in functional assays that require LFA-1-mediated cell adhesion. These results demonstrate that LFA-1-mediated leukocyte adhesion can be antagonized with noncharged, low m.w. molecules and suggest that the potential therapeutic value of adhesion inhibitors can be attained with a small, orally bioavailable compound.     

Induction of tyrosine phosphorylation during ICAM-3 and LFA-1-mediated intercellular adhesion, and its regulation by the CD45 tyrosine phosphatase

Intercellular adhesion molecule (ICAM)-3, a recently described counter- receptor for the lymphocyte function-associated antigen (LFA)-1 integrin, appears to play an important role in the initial phase of immune response. We have previously described the involvement of ICAM-3 in the regulation of LFA-1/ICAM-1-dependent cell-cell interaction of T lymphoblasts. In this study, we further investigated the functional role of ICAM-3 in other leukocyte cell-cell interactions as well as the molecular mechanisms regulating these processes. We have found that ICAM-3 is also able to mediate LFA-1/ICAM-1-independent cell aggregation of the leukemic JM T cell line and the LFA-1/CD18-deficient HAFSA B cell line. The ICAM-3-induced cell aggregation of JM and HAFSA cells was not affected by the addition of blocking mAb specific for a number of cell adhesion molecules such as CD1 1a/CD18, ICAM-1 (CD54), CD2, LFA-3 (CD58), very late antigen alpha 4 (CD49d), and very late antigen beta 1 (CD29). Interestingly, some mAb against the leukocyte tyrosine phosphatase CD45 were able to inhibit this interaction. Moreover, they also prevented the aggregation induced on JM T cells by the proaggregatory anti-LFA-1 alpha NKI-L16 mAb. In addition, inhibitors of tyrosine kinase activity also abolished ICAM-3 and LFA-1- mediated cell aggregation. The induction of tyrosine phosphorylation through ICAM-3 and LFA-1 antigens was studied by immunofluorescence, and it was found that tyrosine-phosphorylated proteins were preferentially located at intercellular boundaries upon the induction of cell aggregation by either anti-ICAM-3 or anti-LFA-1 alpha mAb. Western blot analysis revealed that the engagement of ICAM-3 or LFA-1 with activating mAb enhanced tyrosine phosphorylation of polypeptides of 125, 70, and 38 kD on JM cells. This phenomenon was inhibited by preincubation of JM cells with those anti-CD45 mAb that prevented cell aggregation. Altogether these results indicate that CD45 tyrosine phosphatase plays a relevant role in the regulation of both intracellular signaling and cell adhesion induced through ICAM-3 and beta 2 integrins.     

Regulation of locomotion and cell-cell contact area by the LFA-1 and ICAM-1 adhesion receptors.

We demonstrate complementary differences in the behavior of B lymphoblastoid cells adhering to LFA-1 or its counter-receptor ICAM-1. The interaction of B lymphoblastoid cells with glass-supported planar bilayers bearing LFA-1 or ICAM-1 was observed by time-lapse video microscopy, and the distribution of adhesion receptors on cells interacting with the planar bilayers was studied by immunofluorescence microscopy. B lymphoblasts formed a large contact area and crawled rapidly (up to 25 microns/min) on planar bilayers bearing ICAM-1. In contrast, these cells attached to planar bilayers bearing LFA-1 through a fixed point about which the cells actively pivoted, using a single stalk-like projection. Phorbol ester-stimulated lymphoblasts, which adhere more strongly to ICAM-1-bearing substrates than unstimulated lymphoblasts, were still capable of locomotion on ICAM-1. Phorbol ester stimulation of B lymphoblasts on planar bilayers bearing LFA-1 promoted a rapid conversion from "stalk" attachment to symmetrical spreading of the cell on the substrate. Cellular LFA-1 remained uniformly distributed on the cell surface during interaction with bilayers bearing purified ICAM-1 as determined by immunofluorescence. In contrast, ICAM-1 was concentrated in the stalk-like structure through which the unstimulated B lymphoblasts adhered to LFA-1 in planar bilayers, but ICAM-1 immunofluorescence became more uniformly distributed over the cell surface within minutes of phorbol ester addition. Neither LFA-1 or ICAM-1 colocalized with the prominent staining of filamentous actin in the ruffling membrane regions. Interaction through cell surface LFA-1 and ICAM-1, 2, or 3 promotes different cellular morphologies and behaviors, the correlation of which with previously observed patterns of lymphocyte interaction with different cell types is discussed.     

Structure of an allosteric inhibitor of LFA-1 bound to the I-domain studied by crystallography, NMR, and calorimetry

LFA-1 (lymphocyte function-associated antigen-1) plays a role in intercellular adhesion and lymphocyte trafficking and activation and is an attractive anti-inflammatory drug target. The alpha-subunit of LFA-1, in common with several other integrins, has an N-terminally inserted domain (I-domain) of approximately 200 amino acids that plays a central role in regulating ligand binding to LFA-1. An additional region, termed the I-domain allosteric site (IDAS), has been identified exclusively within the LFA-1 I-domain and shown to regulate the function of this protein. The IDAS is occupied by small molecule LFA-1 inhibitors when cocrystallized or analyzed by (15)N-(1)H HSQC (heteronuclear single-quantum coherence) NMR (nuclear magnetic resonance) titration experiments. We report here a novel arylthio inhibitor that binds the I-domain with a K(d) of 18.3 nM as determined by isothermal titration calorimetry (ITC). This value is in close agreement with the IC(50) (10.9 nM) derived from a biochemical competition assay (DELFIA) that measures the level of inhibition of binding of whole LFA-1 to its ligand, ICAM-1. Having established the strong affinity of the arylthio inhibitor for the isolated I-domain, we have used a range of techniques to further characterize the binding, including ITC, NMR, and X-ray crystallography. We have first developed an effective ITC binding assay for use with low-solubility inhibitors that avoids the need for ELISA-based assays. In addition, we utilized a fast NMR-based assay for the generation of I-domain-inhibitor models. This is based around the collection of HCCH-TOCSY spectra of LFA-1 in the bound form and the identification of a subset of side chain methyl groups that give chemical shift changes upon binding of LFA-1 inhibitors. This subset was used in two-dimensional (13)C-(15)N and (15)N-filtered and -edited two-dimensional NMR experiments to identify a minimal set of intraligand and ligand-protein NOEs, respectively (nuclear Overhauser enhancements). Models from the NMR data were assessed by comparison to an X-ray crystallographic structure of the complex, confirming that the method correctly predicted the essential features of the bound ligand.     

Discovery and SAR of diarylsulfide cyclopropylamide LFA-1/ICAM-1 interaction antagonists

Diarylsulfide cyclopropylamides were synthesized and evaluated as LFA-1/ICAM-1 interaction antagonists. A substituent pattern was identified which maximized potency and minimized protein binding as exemplified by antagonist 30 (IC50 = 5 nM).     

NMR solution structure of a potent cyclic nonapeptide inhibitor of ICAM-1-mediated leukocyte adhesion produced by homologous amino acid substitution.

We have previously described a disulfide-linked cyclic nonapeptide (inhibitory peptide-01, IP01), with the sequence CLLRMRSIC, which binds to intercellular adhesion molecule-1 (ICAM-1), and blocks binding to its counter-structure, the integrin alphaLbeta2 (leukocyte functional antigen-1, LFA-1) (Sillerud et al., J. Peptide Res. 62, 2003: 97). We now report the optimization of this peptide by means of single homologous amino acid substitutions to yield a new peptide (IP02-K6; CLLRMKSAC) which shows an approximately sixfold improvement in inhibitory activity of multivalent leukocyte binding (inhibition constant for 50% inhibition, IC50 = 90 microm) compared with IP01 (IC50 = 580 microm). This improvement in activity gives IP02-K6 potent in vivo activity in a murine model of ischemia reperfusion injury (Merchant et al., Am. J. Physiol. Heart Circ. 284, 2003: H1260). In order to determine the structural features relevant to ICAM-1-binding, we have determined the structure of IP02-K6 using proton nuclear magnetic resonance (NMR) spectroscopy and restrained molecular modeling. In our previously reported study of solution models of IP01, we observed three interconverting conformations during low-temperature molecular dynamics simulation. In the present study, we find a single conformation of IP02-K6 similar to one of the previously found conformations of IP01 (family C). In particular, an R4-S7 beta-turn is present in similar proportions in both conformation C of IP01 and in IP02-K6; this motif is important in binding to ICAM-1 because this turn enables the IP02-K6 backbone to drape over proline-36 on ICAM-1. The NMR-derived solution model of IP02-K6 was found to dock at the alphaLbeta2-binding site on ICAM-1 with no changes in peptide backbone conformation. This docking model displaced five of the 15 alphaLbeta2 residues at the ICAM-1-binding site and provided a rationale for understanding the quantitative relationship between IP02-K6 structure and biologic activity.     

CD40 signaling activates CD11a/CD18 (LFA-1)-mediated adhesion in B cells.

Cell-cell adhesion events play critical roles in the sequential migrations and multiple specific cell-cell interactions which B cells undergo during normal development and function. We have observed that mAb to several B cell-associated molecules, including mAb to CD19, CD37, and CD40, induce homotypic aggregation of freshly isolated human B cells. The aggregation of B cells induced by CD40 mAb was due to activation of a cell-cell adhesion system, and not due to agglutination by mAb, because 1) in addition to being energy dependent and cation dependent, the aggregation was blocked by inhibitors of messenger RNA and protein synthesis; and 2) a mouse B cell line transformed with intact human CD40 aggregated in response to CD40 mAb, whereas a line expressing surface CD40, but lacking the cytoplasmic tail and previously shown incapable of transmitting a signal from the cell surface, did not aggregate. The aggregation, although of slow onset, was persistent and of high avidity. In addition, CD40 mAb induced increased surface expression of intercellular adhesion molecule-1 (CD54), a ligand for CD11a/CD18 (LFA-1), and CD18 mAb blocked aggregation. CD40 mAb also augmented the ability of dense B cells to stimulate the proliferation of allogeneic T cells via a CD18-dependent process. We conclude that signaling through CD40, elicited by cross-linking the CD40 protein on the cell surface, activates the CD18/intercellular adhesion molecule adhesion system; in addition, CD40 cross-linking may activate a second adhesion system since CD40 mAb induced aggregation of the B cell line Ramos, which does not express surface CD18. B cell adhesion may be triggered by signaling through multiple surface proteins, thereby lending specificity of activation to adhesion systems which are broadly expressed.     

Down-regulation of cell adhesion molecules LFA-1 and ICAM-1 after in vitro treatment with the anti-TNF-alpha agent thalidomide.

The tumor necrosis factor-alpha (TNF-alpha) inhibitor thalidomide is known to be a potent modulator of host immunity, a potential treatment for autoimmune disorders such as rheumatoid arthritis (RA) and a treatment for complications of HIV-1 infection. RA is an autoimmune disease of the joints that has been associated with hyperactivity of lymphocytes and other leukocytes, over-expression of pro-inflammatory cytokines (TNF-alpha and IL-1) and chronic debilitating inflammation. Thalidomide may play a role in RA treatment by altering leukocyte function through down-modulation of cell adhesion molecules necessary for leukocyte migration to inflammatory sites. The present study investigates down-regulation of cell adhesion molecules (ICAM-1 and LFA-1) and decreases in cell-cell contacts between human T leukemic (CEM) cells and human umbilical vein endothelial cells (HUVEC) after thalidomide exposure. CEM cells were cultured in RPMI 1640 medium with 0, 10 or 50 microg/ml thalidomide, stained with fluorescent monoclonal antibodies specific to ICAM-1 and LFA-1 and expression was measured with flow cytometry. For cell-cell adhesion measurements, monolayers of HUVEC cultured in Kaign's F-12 medium were incubated with thalidomide treated CEM cells stained with calcein AM. Specific cell adhesion between the two cell types was visualized with fluorescence microscopy. Thalidomide treatment significantly reduced cell adhesion molecule expression in a dose-dependent fashion and inhibited HUVEC/CEM cell adhesion. These data support the hypothesis that thalidomide has modulatory actions on leukocyte functions through expression of cell adhesion molecules.     

Motility and ultrastructure of large granular lymphocytes on lipid bilayers reconstituted with adhesion receptors LFA-1, ICAM-1, and two isoforms of LFA-3

Large granular lymphocytes, mediators of NK activity, bind to other cells using both the LFA (lymphocyte function-associated)-1-ICAM and the CD2-LFA-3 adhesion pathways. Here we have studied the motility and ultrastructure of large granule lymphocyte (LGL) on lipid bilayers containing purified LFA-1, ICAM-1, and the transmembrane and glycophosphatidylinositol isoforms of LFA-3. LGLs moved at 8 microns/min on ICAM-1 but poorly (less than 1 microns/min) on its receptor pair LFA-1. TM-LFA-3 promoted locomotion at a rate close to ICAM-1, whereas the cells were less motile on GPI-LFA-3. The difference in the rates of locomotion on the two isoforms of LFA-3 is presumably attributable to their difference in anchoring and lateral mobility in the bilayer. In spite of the variation in motility the ultrastructure of the adhering cells was similar on all four ligands. LGLs contacted the membrane variably, i.e., cells adhering only in a few small areas or in larger areas were detected on each ligand. The relative percentage of the plasma membrane facing the lipid bilayer was greatest on ICAM-1 and least on the transmembrane isoform of LFA-3, demonstrating no correlation with motility. The ratio of adjacent plasma membrane to lipid bilayer was virtually constant for all four ligands. Activation of the LGLs with a combination of CD2 mAb T11(2) and T11(3) (T11(2/3) mAb) reduced the movement on ICAM-1 and virtually immobilized the cells on the other bilayers. In the presence of T11(2/3) mAb, the area of cell membrane attaching to bilayers containing ICAM-1 and GPI-LFA-3 was decreased and the percentage of plasma membrane facing other cells was increased. No preferential orientation of the Golgi apparatus or degranulation was detected in the absence or presence of T11(2/3) mAb, but a significantly lower percentage of LGLs on ICAM-1 contained a profile of the Golgi apparatus after exposure to T11(2/3) mAb. The results demonstrate that the motility of LGLs depends on the type of receptor in the opposing bilayer, the receptor mobility in the bilayer, and the activation of the cells. The ultrastructure of LGLs binding to any of the adhesion molecules does not have the characteristics of LGLs in cytolytic contact with target cells, suggesting that the mediation of an attack on a target requires more complex stimulus than any one of the single adhesion proteins tested here.     

Lymphocyte-fibroblast adhesion. A useful model for analysis of the interaction of the leucocyte integrin LFA-1 with ICAM-1.

The adhesion of human T lymphoblasts to ICAM-1-expressing normal dermal fibroblasts has been assessed as a sensitive model system for the analysis of the interaction of the leucocyte integrin LFA-1 with its counter-receptor ICAM-1. Using this model system, the effects of factors known to regulate the activity of LFA-1 have been quantitated: temperature; concentration of divalent cations; and exposure to phorbol esters. We show here that under the appropriate assay conditions, this model system represents a useful and simple alternative to the detection of leucocyte binding to purified ICAM-1 and also has the additional advantage of permitting more sensitive quantification than is possible using the homotypic adhesion assay.     

Dynamic regulation of LFA-1 activation and neutrophil arrest on intercellular adhesion molecule 1 (ICAM-1) in shear flow

Neutrophil recruitment during acute inflammation is triggered by G-protein-linked chemotactic receptors that in turn activate beta(2) integrin (CD18), deemed a critical step in facilitating cell capture and arrest under the shear force of blood flow. A conformational switch in the I domain allosteric site (IDAS) and in CD18 regulates LFA-1 affinity for endothelial ligands including intercellular adhesion molecule 1 (ICAM-1). We examined the dynamics of CD18 activation in terms of the efficiency of neutrophil capture of ICAM-1, and we correlated this with the membrane topography of 327C, an antibody that recognizes the active conformation of CD18 I-like domain. Adhesion increased in direct proportion to chemotactic stimulus rising 7-fold over a log range of interleukin-8 (IL-8). A threshold dose of approximately 75 pm IL-8, corresponding to ligation of only approximately 10-100 receptors, was sufficient to activate approximately 20,000 CD18 and a rapid boost in the capture efficiency on ICAM-1. This was accompanied by a rapid redistribution of active LFA-1, but not Mac-1, into membrane patches, a necessary component for optimum adhesion efficiency. Shear-resistant arrest on a monolayer of ICAM-1 was reversed within minutes of chemotactic stimulation correlating with a shift from high to low affinity CD18 and dispersal of patches of active CD18. Mobility of active CD18 into high avidity patches was dependent on phosphatidylinositol 3-kinase activity and not F-actin polymerization. The data reveal that the number of chemotactic receptors bound and the topography and lifetime of high affinity LFA-1 tightly regulate the efficiency of neutrophil capture on ICAM-1.     

Ultrastructural identification and distribution of the adhesion molecules ICAM-1 and LFA-1 in the vascular and extravascular compartments of the human palatine tonsil

Summary Immunohistological analysis of sections prepared from human palatine tonsils revealed marked differences in the distribution of the adhesion molecule, leucocyte function antigen-1 (LFA-1) and its counter receptor, intercellular adhesion molecule-1 (ICAM-1). Light microscopy showed that LFA-1 was restricted to the leucocytes, particularly the lymphocytes. In contrast, staining of ICAM-1 was predominantly confined to the vascular endothelium with the greatest expression seen on the morphologically distinct high endothelial venules in the parafollicular areas; these are the sites that appear to support lymphocyte migration. Electron microscopy revealed that ICAM-1 was present on the luminal and lateral surfaces of the high endothelium and absent from the abluminal surface supported by basal lamina. The ICAM-1 was also absent from those surfaces of the endothelium that were in close contact with intravascular lymphocytes. Other cells stained by the anti-ICM-1 antibody included dendritic cells, plasma cells and epithelial cells in the reticulated crypt epithelium and in the upper strata of the non-keratinised stratified squamous epithelium. The high expression of LFA-1 was most prominent on lymphocytes, low on antigen-presenting cells and activated lymphoid cells, and not detectable on plasma cells, epithelial and endothelial cells. We propose that LFA-1/ICAM-1 binding participates in mediating the transendothelial migration of lymphocytes across the high endothelial venules of palatine tonsil.