Ravi M. Rao

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 transendo-thelial migration in response to fractalkine (FKN; FKN/CX3CL1) and stromal-derived factor 1α (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.

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-1β 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.

Endothelial-Dependent Mechanisms of Leukocyte Recruitment to the Vascular Wall

Inflammation is a fundamental process that protects organisms by removing or neutralizing injurious agents. A key event in the inflammatory response is the localized recruitment of various leukocyte subsets. Here we address the cellular and regulatory mechanisms of leukocyte recruitment to the vessel wall in cardiovascular disease and discuss our evolving understanding of the role of the vascular endothelium in this process. The vascular endothelium is the continuous single-cell lining of the cardiovascular system that forms a critical interface between the blood and its components on one side and the tissues and organs on the other. It is heterogeneous and has many synthetic and metabolic functions including secretion of platelet-derived growth factor, von Willebrand factor, prostacyclin, NO, endothelin-1, and chemokines and the expression of adhesion molecules. It also acts as a nonthrombogenic and selective permeable barrier. Endothelial cells also interact closely with the extracellular matrix and with adjacent cells including pericytes and smooth muscle cells within the vessel wall. A central question in vascular biology is the role of the endothelium in the initiation of inflammatory response, the extent of its “molecular conversations” with recruited leukocytes, and its influence on the extent and/or outcome of this response.

Targeting platelet-leukocyte interactions: Identification of the integrin Mac-1 binding site for the platelet counter receptor glycoprotein Ibα

The firm adhesion and transplatelet migration of leukocytes on vascular thrombus are dependent on the interaction of the leukocyte integrin Mac-1 (αMβ2, CD11b/CD18) and the platelet counter receptor glycoprotein (GP) Ibα. Previous studies have established a central role for the I domain, a stretch of ∼200 amino acids within the αM subunit, in the binding of GP Ibα. This study was undertaken to establish the molecular basis of GP Ibα recognition by αMβ2. The P201–K217 sequence, which spans an exposed loop and amphipathic α4 helix in the three-dimensional structure of the αMI domain, was identified as the binding site for GP Ibα. Mutant cell lines in which the αMI domain segments P201–G207 and R208–K217 were switched to the homologous, but non-GP Ibα binding, αL domain segments failed to support adhesion to GP Ibα. 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 αMI domains to GP Ibα. Synthetic peptides duplicating the P201–K217, but not scrambled versions, directly bound GP Ibα and inhibited αMβ2-dependent adhesion to GP Ibα and adherent platelets. Finally, grafting critical amino acids within the P201–K217 sequence onto αL, converted αLβ2 into a GP Ibα binding integrin. Thus, the P201–K217 sequence within the αMI domain is necessary and sufficient for GP Ibα binding. These observations provide a molecular target for disrupting leukocyte–platelet complexes that promote vascular inflammation in thrombosis, atherosclerosis, and angioplasty-related restenosis.

Elastase Release by Transmigrating Neutrophils Deactivates Endothelial-bound SDF-1α and Attenuates Subsequent T Lymphocyte Transendothelial Migration

Leukocyte trafficking to sites of inflammation follows a defined temporal pattern, and evidence suggests that initial neutrophil transendothelial migration modifies endothelial cell phenotype. We tested the hypothesis that preconditioning of human umbilical vein endothelial cells (HUVEC) by neutrophils would also modify the subsequent transendothelial migration of T lymphocytes across cytokine-stimulated HUVEC in an in vitro flow assay. Using fluorescence microscopy, preconditioning of HUVEC by neutrophils was observed to significantly reduce the extent of subsequent stromal cell–derived factor-1α (SDF-1α [CXCL12])-mediated T lymphocyte transendothelial migration, without reducing accumulation. In contrast, recruitment of a second wave of neutrophils was unaltered. Conditioned medium harvested after transendothelial migration of neutrophils or supernatants from stimulated neutrophils mediated a similar blocking effect, which was negated using a specific neutrophil elastase inhibitor. Furthermore, T lymphocyte transendothelial migration was inhibited by treatment of HUVEC with purified neutrophil elastase, which selectively cleaved the amino terminus of HUVEC-bound SDF-1α, which is required for its chemotactic activity. The reduction in T lymphocyte transendothelial migration was not observed using a different chemokine, ELC (CCL19), and was not reversed by replenishment of SDF-1α, indicating endothelial retention of the inactivated chemokine. In summary, transmigrating neutrophils secrete localized elastase that is protected from plasma inhibitors, and thereby modulate trafficking of other leukocyte subsets by altering the endothelial-associated chemotactic activities.

The S128R polymorphism of E-selectin mediates neuraminidase-resistant tethering of myeloid cells under shear flow.

E‐selectin mediates the rolling of circulating leukocytes on vascular endothelial cells. A polymorphism, in which serine is substituted for arginine at position 128 (S128R) in the EGF domain, has been associated with both early‐onset atherosclerosis and SLE. We investigated whether the substitution alters the ligand‐binding properties of E‐selectin under shear flow by studying the capacity of Chinese hamster ovary cell transfectants expressing wild type (WT) or S128R E‐selectin to support interactions of neutrophils, K562 cells or HL60 cells. We initially chose to study non‐fucosylated K562 cells. No interactions were observed on WT E‐selectin, whereas S128R supported a transient tethering interaction of K562 cells, which was resistant to digestion with either neuraminidase or O‐sialoglycoprotein endopeptidase, and, in turn, could result in firm adhesion in the presence of a β2‐integrin. HL60 cells exhibited increased rolling on S128R E‐selectin. Although neuraminidase treatment inhibited all HL60 interactions with WT E‐selectin, it unmasked transient tethers on S128R. We further observed that S128R recruited significantly more neutrophils than WT E‐selectin, without affecting neutrophil rolling velocity. This polymorphism may therefore amplify leukocyte‐endothelial cell interactions and may be a factor linking the S128R polymorphism to vascular disease.

Enhanced Recruitment of Th2 and CLA-Negative Lymphocytes by the S128R Polymorphism of E-Selectin

E-selectin is a cytokine-inducible endothelial cell adhesion molecule that binds a restricted population of T lymphocytes consisting of Th1 memory cells bearing the cutaneous lymphocyte Ag (CLA). A serine to arginine (S128R) polymorphism in E-selectin has been reported at increased frequency in patients with systemic lupus erythematosus and atherosclerosis. Here we tested the hypothesis that the S128R substitution may contribute to increased vascular disease by altering the number and/or phenotype of lymphocytes interacting with E-selectin under shear flow. We observed that CHO cell monolayers transfected with S128R recruited significantly greater numbers of unfractionated lymphocytes than monolayers expressing an equivalent density of wild-type (WT) E-selectin. Depletion of the CLA+ subpopulation or generation of CLA− lymphoblasts abolished rolling and arrest on WT E-selectin, but left a residual population that interacted with S128R. Generation of Th subsets revealed preferential interaction of Th0 and Th2, but not Th1, cells with S128R compared with WT. However, only T cells of a memory phenotype interacted with S128R, since neither monolayer supported rolling of CD45RA+ cells. Our results demonstrate that the S128R polymorphism extends the range of lymphocytes recruited by E-selectin, which may provide a mechanistic link between this polymorphism and vascular inflammatory disease.

Emerging Topics in the Regulation of Leukocyte Transendothelial Migration

This year marks the 50th Anniversary of the founding of the Microcirculation 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 Microcirculation 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.