Selective inhibition of ICAM-1 and E-selectin expression in human endothelial cells. 2. Aryl modifications of 4-(aryloxy)thieno[2,3-c]pyridines with fine-tuning at C-2 carbamides.

The elevated expression of cell adhesion molecules (CAMs) on the lumenal surface of vascular endothelial cells is a critical early event in the complex inflammatory process. The adhesive interactions of these CAMs that include E-selectin, ICAM-1, and VCAM-1 with their counter-receptors on leukocytes, such as integrins of the alpha(L)beta(2) family, result in migration of the leukocytes to the site of inflammation and cause tissue injury. Pharmaceutical agents that could suppress the induced expression of one or more of these cell adhesion molecules would provide a novel mechanism to attenuate the inflammatory responses associated with chronic inflammatory diseases. A-205804 (1), a potent and selective inhibitor of the induced expression of E-selectin and ICAM-1 over VCAM-1, was further modified with emphasis at the C-4 and C-2 positions to identify a more potent drug candidate with a good pharmacokinetic profile and physical properties. Replacement of the C-4 sulfur linkage in 1 with an oxygen atom eliminated one of the two major metabolites for this lead molecule. The para-position of the 4-phenoxy group of the thieno[2,3-c]pyridine lead is found to be very critical for a higher in vitro potency and selectivity of E-selectin and ICAM-1 over VCAM-1 expression. This position is presumably close to the solvent-accessible region of the target protein-inhibitor complex. An attempt to install a water-solubilizing group at the para-position of the phenoxy group to increase the aqueous solubility of this lead series through various linkages failed to provide an ideal inhibitor. Only small substituents such as fluorine are tolerated at the meta- and ortho-positions of the 4-phenoxy to retain a good in vitro potency. Bromo, trifluoromethyl, pyrazol-1-yl, and imidazol-1-yl are among the better substituents at the para-position. With fine-tuning at the C-2 position we discovered a series of very potent (IC(50) < 5 nM for ICAM-1) and selective (>200-fold vs VCAM-1) inhibitors with a good pharmacokinetic profile. Demonstrated efficacy in a rat rheumatoid arthritis model and in a mice asthma model with selected compounds is also reported.

Discovery of inhibitors of cell adhesion molecule expression in human endothelial cells. 1. Selective inhibition of ICAM-1 and E-selectin expression.

A critical early event in the inflammatory cascade is the induced expression of cell adhesion molecules on the lumenal surface of vascular endothelial cells. These adhesion molecules include E-selectin, ICAM-1, and VCAM-1, which serve to recruit circulating leukocytes to the site of the inflammation. These adhesive interactions allow the leukocytes to firmly adhere to and cross the vascular endothelium and migrate to the site of tissue injury. Pharmaceutical agents which would prevent the induced expression of one or more of the cell adhesion molecules on the endothelium might be expected to provide a novel mechanism to attenuate the inflammatory responses associated with chronic inflammatory diseases. A thieno[2,3-d]pyrimidine, A-155918, was identified from a whole-cell high-throughput assay for compounds which inhibited the tumor necrosis factor-alpha (TNFalpha)-induced expression of E-selectin, ICAM-1, or VCAM-1 on human vascular endothelial cells. Traditional medicinal chemistry methods were applied to this low-micromolar inhibitor, resulting in the 2,4-disubstituted thieno[2,3-c]pyridine A-205804, a potent and selective lead inhibitor of E-selectin and ICAM-1 expression (IC(50) = 20 and 25 nM, respectively). The relative position of the nitrogen atom in the thienopyridine isomer was shown to be critical for activity, as was a small amide 2-substituent.

Intercellular adhesion molecule-5 induces dendritic outgrowth by homophilic adhesion.

Intercellular adhesion molecule-5 (ICAM-5) is a dendritically polarized membrane glycoprotein in telencephalic neurons, which shows heterophilic binding to leukocyte beta(2)-integrins. Here, we show that the human ICAM-5 protein interacts in a homophilic manner through the binding of the immunoglobulin domain 1 to domains 4-5. Surface coated ICAM-5-Fc promoted dendritic outgrowth and arborization of ICAM- 5-expressing hippocampal neurons. During dendritogenesis in developing rat brain, ICAM-5 was in monomer form, whereas in mature neurons it migrated as a high molecular weight complex. The findings indicate that its homophilic binding activity was regulated by nonmonomer/monomer transition. Thus, ICAM-5 displays two types of adhesion activity, homophilic binding between neurons and heterophilic binding between neurons and leukocytes.

Binding of T lymphocytes to hippocampal neurons through ICAM-5 (telencephalin) and characterization of its interaction with the leukocyte integrin CD11a/CD18.

Intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a member of the immunoglobulin superfamily expressed on telencephalic neurons, and serves as a ligand for the leukocyte integrin CD11 a/CD18. We studied here the binding site in ICAM-5 for CD11a/CD18. Protein constructs containing the first immunoglobulin domain of ICAM-5 were able to support CD11a/CD18 interaction, while deletion of the first domain abolished binding. Monoclonal antibodies reacting with the first domain of ICAM-5 also completely blocked the interaction. The soluble first domain of ICAM-5 inhibited the binding of T cells to immobilized ICAM-5 at concentrations of 50 nM and higher. Interestingly, the sixth domain of ICAM-5 was also able to support leukocyte binding, but this binding activity may not involve leukocyte integrins. To test the involvement of ICAM-5 in leukocyte-neuron interactions, an assay using human T cells binding to rat hippocampal neurons was established. This binding was blocked by monoclonal antibodies against CD11a/CD18 and ICAM-5. Thus ICAM-5 may act as a major adhesion molecule for leukocyte binding to neurons in the central nervous system.

The leukocyte integrin alpha D beta 2 binds VCAM-1: evidence for a binding interface between I domain and VCAM-1.

The trafficking of leukocytes through tissues is supported by an interaction between the beta 2 (CD18) integrins CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) and their ligand ICAM-1. The most recently identified and fourth member of the beta 2 integrins, alpha D beta 2, selectively binds ICAM-3 and does not appear to bind ICAM-1. We have reported recently that alpha D beta 2 can support eosinophil adhesion to VCAM-1. Here we demonstrate that expression of alpha D beta 2 in a lymphoid cell that does not express alpha 4 integrins confers efficient binding to VCAM-1. In addition, a soluble form of alpha D beta 2 binds VCAM-1 with greater efficiency relative to ICAM-3. The I domain of alpha D contains a binding site for VCAM-1 since recombinant alpha D I domain binds specifically to VCAM-1. In addition, alpha D mAb that block cellular binding to VCAM-1 bind the alpha D I domain. Using VCAM-1 mutants we have determined that the binding site on VCAM-1 for alpha D beta 2 overlaps with that of alpha 4++ integrins. Substitution of VCAM-1 aspartate at position 40, D40, within the conserved integrin binding site, diminishes binding to alpha D beta 2 and abrogates binding to the alpha D I domain. The corresponding integrin binding site residue in ICAM-3 is also essential to alpha D beta 2 binding. Finally, we demonstrate that alpha D beta 2 can support lymphoid cell adhesion to VCAM-1 under flow conditions at levels equivalent to those mediated by alpha 4 beta 1. These results indicate that VCAM-1 can bind to an I domain and that the binding of alpha D beta 2 to VCAM-1 may contribute to the trafficking of a subpopulation of leukocytes that express alpha D beta 2.

Mapping of the ICAM-5 (telencephalin) gene, a neuronal member of the ICAM family, to a location between ICAM-1 and ICAM-3 on human chromosome 19p13.2.

Intercellular adhesion molecule 5 (ICAM-5, telencephalin) is a cell adhesion molecule expressed on neurons in the most rostral segment of the mammalian brain, the telencephalon. Antibody studies in rodents and rabbits have demonstrated expression of this molecule on the cell body and dendrites of these neurons. We have examined the expression pattern in human brain by Northern blot analysis of 16 human brain segments. This analysis has confirmed the unique expression pattern of this ICAM in human. In addition, we report the mapping of the human ICAM-5 gene to an 80-kb region on chromosome 19p13.2 that also contains ICAM-1 and ICAM-3.

AKAP79 inhibits calcineurin through a site distinct from the immunophilin-binding region.

Targeting of protein kinases and phosphatases provides additional specificity to substrate selectivity in cellular signaling. In the case of the Ca2+/calmodulin-dependent protein phosphatase calcineurin, AKAP79 has been shown to bind calcineurin and inhibit its activity in vitro (Coghlan, V., Perrino, B. A., Howard, M., Langeberg, L. K., Hicks, J. B., Gallatin, W. M., and Scott, J. D. (1995) Science 267, 108-111). In the present study, we characterized the binding regions on calcineurin A (CnA) and AKAP79 that are important for this interaction. Residues 30-98 and 311-336 on CnA, and residues 108-280 on AKAP79 were found to be important for binding. The binding of CnA by AKAP79 does not require the calcineurin B subunit, and occurs in a region distinct from where the immunosuppressant-immunophilin complex bind. AKAP79 also bound to CnA in cells transfected with AKAP79 and CnA. To determine the function of AKAP79-calcineurin interaction in intact cells, we measured the dephosphorylation and subsequent activation of NFAT, a transcription factor that is a substrate for calcineurin. Overexpression of AKAP79 inhibited NFAT dephosphorylation, resulting in a decrease in NFAT activation. These results demonstrated that AKAP79 can bind to and inhibit calcineurin activity in vivo, suggesting a physiological role for AKAP79-calcineurin interaction in NFAT-mediated signaling.

The leukointegrin alpha d/beta 2 (alpha d/CD18): specific changes in surface expression in patients on hemodialysis.

Alpha d/CD18 is a newly discovered leukocyte adhesion molecule with sequence homology to CD11a, b and c of the beta 2 integrin family. Little is known about alpha d expression in vivo, particularly how it compares with the other beta 2 integrins. Previous studies have demonstrated that beta 2 integrin expression, particularly CD11b, is upregulated in vivo during hemodialysis (HD) with complement activating membranes. These changes may contribute to the immunologic abnormalities seen in HD patients. Given the well described changes of beta 2 integrins in these patients, we hypothesized that alpha d expression could also be altered by HD. Using flow cytometry with two specific antibodies to alpha d, alpha d expression in healthy adults (n = 16) was compared on macrophages (MO) > polymorphonuclear cells (PMNs) > lymphocytes (LY). Phorbol ester treatment of leukocytes in vitro significantly increased expression on MO and PMN, but not LY. Chronic HD patients at baseline (n = 15) had elevated (P < 0.05) alpha d mean channel fluorescence (MCF) on MOs, PMNs and LYs compared to normals. PMN alpha d MCF increased at 15 min into HD, but then returned to baseline levels at 180 min. Alpha d MCF for LYs decreased at 180 min, while MOs levels were unchanged. Alpha d expression is increased in chronic renal failure and further regulated by hemodialysis, but with unique characteristics compared to the other beta 2 integrins. Alpha d may be important in abnormal cell-cell contacts in renal failure.

The intercellular adhesion molecule (ICAM) family of proteins. New members and novel functions.

Macromolecular adhesive associations between cells are important for transmitting spatial and temporal information that is critical for immune system function. One such group of proteins, the intercellular adhesion molecules (ICAMs), has grown as newly identified members are revealed. In addition, the functions of the ICAMs, in general, have begun to be better understood, including intracellular signaling events. This information has led to the design of novel therapeutic agents that may prove effective in a variety of disease states.

Requirements for alpha d in IgG immune complex-induced rat lung injury.

Alpha d is a newly cloned adhesion molecule that forms a heterodimer with CD18. The requirement for alpha d in IgG immune complex-induced lung injury in rats has been evaluated by the use of blocking polyclonal and monoclonal antibodies to rat alpha d. Using whole lung extracts, Northern and Western blot analyses have revealed up-regulation of mRNA and alpha d protein in inflamed lungs. Immunostaining has revealed the presence of alpha d in lung tissue and in alveolar macrophages as early as 1 h after initiation of the inflammatory reaction. When polyclonal rabbit Ab to rat alpha d was coinstilled into lung together with Ab to BSA, lung injury (as determined by leakage of [125I]albumin into lung parenchyma) was significantly diminished. In parallel, there was reduced accumulation of neutrophils recoverable in bronchoalveolar lavage (BAL) fluids. These findings were associated with reduced levels of TNF-alpha as well as NO2-/NO3- in BAL fluids. A hamster mAb to rat alpha d was also protective in this lung injury model. Anti-alpha d inhibited in vitro production of NO2-/NO3- by rat alveolar macrophages (stimulated with LPS and IFN-gamma) by approximately 60%. These data suggest that, in the lung inflammatory model employed, alpha d up-regulation occurs in lung macrophages and is necessary for expression of TNF-alpha, recruitment of neutrophils, and full development of lung injury.

Beta2 integrin/ICAM expression in Crohn's disease.

We have previously reported on the expression of the beta2 integrin family of adhesion molecules and their ligands, the ICAM molecules, in the normal human intestine. These molecules likely have a role to play in the inflammatory response and, therefore, were studied in a group of patients with Crohn's disease. A comprehensive study was undertaken in both colon (n = 8) and ileum (n = 10) specimens from 15 patients who underwent surgical resections. Immunohistochemistry was performed for CD18, CD11a, CD11b, CD11c, alphad, ICAM-1, ICAM-2, and ICAM-3. Each of the mucosal, submucosal, muscle, and adventitial layers were scored for expression. Specimens from normal colon (n = 15), normal ileum (n = 6), and ulcerative colitis (n = 7) were used for comparisons. Compared with normal, the expression in the colon mucosa and submucosa in Crohn's disease was increased for all beta2 integrins. Mucosal CD11c expression was significantly greater in Crohn's disease than in ulcerative colitis. In the colon muscle and adventitial layers the expression in Crohn's disease was similar to normal but increased compared with ulcerative colitis. In Crohn's disease ileum, the beta2 integrin mucosal and submucosal expression was similar to normal; however, muscle and adventitial expression was increased, particularly for CD11c. Colon ICAM-1, ICAM-2, and ICAM-3 expression in Crohn's disease was similar to that seen in ulcerative colitis. ICAM-1 was predominantly expressed on endothelium but in the inflammatory bowel diseases was also evident on mucosal mononuclear cells. ICAM-1 and ICAM-2 expression was increased in Crohn's disease colon and ileum compared with normals. This was most notable in ileal mucosa since ICAM-2 is typically absent in normal ileal mucosa. In summary, we are reporting a comprehensive immunohistochemical study of the differential expression of beta2 integrins, including the newly described alphad molecule, and the ICAM molecules in all layers of the colon and ileum from patients with Crohn's disease. The increased expression of these molecules may have implications for therapeutic interventions in Crohn's disease.

Functional mapping of the cytoplasmic region of intercellular adhesion molecule-3 reveals important roles for serine residues.

Intercellular adhesion molecule-3 (ICAM-3), a ligand for beta2 integrins, elicits a variety of activation responses in lymphocytes. We describe a functional mapping study that focuses on the 37-residue cytoplasmic region of ICAM-3. Carboxyl-terminal truncations delineated portions involved in T cell antigen receptor costimulation, homotypic aggregation, and cellular spreading. Truncation of the membrane distal 25 residues resulted in loss of T cell antigen receptor costimulation as determined by interleukin 2 secretion. Aggregation and cell spreading were sensitive to truncation of the membrane distal and proximal thirds of the cytoplasmic portion. Phosphoamino acid analysis revealed that ICAM-3 from activated cells contained phosphoserine and phosphopeptide mapping identified Ser489 as a site of phosphorylation in vivo. Mutation of Ser489 or Ser515 to alanine blocked interleukin 2 secretion, aggregation and cell spreading, while mutation of other serine residues affected only a subset of functions. Ser489 was a phosphorylation site in vitro for recombinant protein kinase Ctheta. Finally, treatment of Jurkat cells with chelerythrine chloride, a protein kinase C inhibitor, prevented ICAM-3-triggered spreading. This study delineates separable regions and amino acid residues within the cytoplasmic portion of ICAM-3 that are important for T cell function.

Distribution of immunoglobulin superfamily members ICAM-1, -2, -3, and the beta 2 integrin LFA-1 in multiple sclerosis lesions.

To identify potential molecular substrates for leukocyte trafficking and activation in multiple sclerosis (MS) brain, we determined the immunocytochemical distribution of the beta, integrin lymphocyte-function-associated antigen-1 (LFA-1) and its major ligands, intercellular adhesion molecule (ICAM)-1, ICAM-2, and ICAM-3 in MS tissue. Colocalization of these adhesion molecules with lineage-specific markers was analyzed by dual-labeling immunocytochemistry and confocal microscopy. ICAM-1 and ICAM-2 were detected on endothelial cells, and ICAM-3 immunoreactivity was restricted to infiltrating leukocytes. In control brain, 10% of glucose transporter-1 positive vessels contained ICAM-1 immunoreactivity on their luminal surface and 21% were ICAM-2-positive. A significant increase in ICAM-1-positive vessels was found in MS brains. This increase was greater in MS lesions (81% of vessels) than in nonlesion areas (37% of vessels). A significant increase in ICAM-1-positive vessels was found in encephalitis (55% of vessels) but not in Parkinson's (17% of vessels) brains. The percentage of vessels expressing ICAM-2 was not increased in MS, encephalitis, or Parkinson's brains. Both ICAM-3 and LFA-1 were detected on the vast majority of infiltrating lymphocytes and monocytes in and near MS lesions, and these cells were often closely apposed to each other. In addition, LFA-1 was detected on activated microglia located close to the edge of demyelinating lesions. ICAM-3-positive leukocytes were often closely apposed to LFA-1-positive microglia. These results suggest a role for ICAM-1, -2, and LFA-1 in the transendothelial migration of leukocytes into MS brain and a role for ICAM 3/LFA-1 interactions in the activation of lymphocytes, monocytes, and microglia in MS lesions.

Beta 2-integrin/intercellular adhesion molecule (ICAM) expression in the normal human intestine.

The beta 2-integrin family of adhesion molecules and their ligands, the ICAM molecules, probably have an important role to play in the intestinal immune response. To date, any data published regarding the intestinal expression of beta 2-integrins and ICAMs have mostly described only mucosal expression. The presence in the intestine of alpha d, a newly described beta 2-integrin molecule, has not yet been determined. To understand further the expression of these molecules in all layers of the intestine, a comprehensive study was undertaken in both normal colon and normal small intestine specimens from 15 patients (colon n = 15, and ileum n = 6) who underwent surgical resections. Immunohistochemistry was performed for CD18, CD11a, CD11b, CD11c, alpha d, ICAM-1, ICAM-2, and ICAM-3. The colon and small intestine cases were grouped separately and each of the mucosal, submucosal, muscle and adventitial layers were scored for expression. The beta 2-integrin molecules were expressed by mononuclear cells in all layers of the bowel, predominantly in the mucosa and adventitia. CD11a showed the greatest mucosal expression and CD11b showed the greatest adventitial expression. alpha d exhibited expression in all bowel layers, as well, with most intense expression in the mucosa and adventitia. CD11c exhibited the least expression of all alpha subunit molecules. The expression of these alpha-chains at times appeared to be greater than that seen for CD18 (beta 2), raising the possibility of non-beta 2-containing complexes (i.e. alpha-chains associating with an alternate beta-chain). ICAM-1 was expressed on endothelium, particularly in the mucosa and rarely on mucosal mononuclear cells. ICAM-2 was predominantly on submucosal endothelium and rarely expressed in colon mucosa and never expressed in ileal mucosa. ICAM-3 was expressed by mononuclear cells throughout the bowel wall, but also on adventitial endothelium in selected cases. In summary, we are reporting a comprehensive immunohistochemical study of the differential expression of beta 2-integrins, including the newly described alpha d molecule, and the ICAM molecules in all layers of normal human colon and ileum. We raise the possibility that a second beta 2-integrin beta subunit may exist, and we report that ICAM-3 is expressed on endothelium, particularly in the adventitial layer.

Intercellular adhesion molecule-3 on endothelial cells. Expression in tumors but not in inflammatory responses.

Intercellular adhesion molecule-3 (ICAM-3) was identified as the third counter-receptor for lymphocyte function-associated antigen-1. ICAM-3 is absent on endothelial cells in normal tissues but found on endothelial cells in lymphomas. Here, we examined ICAM-3 expression on vascular endothelial cells in lymphomas, nonlymphoid malignancies, benign tumors, and inflammatory diseases. We compared the expression of ICAM-3 on endothelial cells with the severity of inflammatory infiltrates and with the presence of E-selectin and VCAM-1. We found that ICAM-3 expression on endothelial cells was high on both benign and malignant tumors whereas it was low in inflammatory diseases. In contrast to E-selectin, ICAM-3 expression on endothelial cells was not correlated to the severity of inflammatory infiltrates. In hemangiomas, we showed by Northern blot analysis and immunocytochemistry that ICAM-3 expression was induced and that it was localized in immature areas that sustain the early stages of angiogenesis. Therefore, expression of ICAM-3 on blood vessels does not seem to play a role in the recruitment of leukocytes during inflammation but rather is correlated with angiogenesis and tumor development.

Elevated levels of circulating intercellular adhesion molecule-3 (cICAM-3) in Psoriasis.

Intercellular adhesion molecule (ICAM)-3 is important in regulating leukocyte function and T-lymphocyte-antigen presenting cell interactions. Soluble, circulating forms of ICAM-3 and ICAM-1 (cICAM-3, cICAM-1) exist in serum, and levels are elevated in a variety of autoimmune diseases. Two types of soluble circulating tumour necrosis factor receptor (cTNF-R1, cTNF-R2) are found in the sera of healthy people. cTNF-R1 binds TNF-alpha and is important in regulating TNF-alpha-mediated inflammation. Psoriasis is a T-lymphocyte-mediated disease, characterized by cutaneous expression of ICAM-1, ICAM-3 and TNF-alpha. As it is unknown whether cICAM-3 is increased in sera of patients with psoriasis, we measured serum levels of cICAM-3 and compared them to levels of cICAM-1, cTNF-R1 and clinical severity of psoriasis. Sera was taken from 112 healthy controls and 32 patients with psoriasis. Clinical severity of psoriasis was assessed using the psoriasis area severity index (PASI). cICAM-1, cICAM-3 and cTNF-R1 in serum were quantitated using a dual antibody, solid phase ELISA, Levels of cICAM-3, cICAM-1 and cTNF-R1 were significantly increased in sera of patients with psoriasis as compared with controls, and these elevated levels correlated with clinical severity of psoriasis as assessed by the PASI. Also, there were good correlations between serum levels of cICAM-3, cICAM-1 and cTNF-R1 in psoriasis. These results demonstrate, for the first time, that circulating levels of cICAM-3 are increased in psoriasis and that these levels correlate both with disease severity and with elevated levels of cICAM-1 and cTNF-R1. The exact physiologic roles of circulating, soluble adhesion molecules and cTNF-R1 are unknown, but it is hypothesised that elevation of their circulating levels, as observed in psoriasis, may play a role in modulating the inflammatory reactions occurring in this disease.

The ICAM-3/LFA-1 interaction is critical for epidermal Langerhans cell alloantigen presentation to CD4+ T cells.

Intercellular adhesion molecule (ICAM)-3 is a recently described member of the immunoglobulin superfamily and, as such, is closely related to ICAM-1 and ICAM-2. All three ICAMS are cognate for the counter-receptor lymphocyte function associated antigen-1 (LFA-1, CD11a/CD18). Unlike ICAM-1 and ICAM-2, ICAM-3 is constitutively expressed at high levels on resting leucocytes. We investigated the expression and function of ICAM-3 in normal skin (n = 5), as well as its expression in psoriasis (n = 4), atopic eczema (n = 4), allergic (rhus) contact dermatitis (n = 3), and cutaneous T-cell lymphoma (CTCL, n = 2). Five-micrometre cryostat sections of skin were stained using monoclonal antibodies to ICAM-3 and a well characterized immunoperoxidase technique. In normal skin, ICAM-3 was expressed by all cutaneous leucocytes but most striking was the strong expression of ICAM-3 by Langerhans cells within both epidermis and dermis. This observation was confirmed by double-labelling with CD1a and negative staining with an IgG1 isotype control. In psoriasis, atopic eczema, allergic contact dermatitis, and CTCL, ICAM-3 was co-expressed on all CD1a+ cells, although, in psoriasis, the intensity of ICAM-3 expression was reduced. Functional blocking experiments were performed to determine whether the observed ICAM-3 expression on Langerhans cells was functionally important in antigen presentation. CD4+ T cells were prepared from peripheral blood and 10(5) CD4+ T cells combined with 10(5) epidermal cells harvested from keratome biopsies of normal skin of an individual allogeneic to the T-cell donor. Addition of 50 micrograms anti-ICAM-3 to the co-culture resulted in a consistent (50%) reduction in degree of alloantigen presentation by Langerhans cells to T cells. Inhibition was 77% of that produced by the addition of anti-LFA-1. These data indicate that ICAM-3 is constitutively expressed by Langerhans cells and is a major ligand for LFA-1 on CD4+ T cells during their response to Langerhans cells. Because fresh Langerhans cells constitutively express little ICAM-1, whereas ICAM-3 is constitutively expressed at high levels, it would appear that ICAM-3 is the dominant functional ICAM on in situ Langerhans cells in the normal epidermis.

A novel leukointegrin, alpha d beta 2, binds preferentially to ICAM-3.

The leukocyte-restricted beta 2 (CD18) integrins mediate cell adhesion in a variety of events essential for normal immune function. Despite extensive research in this field, only three members of this integrin subfamily have been described: CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), and CD11c/CD18 (p150,95). We have identified a cDNA encoding a fourth alpha chain, alpha d, that associates with CD18. The alpha d subunit is more closely related to CD11b and CD11c than to CD11a. This integrin is expressed at moderate levels on myelomonocytic cell lines and subsets of peripheral blood leukocytes, and more strongly on tissue-compartmentalized cells such as foam cells, specialized macrophages found in aortic fatty streaks that may develop into atherosclerotic lesions. The alpha d/CD18 molecule exhibits preferential recognition of ICAM-3 over ICAM-1.

Association of protein kinase A and protein phosphatase 2B with a common anchoring protein.

Specificity of protein kinases and phosphatases may be achieved through compartmentalization with preferred substrates. In neurons, adenosine 3', 5'-monophosphate (cAMP)-dependent protein kinase (PKA) is localized at postsynaptic densities by association of its regulatory subunit with an A kinase anchor protein, AKAP79. Interaction cloning experiments demonstrated that AKAP79 also binds protein phosphatase 2B, or calcineurin (CaN). A ternary complex of PKA, AKAP, and CaN was isolated from bovine brain, and colocalization of the kinase and the phosphatase was established in neurites of cultured hippocampal neurons. The putative CaN-binding domain of AKAP79 is similar to that of the immunophilin FKBP-12, and AKAP79 inhibited CaN phosphatase activity. These results suggest that both PKA and CaN are targeted to subcellular sites by association with a common anchor protein and thereby regulate the phosphorylation state of key neuronal substrates.