Tatiana P. Ugarova

Molecular determinants of acute inflammatory responses to biomaterials.

The frequent inflammatory responses to implanted medical devices are puzzling in view of the inert and nontoxic nature of most biomaterials. Because implant surfaces spontaneously adsorb host proteins, this proteinaceous film is probably important in the subsequent attraction of phagocytes. In fact, earlier we found that acute inflammatory responses to experimental polyethylene terephthalate implants in mice require the precedent adsorption of one particular host protein, fibrinogen. The present investigations were aimed at defining the molecular determinants of fibrinogen-mediated acute inflammatory responses to implanted biomaterials. We find: (a) plasmin degradation of purified fibrinogen into defined domains reveals that the proinflammatory activity resides within the D fragment, which contains neither the fibrin cross-linking sites nor RGD sequences; (b) the major (and, perhaps, exclusive) proinflammatory sequence appears to be fibrinogen gamma 190-202, previously shown to interact with CD11b/CD18 (Mac-1). The chemically synthesized peptide, cross-linked to albumin (which itself does not promote inflammatory responses), mimics the proinflammatory effect of adsorbed native fibrinogen; and (c) this sequence probably promotes inflammatory responses through interactions with Mac-1 because phagocyte accumulation on experimental implants is almost completely abrogated by administration of recombinant neutrophil inhibitory factor (which blocks CD11b-fibrin(ogen) interaction). We conclude that improved knowledge of such surface-protein-phagocyte interactions may permit the future development of more biocompatible implantable materials.

Modulating fibroblast adhesion, spreading, and proliferation using self‐assembled monolayer films of alkylthiolates on gold

Ultrathin, highly organized functionalized alkylthiol monolayers were applied as model substrates for cell growth and protein adsorption studies. The aim of this approach was to improve the understanding of molecular surface determinants required for adhesion-dependent cell growth and proliferation using well-controlled surface chemistry. Carboxyl- and methyl-terminated alkylthiol monolayers on gold were used to monitor Swiss 3T3 fibroblast adhesion, spreading, and growth. Stress fiber and focal contact formation were determined by immunostaining of actin filaments and paxillin. Fibronectin deposition and conformation on these surface chemistries in the presence and absence of competing proteins were also determined. The relative levels of adsorbed fibronectin were assessed using radiolabeled proteins. Exposure of the 10th type III cell integrin binding domain of fibronectin was assessed using a radiolabeled monoclonal antibody. Distinct alkylthiol substrate chemistry-dependent differences were observed in fibroblast adhesion, spreading, and growth. The formation of focal contacts and stress fibers was enhanced on the carboxyl-terminated surface relative to the methyl surface. Relative deposition and conformations of adsorbed fibronectin were shown to be dependent on surface chemistry in both the presence and absence of competing proteins. The results indicated that well-controlled culture surfaces modulate differential cell adhesion, spreading, and growth through modulations of the amounts and conformations of adsorbed extracellular matrix molecules (e.g., fibronectin).

Minimally modified low-density lipoprotein induces monocyte adhesion to endothelial connecting segment-1 by activating β1 integrin

We have shown previously that treatment of human aortic endothelial cells (HAECs) with minimally modified low-density lipoprotein (MM-LDL) induces monocyte but not neutrophil binding. This monocyte binding was not mediated by endothelial E-selectin, P-selectin, vascular cell adhesion molecule-I, or intercellular adhesion molecule-I, suggesting an alternative monocyte-specific adhesion molecule. We now show that moncytic alpha4beta1 integrins mediate binding to MM-LDL-treated endothelial cells. We present data suggesting that the expression of the connecting segment-1 (CS-1) domain of fibronectin (FN) is induced on the apical surface of HAEC by MM-LDL and is the endothelial alpha4beta1 ligand in MM-LDL-treated cells. Although the levels of CS-1 mRNA and protein were not increased, we show that MM-LDL treatment causes deposition of FN on the apical surface by activation of beta1integrins, particularly those associated with alpha5 integrins. Activation of beta1 by antibody 8A2 also induced CS-1-mediated monocyte binding. Confocal microscopy demonstrated the activated beta1 and CS-1colocalize in concentrated filamentous patches on the apical surface of HAEC. Both anti-CS-1 and an antibody to activated beta1 showed increased staining on the luminal endothelium of human coronary lesions with active monocyte entry. These results suggest the importance of these integrin ligand interactions in human atherosclerosis.

Identification of a novel recognition sequence for integrin α(M)β2 within the γ-chain of fibrinogen

The interaction of leukocyte integrin αMβ2 (CD11b/CD18, Mac-1) with fibrinogen has been implicated in the inflammatory response by contributing to leukocyte adhesion to the endothelium and subsequent transmigration. Previously, it has been demonstrated that a peptide, P1, corresponding to residues 190–202 in the γ-chain of fibrinogen, binds to αMβ2 and blocks the interaction of fibrinogen with the receptor and that Asp199 within P1 is important to activity. We have demonstrated, however, that a double mutation of Asp199-Gly200 to Gly-Ala in the recombinant γ-module of fibrinogen, spanning region 148–411, did not abrogate αMβ2 recognition and considered that other binding sites in the γ-module may participate in the receptor recognition. We have found that synthetic peptide P2, duplicating γ377–395, inhibited adhesion of αMβ2-transfected cells to immobilized D100 fragment of fibrinogen in a dose-dependent manner. In addition, immobilized P2 directly supported efficient adhesion of the αMβ2-expressing cells, including activated and non-activated monocytoid cells. The I domain of αMβ2 was implicated in recognition of P2, as the biotinylated recombinant αMI domain specifically bound to both P2 and P1 peptides. Analysis of overlapping peptides spanning P2 demonstrated that it may contain two functional sequences: γ377–386 (P2-N) and γ383–395 (P2-C), with the latter sequence being more active. In the three-dimensional structure of the γ-module, γ190–202 and γ377–395 reside in close proximity, forming two antiparallel β strands. The juxtapositioning of these two sequences may form an unique and complex binding site for αMβ2.

Origin of the Nonadhesive Properties of Fibrinogen Matrices Probed by Force Spectroscopy

The deposition of a multilayered fibrinogen matrix on various surfaces results in a dramatic reduction of integrin-mediated cell adhesion and outside-in signaling in platelets and leukocytes. The conversion of a highly adhesive, low-density fibrinogen substrate to the nonadhesive high-density fibrinogen matrix occurs within a very narrow range of fibrinogen coating concentrations. The molecular events responsible for this transition are not well understood. Herein, single-cell and molecular force spectroscopy were used to determine the early steps in the formation of nonadhesive fibrinogen substrates. We show that the adsorption of fibrinogen in the form of a molecular bilayer coincides with a several-fold reduction in the adhesion forces generated between the AFM tip and the substrate as well as between a cell and the substrate. The subsequent deposition of new layers at higher coating concentrations of fibrinogen results in a small additional decrease in adhesion forces. The poorly adhesive fibrinogen bilayer is more extensible under an applied tensile force than is the surface-bound fibrinogen monolayer. Following chemical cross-linking, the stabilized bilayer displays the mechanical and adhesive properties characteristic of a more adhesive fibrinogen monolayer. We propose that a greater compliance of the bi- and multilayer fibrinogen matrices has its origin in the interaction between the molecules forming the adjacent layers. Understanding the mechanical properties of nonadhesive fibrinogen matrices should be of importance in the therapeutic control of pathological thrombosis and in biomaterials science.

Recognition of Fibrinogen by Leukocyte Integrins

Abstract: Numerous studies have provided evidence that fibrinogen plays a multifaceted role in the immune and inflammatory response. The ability of fibrinogen to participate in the inflammatory response depends on its specific interaction with leukocyte cell surface adhesion receptors, integrins. Two leukocyte integrins, αMβ2 (CD11b/CD18, Mac‐1) and αXβ2 (CD11c/CD18, p150,95), are the main fibrinogen receptors expressed on neutrophils, monocytes, macrophages and several subsets of lymphocytes. The recognition site for αMβ2 has been previously mapped to the carboxyl‐terminal globular γC domains (γ143–411) and two sequences, γ190–202 (P1) and γ377–395 (P2), were implicated as the putative binding sites. We now demonstrate that a second leukocyte integrin, αXβ2, which is highly homologous to αMβ2, mediates adhesion of the αXβ2‐bearing cells to the D fragment and to the recombinant γ‐module, γ143–411. Within the γC domain, αXβ2 may recognize P1 and P2 sequences since synthetic peptides duplicating these sequences effectively inhibits adhesion of the αXβ2‐expressing cells to the D fragment. In addition, neutrophil inhibitory factor, NIF, a potent inhibitor of αXβ2, also inhibited αXβ2‐mediated cell adhesion. These data suggest that recognition of the γC domain of fibrinogen by αMβ2 and αXβ2 may have common structural requirements.

A Molecular Basis for Integrin αMβ2 Ligand Binding Promiscuity

The leukocyte integrin αMβ2 is a highly promiscuous leukocyte receptor capable of binding a multitude of unrelated ligands. To understand the molecular basis for the broad ligand recognition of αMβ2, the inter-integrin chimera was created. In the chimeric integrin, the βd-α5 loop-α5 helix segment comprised of residues Lys245–Arg261 from the αMI domain of αMβ2 was inserted into the framework of αLβ2. The construct was expressed in HEK 293 cells, and the ability of generated cells to adhere to fibrinogen and its derivatives was characterized first. Grafting the αM(Lys245–Arg261) sequence converted αLβ2 into a fibrinogen-binding protein capable of mediating efficient and specific adhesion similar to that of wild-type αMβ2. Verifying a switch in the binding specificity of αLβ2, the chimeric receptor became competent to support cell migration to fibrinogen. Mutations at positions Phe246, Asp254, and Pro257 within Lys245–Arg261 of αMβ2 produced significant decreases in cell adhesion, illustrating the critical role of these residues in ligand binding. The insertion of αM(Lys245–Arg261) imparted to the chimeric integrin the ability to recognize many typical αMβ2 protein ligands. Furthermore, cells expressing the chimeric receptor, but not αLβ2, were able to stick to uncoated plastic, which represents the hallmark of wild-type αMβ2. These results suggest that αM(Lys245–Arg261) serves as a consensus binding site for interaction with a variety of distinct molecules and, thus, may define the degenerate recognition properties inherent to αMβ2.

Identification of the Binding Site for Fibrinogen Recognition Peptide γ383–395 within the αMI-Domain of Integrin αMβ2

The leukocyte integrin αMβ2 (Mac-1, CD11b/CD18) is a cell surface adhesion receptor for fibrinogen. The interaction between fibrinogen and αMβ2 mediates a range of adhesive reactions during the immune-inflammatory response. The sequence γ383TMKIIPFNRLTIG395, P2-C, within the γ-module of the D-domain of fibrinogen, is a recognition site for αMβ2 and αXβ2. We have now identified the complementary sequences within the αMI-domain of the receptor responsible for recognition of P2-C. The strategy to localize the binding site for P2-C was based on distinct P2-C binding properties of the three structurally similar I-domains of αMβ2, αXβ2, and αLβ2,i.e. the αMI- and αXI-domains bind P2-C, and the αLI-domain did not bind this ligand. The Lys245-Arg261 sequence, which forms a loop βD-α5 and an adjacent helix α5 in the three-dimensional structure of the αMI-domain, was identified as the binding site for P2-C. This conclusion is supported by the following data: 1) mutant cell lines in which the αMI-domain segments 245KFG and Glu253-Arg261 were switched to the homologous αLI-domain segments failed to support adhesion to P2-C; 2) synthetic peptides duplicating the Lys245-Tyr252 and Glu253-Arg261 sequences directly bound the D fragment and P2-C derivative, γ384–402, and this interaction was blocked efficiently by the P2-C peptide; 3) mutation of three amino acid residues within the Lys245-Arg261 segment, Phe246, Asp254, and Pro257, resulted in the loss of the binding function of the recombinant αMI-domains; and 4) grafting the αM(Lys245-Arg261) segment into the αLI-domain converted it to a P2-C-binding protein. These results demonstrate that the αM(Lys245-Arg261) segment, a site of the major sequence and structure difference among αMI-, αXI-, and αLI-domains, is responsible for recognition of a small segment of fibrinogen, γThr383-Gly395, by serving as ligand binding site.

The interplay between integrins αmβ2 and α5β1 during cell migration to fibronectin

A directed migration of leukocytes through the extracellular matrix requires the regulated engagement of integrin cell adhesion receptors. The integrin alpha(M)beta(2) (CD11b/CD18, Mac-1) is progressively upregulated to high levels on migrating phagocytic leukocytes in response to inflammatory stimuli and is able to bind numerous ligands in the interstitial matrix. The role of alpha(M)beta(2) in migration of leukocytes through the extracellular matrix and its cooperation with other leukocyte integrins during migration are not understood. Using a model system consisting of cells that express different levels of alpha(M)beta(2) and an invariable level of endogenous integrin alpha(5)beta(1), we have explored a situation relevant to migrating neutrophils when alpha(M)beta(2) and alpha(5)beta(1) engage the same ligand, fibronectin. We show that fibronectin is a ligand for alpha(M)beta(2) and that both alpha(M)beta(2) and alpha(5)beta(1) on the alpha(M)beta(2)-expressing cells contribute to adhesion to fibronectin. However, migration of these cells to fibronectin is mediated by alpha(5)beta(1), whereas alpha(M)beta(2) retards migration. The decrease in migration correlates directly with the increased alpha(M)beta(2) density. Ligation of alpha(M)beta(2) with function-blocking antibodies can reverse this effect. The restorative effects of antibodies are caused by the removal of restraint imposed by the excess of alpha(M)beta(2)-fibronectin adhesive bonds. These findings indicate that alpha(M)beta(2) can increase general cell adhesiveness which results in braking of cell migration mediated by integrin alpha(5)beta(1). Because alpha(M)beta(2) binds numerous proteins in the extracellular matrix with a specificity overlapping that of the beta(1) integrins, the results suggest that alpha(M)beta(2) can affect the beta(1) integrin-mediated cell migration.

The Role of Integrin αDβ2 (CD11d/CD18) in Monocyte/Macrophage Migration

Integrin alpha(D)beta(2) (CD11d/CD18) is a multiligand macrophage receptor with recognition specificity identical to that of the major myeloid cell-specific integrin alpha(M)beta(2) (CD11b/CD18, Mac-1). Despite its prominent upregulation on inflammatory macrophages, the role of alpha(D)beta(2) in monocyte and macrophage migration is unknown. In this study, we have generated model and natural cell lines expressing different densities of alpha(D)beta(2) and examined their migration to various extracellular matrix proteins. When expressed at a low density, alpha(D)beta(2) on the surface of recombinant HEK293 cells and murine IC-21 macrophages cooperates with beta(1)/beta(3) integrins to support cell migration. However, its increased expression on the alpha(D)beta(2)-expressing HEK293 cells and its upregulation by PMA on the IC-21 macrophages result in increased cell adhesiveness and inhibition of cell migration. Furthermore, ligation of alpha(D)beta(2) with anti-alpha(D) blocking antibodies restores beta(1)/beta(3)-driven cell migration by removing the excess alpha(D)beta(2)-mediated adhesive bonds. Consistent with in vitro data, increased numbers of inflammatory macrophages were recovered from the inflamed peritoneum of mice after the administration of anti-alpha(D) antibody. These results demonstrate that the density of alpha(D)beta(2) is critically involved in modulating macrophage adhesiveness and their migration, and suggest that low levels of alpha(D)beta(2) contribute to monocyte migration while alpha(D)beta(2) upregulation on differentiated macrophages may facilitate their retention at sites of inflammation.