Targeting platelet-leukocyte interactions: identification of the integrin Mac-1 binding site for the platelet counter receptor glycoprotein Ibalpha.

The firm adhesion and transplatelet migration of leukocytes on vascular thrombus are dependent on the interaction of the leukocyte integrin Mac-1 (alphaMbeta2, CD11b/CD18) and the platelet counter receptor glycoprotein (GP) Ibalpha. Previous studies have established a central role for the I domain, a stretch of approximately 200 amino acids within the alphaM subunit, in the binding of GP Ibalpha. This study was undertaken to establish the molecular basis of GP Ibalpha recognition by alphaMbeta2. The P201-K217 sequence, which spans an exposed loop and amphipathic alpha4 helix in the three-dimensional structure of the alphaMI domain, was identified as the binding site for GP Ibalpha. Mutant cell lines in which the alphaMI domain segments P201-G207 and R208-K217 were switched to the homologous, but non-GP Ibalpha binding, alphaL domain segments failed to support adhesion to GP Ibalpha. Mutation of amino acid residues within P201-K217, H210-A212, T213-I215, and R216-K217 resulted in the loss of the binding function of the recombinant alphaMI domains to GP Ibalpha. Synthetic peptides duplicating the P201-K217, but not scrambled versions, directly bound GP Ibalpha and inhibited alphaMbeta2-dependent adhesion to GP Ibalpha and adherent platelets. Finally, grafting critical amino acids within the P201-K217 sequence onto alphaL, converted alphaLbeta2 into a GP Ibalpha binding integrin. Thus, the P201-K217 sequence within the alphaMI domain is necessary and sufficient for GP Ibalpha binding. These observations provide a molecular target for disrupting leukocyte-platelet complexes that promote vascular inflammation in thrombosis, atherosclerosis, and angioplasty-related restenosis.

Fractalkine preferentially mediates arrest and migration of CD16+ monocytes.

CD16+ monocytes represent 5-10% of peripheral blood monocytes in normal individuals and are dramatically expanded in several pathological conditions including sepsis, human immunodeficiency virus 1 infection, and cancer. CD16+ monocytes produce high levels of proinflammatory cytokines and may represent dendritic cell precursors in vivo. The mechanisms that mediate the recruitment of CD16+ monocytes into tissues remain unknown. Here we investigate molecular mechanisms of CD16+ monocyte trafficking and show that migration of CD16+ and CD16- monocytes is mediated by distinct combinations of adhesion molecules and chemokine receptors. In contrast to CD16- monocytes, CD16+ monocytes expressed high CX3CR1 and CXCR4 but low CCR2 and CD62L levels and underwent efficient transendothelial migration in response to fractalkine (FKN; FKN/CX3CL1) and stromal-derived factor 1 alpha (CXCL12) but not monocyte chemoattractant protein 1 (CCL2). CD16+ monocytes arrested on cell surface-expressed FKN under flow with higher frequency compared with CD16- monocytes. These results demonstrate that FKN preferentially mediates arrest and migration of CD16+ monocytes and suggest that recruitment of this proinflammatory monocyte subset to vessel walls via the CX3CR1-FKN pathway may contribute to vascular and tissue injury during pathological conditions.

Coordinated redistribution of leukocyte LFA-1 and endothelial cell ICAM-1 accompany neutrophil transmigration.

The leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1) and its endothelial ligand intercellular adhesion molecule (ICAM)-1 play an important role in transmigration as demonstrated by in vivo and in vitro models of inflammation. Despite the prominent role, little is known concerning the distribution and dynamic behavior of these adhesion molecules during leukocyte transmigration. Therefore, we examined the spatial and temporal distribution of LFA-1 on neutrophils actively transmigrating tumor necrosis factor-alpha-activated human umbilical vein endothelial monolayers under shear flow. Upon neutrophil arrest, LFA-1 was evenly distributed. However, once neutrophils initiated transmigration, LFA-1 rapidly redistributed to form a ringlike cluster at the neutrophil-endothelial junctional interface through which transmigration occurred. As transmigration was completed, LFA-1 redistributed to the neutrophil uropod. Endothelial ICAM-1 and JAM-A both colocalized with the ringlike LFA-1 cluster. Further analysis of PMA-stimulated neutrophils, which increase mobility of LFA-1, showed a rapid redistribution of LFA-1 and ICAM-1, but not endothelial JAM-A. Thus, endothelial JAM-A does not appear to contribute to adhesion or transmigration in this system. This is the first demonstration that neutrophil LFA-1 rapidly redistributes to form a ringlike structure that coclusters with endothelial ICAM-1 as the neutrophil transmigrates.

KLF2 Is a novel transcriptional regulator of endothelial proinflammatory activation.

The vascular endothelium is a critical regulator of vascular function. Diverse stimuli such as proinflammatory cytokines and hemodynamic forces modulate endothelial phenotype and thereby impact on the development of vascular disease states. Therefore, identification of the regulatory factors that mediate the effects of these stimuli on endothelial function is of considerable interest. Transcriptional profiling studies identified the Kruppel-like factor (KLF)2 as being inhibited by the inflammatory cytokine interleukin-1beta and induced by laminar shear stress in cultured human umbilical vein endothelial cells. Overexpression of KLF2 in umbilical vein endothelial cells robustly induced endothelial nitric oxide synthase expression and total enzymatic activity. In addition, KLF2 overexpression potently inhibited the induction of vascular cell adhesion molecule-1 and endothelial adhesion molecule E-selectin in response to various proinflammatory cytokines. Consistent with these observations, in vitro flow assays demonstrate that T cell attachment and rolling are markedly attenuated in endothelial monolayers transduced with KLF2. Finally, our studies implicate recruitment by KLF2 of the transcriptional coactivator cyclic AMP response element-binding protein (CBP/p300) as a unifying mechanism for these various effects. These data implicate KLF2 as a novel regulator of endothelial activation in response to proinflammatory stimuli.

Ly-6Chi monocytes dominate hypercholesterolemia-associated monocytosis and give rise to macrophages in atheromata.

Macrophage accumulation participates decisively in the development and exacerbation of atherosclerosis. Circulating monocytes, the precursors of macrophages, display heterogeneity in mice and humans, but their relative contribution to atherogenesis remains unknown. We report here that the Ly-6C(hi) monocyte subset increased dramatically in hypercholesterolemic apoE-deficient mice consuming a high-fat diet, with the number of Ly-6C(hi) cells doubling in the blood every month. Ly-6C(hi) monocytes adhered to activated endothelium, infiltrated lesions, and became lesional macrophages. Hypercholesterolemia-associated monocytosis (HAM) developed from increased survival, continued cell proliferation, and impaired Ly-6C(hi) to Ly-6C(lo) conversion and subsided upon statin-induced cholesterol reduction. Conversely, the number of Ly-6C(lo) cells remained unaffected. Thus, we believe that Ly-6C(hi) monocytes represent a newly recognized component of the inflammatory response in experimental atherosclerosis.

Absence of TRAM restricts Toll-like receptor 4 signaling in vascular endothelial cells to the MyD88 pathway.

Mammalian cells respond to bacterial lipopolysaccharide (LPS) through a cognate receptor: Toll-like receptor 4 (TLR4). The signaling pathways, which link TLR4 to the proinflammatory transcription factor nuclear factor kappaB (NF-kappaB), occur through the intracellular docking proteins MyD88 and Trif. We hypothesize that unlike antigen-presenting cells, vascular endothelial cells (ECs) lack the Trif protein TRAM and are therefore incapable of eliciting Trif-dependent immune responses to LPS. Stimulation of wild-type mice with LPS leads to the activation of NF-kappaB in ECs and macrophages in vitro and in vivo. In contrast to macrophages, LPS did not activate endothelial NF-kappaB or NF-kappaB-dependent genes in MyD88(-/-) mice, suggesting the absence of a functional Trif pathway in vascular ECs. Indeed, the Trif-dependent gene cxcl10 was not expressed in ECs after LPS stimulation. This correlated with diminished expression of the Trif accessory TIR protein TRAM in ECs. Overexpression of TRAM cDNA in ECs reconstituted LPS-induced Trif-dependent NF-kappaB activation and cxcl10 promoter activity. The functional absence of TRAM in vascular ECs restricts TLR4 signaling to MyD88-dependent pathway. This is in contrast to macrophages, which respond to LPS via both Trif- and MyD88-dependent pathways. These findings indicate that vascular ECs do not express the Trif-dependent gene subset. This implies that these genes may be dispensable for the endothelial response to bacterial infection and play no role in the endothelial contribution to the development of atherosclerosis.

A Lupus-Associated Mac-1 Variant Has Defects in Integrin Allostery and Interaction with Ligands under Force.

Leukocyte CD18 integrins increase their affinity for ligand by transmitting allosteric signals to and from their ligand-binding αI domain. Mechanical forces induce allosteric changes that paradoxically slow dissociation by increasing the integrin/ligand bond lifetimes, referred to as catch bonds. Mac-1 formed catch bonds with its ligands. However, a Mac-1 gene (ITGAM) coding variant (rs1143679, R77H), which is located in the ß-propeller domain and is significantly associated with systemic lupus erythematosus risk, exhibits a marked impairment in 2D ligand affinity and affinity maturation under mechanical force. Targeted mutations and activating antibodies reveal that the failure in Mac-1 R77H allostery is rescued by induction of cytoplasmic tail separation and full integrin extension. These findings demonstrate roles for R77, and the ß-propeller in which it resides, in force-induced allostery relay and integrin bond stabilization. Defects in these processes may have pathological consequences, as the Mac-1 R77H variant is associated with increased susceptibility to lupus.

Assays of transendothelial migration in vitro.

The inflammatory response is critical for our ability to heal wounds and fight off foreign microorganisms. Uncontrolled inflammation is also at the root of most pathologic conditions. Recruitment of leukocytes to the site of inflammation plays a defining role in the inflammatory response, and migration of leukocytes across endothelium is arguably the point of no return of the inflammatory response. Assays to study the transmigration of leukocytes have and will continue to shed light on the regulation of this vital response. Assays of transendothelial migration in vitro allow the controlled observation of this phenomenon, as well as experiments to study its regulation. In this chapter, we describe in vitro assays of transendothelial migration that have been used successfully in the authors' laboratories for decades and have proven to be reproducible, reliable, and predictive of the behavior of leukocytes and endothelial cells in models of inflammation in vivo.

Emerging topics in the regulation of leukocyte transendothelial migration.

This year marks the 50th Anniversary of the founding of the Microcirculatory Society. Since the formation of this society this field has witnessed tremendous progress in understanding the process of leukocyte recruitment during inflammation, injury, and immune reactions. This topic has been an important focus of many of the members of the Microcirculatory Society as well as our colleagues worldwide. The goal of this brief review is to bring attention to a few emerging topics in inflammation research. Here the focus is on one particular model of how one leukocyte type (PMN) can regulate the recruitment of a second different leukocyte type (T cell) and provide an outline of other aspects that bear on spatial and temporal behavior of specific leukocyte and endothelial cell adhesion molecules during leukocyte transmigration under dynamic shear flow in vitro.

T-bet is required for optimal proinflammatory CD4+ T-cell trafficking.

Inflammatory responses are controlled by T helper 1 (Th1) lymphocytes. An important function of this polarity is the ability of T cells to traffick appropriately in vivo. This differential trafficking is dependent upon the binding of P-selectin glycoprotein ligand-1 to P- and E-selectin on inflamed endothelium as well as the expression of specific chemokine receptors. Here we show that in the absence of T-box expressed in T cells (T-bet), selective migration of T cells in vivo is completely abrogated and that T-bet regulates the binding of CD4(+) T cells to P-selectin. T-bet is also required for the expression of the chemokine receptor CXCR3. Thus, T-bet controls Th1-cell migration to inflammatory sites, which has fundamental consequences for the control of immunologic disease.