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Carl G Gahmberg

Publications and source records attributed to Carl G Gahmberg.

At least 19 recordsLinked to original sources
PubMed2006

DC-SIGN binds ICAM-3 isolated from peripheral human leukocytes through Lewis x residues.

Intercellular adhesion molecule-3 (ICAM-3) binds to the alpha(L)beta(2) integrin and mediates the contact between T cells and antigen-presenting cells. It has been suggested that dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN), a C-type lectin of macrophages and DCs, is an additional ligand of ICAM-3. So far, the glycan structure mediating the interaction of native ICAM-3 with DC-SIGN is undefined. Here, we demonstrate that native ICAM-3 from human peripheral leukocytes binds recombinant DC-SIGN, is recognized by monoclonal Lewis x antibodies, and specifically interacts with DC-SIGN on immature DCs. The presence of Lewis x residues on ICAM-3 was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy. Investigations on different peripheral blood cell populations revealed that only ICAM-3 from granulocytes bound DC-SIGN. Cotransfection studies demonstrated that fucosyltransferase (FUT) IX and, to a significantly lesser extent, FUT IV, but not FUTs III and VII, mediate the synthesis of Lewis x residues on ICAM-3. These findings indicate that FUT IX is the main FUT mediating the synthesis of Lewis x residues of ICAM-3 in cells of the myeloid lineage, and that these residues bind DC-SIGN. The results suggest that ICAM-3 assists in the interaction of granulocytes with DC-SIGN of DCs.

Antigens, CD↗
PubMed2006

Phosphorylation of the LFA-1 integrin beta2-chain on Thr-758 leads to adhesion, Rac-1/Cdc42 activation, and stimulation of CD69 expression in human T cells.

Phosphorylation of the leukocyte function-associated antigen-1 (LFA-1) integrin beta2-chain on Thr-758 occurs after T cell receptor stimulation and leads to 14-3-3 recruitment to the integrin, actin cytoskeleton reorganization, and increased adhesion. Here, we have investigated the signaling effects of beta2 integrin Thr-758 phosphorylation. A penetratin-coupled phospho-Thr-758-beta2 peptide (mimicking the part of the integrin beta-chain surrounding Thr-758) stimulated adhesion of human T cells to the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1). Additionally, the peptide activated the small GTPases Rac-1 and Cdc42 in T cells. Constitutively active forms of Rac-1 and Cdc42, but not Rho, could compensate for the reduction of cell adhesion to ICAM-1 caused by the T758A mutation in the beta2 integrin. Additionally, the active GTPases salvaged the cell-spreading defect of T758A integrin-transfected cells on coated ICAM-1. A dominant negative form of Cdc42, on the other hand, significantly reduced wild-type beta2 integrin-mediated cell adhesion and spreading. In a T cell stimulation system, the pThr-758 penetratin peptide acted in a similar manner to coated ICAM-1 to increase T cell receptor-induced CD69 expression. These results show that Thr-758-phosphorylated LFA-1 is upstream of Rac-1/Cdc42, cell adhesion, and costimulatory activation of human T cells, thus identifying phosphorylation of Thr-758 in beta2 as a proximal element in LFA-1 signaling.

Animals↗
PubMed2006

Red-cell ICAM-4 is a ligand for the monocyte/macrophage integrin CD11c/CD18: characterization of the binding sites on ICAM-4.

Intercellular adhesion molecule 4 (ICAM-4) is a unique member of the ICAM family because of its specific expression on erythroid cells and ability to interact with several types of integrins expressed on blood and endothelial cells. The first reported receptors for ICAM-4 were CD11a/CD18 and CD11b/CD18. In contrast to these 2, the cellular ligands and the functional role of the third beta2 integrin, CD11c/CD18, have not been well defined. Here, we show that ICAM-4 functions as a ligand for the monocyte/macrophage-specific CD11c/CD18. Deletion of the individual immunoglobulin domains of ICAM-4 demonstrated that both its domains contain binding sites for CD11c/CD18. Analysis of a panel of ICAM-4 point mutants identified residues that affected binding to the integrin. By molecular modeling the important residues were predicted to cluster in 2 distinct but spatially close regions of the first domain with an extension to the second domain spatially distant from the other residues. We also identified 2 peptides derived from sequences of ICAM-4 that are capable of modulating the binding to CD11c/CD18. CD11c/CD18 is expressed on macrophages in spleen and bone marrow. Inhibition of erythrophagocytosis by anti-ICAM-4 and anti-integrin antibodies suggests a role for these interactions in removal of senescent red cells.

Binding Sites↗
PubMed2006

P-selectin glycoprotein ligand 1 and beta2-integrins cooperate in the adhesion of leukocytes to von Willebrand factor.

Von Willebrand factor (VWF) is an essential component of hemostasis. However, animal studies using VWF-deficient mice suggest that VWF may also contribute to inflammation. In the present study, we demonstrate that VWF was able to interact with polymorphonuclear leukocytes (PMNs) and monocytes under static and flow conditions. Adhesion under flow was dominated by short-lasting contact with resting PMNs, whereas adhesion of phorbol-12-myristate-13-acetate (PMA)-stimulated PMNs was characterized by firm adhesion. Transient binding of PMNs to VWF appeared to be mediated by P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, recombinant PSGL-1 protein and cell surface-expressed PSGL-1 directly interacted with VWF. As for stable adhesion by PMA-stimulated PMNs, we observed that static adhesion and adhesion under flow were strongly inhibited (greater than 75%) by neutrophil-inhibitory factor, an inhibitor of beta2-integrin function. In addition, the isolated I-domain of alphaMbeta2 bound to VWF, and cell lines expressing alphaLbeta2 or alphaXbeta2 adhered efficiently to VWF. Taken together, our data showed that VWF can function as an adhesive surface for various leukocyte subsets (monocytes, PMNs). Analogous to VWF-platelet interaction, VWF provided binding sites for leukocyte receptors involved in rolling (PSGL-1) and stable (beta2-integrins) adhesion. VWF is unique in its intrinsic capacity to combine the rolling and the stable adhesion step in the interaction with leukocytes.

Animals↗
PubMed2006

alpha-Chain phosphorylation of the human leukocyte CD11b/CD18 (Mac-1) integrin is pivotal for integrin activation to bind ICAMs and leukocyte extravasation.

The promiscuous CD11b/CD18 (Mac-1) integrin has important roles in regulating many immunologic functions such as leukocyte adhesion and emigration from the bloodstream via interactions with the endothelial ligands ICAM-1 and ICAM-2, iC3b-mediated phagocytosis, and apoptosis. However, the mechanisms for Mac-1 inside-out activation have remained poorly understood. Phosphorylation of integrin cytoplasmic domains is emerging as an important mechanism of regulating integrin functions. Here, we have studied phosphorylation of human CD11b, which takes place on the cytoplasmic Ser1126 in neutrophils. We show that mutation of the serine phosphorylation site leads to inability of Mac-1 to become activated to bind the cellular ligands ICAM-1 and ICAM-2. However, CD11b-mutant cells are fully capable of binding other studied CD11b ligands (ie, iC3b and denatured BSA). Activation epitopes expressed in the extracellular domain of the integrin and affinity for soluble ICAM ligands were decreased for the mutated integrin. Additionally, the mutation resulted in inhibition of chemokine-induced migration in a transendothelial assay in vitro and significantly reduced the accumulation of intravenously administered cells in the spleen and lungs of Balb/c mice. These results characterize a novel selective mechanism of Mac-1-integrin activation, which mediates leukocyte emigration from the bloodstream to the tissues.

Animals↗
PubMed2006

alpha-Actinin-dependent cytoskeletal anchorage is important for ICAM-5-mediated neuritic outgrowth.

Intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a dendrite-expressed membrane glycoprotein of telencephalic neurons in the mammalian brain. By deletion of the cytoplasmic and membrane-spanning domains of ICAM-5, we observed that the membrane distribution of ICAM-5 was determined by the cytoplasmic portion. Therefore we have characterized the intracellular associations of ICAM-5 by using a bacterially expressed glutathione S-transferase (GST) fusion protein encompassing the cytoplasmic part of ICAM-5. One of the main proteins in the neuronal cell line Paju that bound to the ICAM-5 cytodomain was alpha-actinin. ICAM-5 expressed in transfected Paju cells was found in alpha-actinin immunoprecipitates, and ICAM-5 colocalized with alpha-actinin both in Paju cells and in dendritic filopodia and spines of primary hippocampal neurons. We were also able to coprecipitate alpha-actinin from rat brain homogenate. Binding to alpha-actinin appeared to be mediated mainly through the N-terminal region of the ICAM-5 cytodomain, as the ICAM-5(857-861) cytoplasmic peptide (KKGEY) mediated efficient binding to alpha-actinin. Surface plasmon resonance analysis showed that the turnover of the interaction was rapid. In a mutant cell line, Paju-ICAM-5-KK/AA, the distribution was altered, which implies the importance of the lysines in the interaction. Furthermore, we found that the ICAM-5/alpha-actinin interaction is involved in neuritic outgrowth and the ICAM-5(857-861) cytoplasmic peptide induced morphological changes in Paju-ICAM-5 cells. In summary, these results show that the interaction between ICAM-5 and alpha-actinin is mediated through binding of positively charged amino acids near the transmembrane domain of ICAM-5, and this interaction may play an important role in neuronal differentiation.

Actinin↗
PubMed2006

Lipoprotein(a) in atherosclerotic plaques recruits inflammatory cells through interaction with Mac-1 integrin.

Lipoprotein(a) [Lp(a)], consisting of LDL and the unique constituent apolipoprotein(a) [apo(a)], which contains multiple repeats resembling plasminogen kringle 4, is considered a risk factor for the development of atherosclerotic disorders. However, the underlying mechanisms for the atherogenicity of Lp(a) are not completely understood. Here, we define a novel function of Lp(a) in promoting inflammatory cell recruitment that may contribute to its atherogenicity. Through its apo(a) moiety Lp(a) specifically interacts with the beta2-integrin Mac-1, thereby promoting the adhesion of monocytes and their transendothelial migration in a Mac-1-dependent manner. Interestingly, the interaction between Mac-1 and Lp(a) was strengthened in the presence of proatherogenic homocysteine and was blocked by plasminogen/angiostatin kringle 4. Through its interaction with Mac-1, Lp(a) induced activation of the proinflammatory transcription factor NFkappaB, as well as the NFkappaB-related expression of prothrombotic tissue factor. In atherosclerotic coronary arteries Lp(a) was found to be localized in close proximity to Mac-1 on infiltrating mononuclear cells. Taken together, our data demonstrate that Lp(a), via its apo(a) moiety, is a ligand for the beta2-integrin Mac-1, thereby facilitating inflammatory cell recruitment to atherosclerotic plaques. These observations suggest a novel mechanism for the atherogenic properties of Lp(a).

Aged↗
PubMed2006

Interfering with leukocyte integrin activation--a novel concept in the development of anti-inflammatory drugs.

Inflammation is a crucial response against invading pathogens, in which immune cells, including neutrophils and T cells, are recruited into tissue from the bloodstream to help clear infection. However, a prevailing inflammatory response where the immune cells attack healthy tissue is associated with many diseases, including asthma, rheumatoid arthritis, atherosclerosis and multiple sclerosis. Integrins are key players in the recruitment of immune cells from the bloodstream into tissues, and are thus therapeutic targets for intervention with inflammatory responses. Thus far, mainly extracellularly acting therapeutics (monoclonal antibodies) have been developed against integrins, targeting ligand binding sites in these heterodimeric adhesion receptors. However, since these therapeutics nonselectively block all integrin functions, some side effects are expected and have been observed. Therefore, novel concepts need to be developed in the therapeutic targeting of integrins. Recently, major advances have been made in the understanding of integrin biology. Integrin structures have been solved by X-ray crystallography, revealing unexpected data about the activation mechanism of integrins in cells. Additionally, several intracellular factors in the integrin activation process have been identified, providing potential specific targets for therapeutic intervention. Here, we present key events and players in leukocyte integrin activation, and discuss potential new drug targets in the prevention of inflammatory disease.

Animals↗
PubMed2005

Specific integrin alpha and beta chain phosphorylations regulate LFA-1 activation through affinity-dependent and -independent mechanisms.

Integrins are adhesion receptors that are crucial to the functions of multicellular organisms. Integrin-mediated adhesion is a complex process that involves both affinity regulation and cytoskeletal coupling, but the molecular mechanisms behind this process have remained incompletely understood. In this study, we report that the phosphorylation of each cytoplasmic domain of the leukocyte function-associated antigen-1 integrin mediates different modes of integrin activation. alpha Chain phosphorylation on Ser1140 is needed for conformational changes in the integrin after chemokine- or integrin ligand-induced activation or after activation induced by active Rap1 (Rap1V12). In contrast, the beta chain Thr758 phosphorylation mediates selective binding to 14-3-3 proteins in response to inside-out activation through the T cell receptor, resulting in cytoskeletal rearrangements. Thus, site-specific phosphorylation of the integrin cytoplasmic domains is important for the dynamic regulation of these complex receptors in cells.

14-3-3 Proteins↗
PubMed2004

LDL-receptor-related protein regulates beta2-integrin-mediated leukocyte adhesion.

Beta2-integrin clustering on activation is a key event in leukocyte adhesion to the endothelium during the inflammatory response. In the search for molecular mechanisms leading to this clustering, we have identified low-density lipoprotein (LDL) receptor-related protein (LRP) as a new partner for beta2-integrins at the leukocyte surface. Immobilized recombinant LRP fragments served as an adhesive surface for blood-derived leukocytes and the U937 cell line. This adhesion was decreased up to 95% in the presence of antibodies against beta2-integrins, pointing to these integrins as potential partners for LRP. Using purified proteins, LRP indeed associated with the alphaMbeta2 complex and the alphaM and alphaL I-domains (K(d, app) approximately 0.5 microM). Immunoprecipitation experiments and confocal microscopy revealed that endogenously expressed LRP and alphaLbeta2 colocalized in monocytes and U937 cells. Furthermore, activation of U937 cells resulted in clustering of alphaLbeta2 and LRP to similar regions at the cell surface, indicating potential cooperation between both proteins. This was confirmed by the lack of alphaLbeta2 clustering in U937 cells treated by antisense oligonucleotides to down-regulate LRP. In addition, the absence of LRP resulted in complete abrogation of beta2-integrin-dependent adhesion to endothelial cells in a perfusion system, demonstrating the presence of a previously unrecognized link between LRP and leukocyte function.

Binding Sites↗
PubMed2004

Intracellular and cell surface localization of a complex between alphaMbeta2 integrin and promatrix metalloproteinase-9 progelatinase in neutrophils.

We have recently demonstrated that promatrix metalloproteinases (proMMPs), particularly proMMP-9, are potent ligands of the leukocyte beta(2) integrins. We studied here the complex formation between proMMP-9 and alpha(M)beta(2), the major MMP and integrin of neutrophils. On resting neutrophils, the proMMP-9/alpha(M)beta(2) complex was primarily detected in intracellular granules, but after cellular activation it became localized to the cell surface, as demonstrated by immunoprecipitation and double immunofluorescence. Further indication of the complex formation was that neutrophils and alpha(M)beta(2)-transfected L cells, but not the wild-type L cells or leukocyte adhesion deficiency cells, bound to immobilized proMMP-9 or its recombinant catalytic domain in a beta(2) integrin-dependent manner. Peptides that bound to the alpha(M) integrin-I domain and inhibited its complex formation with proMMP-9 prevented neutrophil migration in a transendothelial assay in vitro and in a thioglycolate-elicited peritonitis in vivo. These results suggest that the translocating proMMP-9/alpha(M)beta(2) complex may be part of the cell surface machinery guiding neutrophil migration.

Amino Acid Sequence↗
PubMed2004

P marks the spot: site-specific integrin phosphorylation regulates molecular interactions.

Integrins are heterodimeric adhesion receptors at the cell membrane that function as two-way signaling devices. The short intracellular tails of integrins are devoid of catalytic activity, but are nevertheless important for adhesion and signaling, presumably, through interactions with cytoplasmic molecules. Recently, the structure of the intracellular tails has been investigated using NMR, giving important new insight into how integrins might be regulated, but many questions remain unanswered. Signaling by many cell-surface receptors involves protein phosphorylation; over the past few years, phosphorylation of the integrin tails at specific sites has started to emerge as a dynamic mechanism that regulates molecular interactions between integrins and cytoplasmic molecules. This phosphorylation might give rise to signaling specificity and fine-tuning of the integrin-mediated responses.

Amino Acid Sequence↗
PubMed2003

Identification of a negatively charged peptide motif within the catalytic domain of progelatinases that mediates binding to leukocyte beta 2 integrins.

The alpha M beta 2 integrin of leukocytes can bind a variety of ligands. We screened phage display libraries to isolate peptides that bind to the alpha M I domain, the principal ligand binding site of the integrin. Only one peptide motif, (D/E)(D/E)(G/L)W, was obtained with this approach despite the known ligand binding promiscuity of the I domain. Interestingly, such negatively charged sequences are present in many known beta 2 integrin ligands and also in the catalytic domain of matrix metalloproteinases (MMPs). We show that purified beta 2 integrins bind to pro-MMP-2 and pro-MMP-9 gelatinases and that that the negatively charged sequence of the MMP catalytic domain is an active beta 2 integrin-binding site. Furthermore, a synthetic DDGW-containing phage display peptide inhibited the ability of beta 2 integrin to bind progelatinases but did not inhibit the binding of cell adhesion-mediating substrates such as intercellular adhesion molecule-1, fibrinogen, or an LLG-containing peptide. Immunoprecipitation and cell surface labeling demonstrated complexes of pro-MMP-9 with both the alpha M beta 2 and alpha L beta 2 integrins in leukocytes, and pro-MMP-9 colocalized with alpha M beta 2 in cell surface protrusions. The DDGW peptide and the gelatinase-specific inhibitor peptide CTTHWGFTLC blocked beta 2 integrin-dependent leukocyte migration in a transwell assay. These results suggest that leukocytes may move in a progelatinase-beta 2 integrin complex-dependent manner.

Amino Acid Motifs↗
PubMed2003

Threonine phosphorylation sites in the beta 2 and beta 7 leukocyte integrin polypeptides.

The cytoplasmic domains of integrins play a key role in a variety of integrin-mediated events including adhesion, migration, and signaling. The molecular mechanisms that enhance integrin function are still incompletely understood. Because protein kinases are known to be involved in the signaling and the activation of integrins, the role of phosphorylation has been studied by several groups. The beta(2) leukocyte integrin subunit has previously been shown to become phosphorylated in leukocytes on cytoplasmic serine and functionally important threonine residues. We have now mapped the phosphorylated threonine residues in activated T cells. After phorbol ester stimulation, all three threonine residues (758-760) of the threonine triplet became phosphorylated but only two at a time. CD3 stimulation leads to a strong threonine phosphorylation of the beta(2) integrin, but differed from phorbol ester activation in that phosphorylation occurred only on threonine 758. The other leukocyte-specific integrin, beta(7), has also been shown to need the cytoplasmic domain and leukocyte-specific signal transduction elements for integrin activation. Cell activation with phorbol ester, and interestingly, through the TCR-CD3 complex, caused beta(7) integrin binding to VCAM-1. Additionally, cell activation led to increased phosphorylation of the beta(7) subunit, and phosphoamino acid analysis revealed that threonine residues became phosphorylated after cell activation. Sequence analysis by manual radiosequencing by Edman degradation established that threonine phosphorylation occurred in the same threonine triplet as in beta(2) phosphorylation.

Alanine↗
PubMed2003

Ezrin is a substrate for Lck in T cells.

We evaluated the role of Lck tyrosine kinase, an early effector of T cell activation, in regulation of the membrane-cytoskeleton linker protein ezrin. Ezrin was constitutively tyrosine phosphorylated in wild-type and CD45-deficient Jurkat T cells, but not in Lck-deficient cells. However, phosphorylation was evident in cells, in which Lck activity had been restored by transfection. Phosphorylation was reduced by the Src family kinase inhibitor PP2 and increased by the tyrosine phosphatase inhibitor pervanadate, implying continuous tyrosine phosphorylation and dephosphorylation. Lck phosphorylated ezrin in vitro, and the major phosphotyrosine was identified as Y145. These results identify ezrin as the first cytoskeletal substrate for Lck.

Cell Line↗
PubMed2003

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.

Animals↗