When rat pancreatic polynucleosomes were poly(ADP-ribosyl)ated with purified calfthymus poly(ADP-ribose) polymerase and examined by electron microscopy, a relaxation of their native zigzag structure was observed. At high ionic strengths control nucleosomes condensed into 250-A-thick fibers, but poly(ADP-ribosyl)ated polynucleosomes did not; they showed a close resemblance to chromatin depleted of histone HI. The relaxed state of poly(ADP-ribosyl)ated polynucleosomes was also confirmed by sedimentation velocity analysis. Histone HI was found to be the major histone acceptor of poly(ADP-ribose). Poly(ADP-ribose) linked to histone HI did not seem to cause its dissociation from the chromatin, but it impaired significantly its effect on chromatin condensation.Post-translational modifications of histones-e.g., phosphorylation (1), acetylation (2, 3), and poly(ADP-ribosyl)ation (4,5) have been suggested as possible mechanisms to modulate the nucleosomal structure during DNA transcription and replication.Poly(ADP-ribose) polymerase was suggested to be preferentially located at internucleosomal regions of HeLa cell nucleosomes (6, 7); its specific activity was shown to be highest at selected folded regions of 8-10 nucleosomes in higher-ordered chromatin (8). In addition, the poly(ADP-ribosyl)ation of several chromatin components, including all the histones, was demonstrated (9, 10). All these findings reinforced the suggestions of involvement of poly(ADP-ribosyl)ation in DNA replication, DNA repair, and gene expression through alteration of the chromatin structure.In order to test this hypothesis, pancreatic polynucleosomes (20-40 nucleosomes) were poly(ADP-ribosyl)ated by using purified calf thymus poly(ADP-ribose) polymerase. Changes of nucleosome structure were examined by electron microscopy and by ultracentrifugation and the poly(ADP-ribosyl)ated histones were characterized.MATERIALS AND METHODS Preparation ofPolynucleosomes. Rat pancreatic polynucleosomes were isolated as described (11). Nuclei were digested with micrococcal nuclease (Sigma) at 30°C for 2 min, with 0.5 unit of nuclease per mg of DNA. The polynucleosomes (20-40 nucleosomes) were isolated on linear 5-29% sucrose gradients by centrifugation at 260,000 X g for 150 min in a Beckman SW 41 rotor and were characterized by electron microscopy and circular dichroism.Poly(ADP-ribosyl)ation of Polynucleosomes. Polynucleosomes were poly(ADP-ribosyl)ated by using purified calf thymus poly(ADP-ribose) polymerase at 25°C in an incubation medium of 500 utl containing 8 jig of DNA-independent enzyme (12,13) Electron Microscopy. Chromatin samples diluted to 0.5 ,g/ ml (expressed as DNA concentration) with a buffer containing 5 mM triethanolamine at pH 7.4, 0.2 mM EDTA, and the appropriate concentration of NaCl (see figure legends) were fixed in 0.1% glutaraldehyde (Balzers, Lichtenstein) for 1 hr at room temperature. Adsorption of the specimens onto positively charged carbon-coated grids (400 mesh) was performed as described (14). The specimens were stained w...

Atherosclerotic lesions develop in regions of arterial curvature and branch points, which are exposed to disturbed blood flow and have unique gene expression patterns. The cellular and molecular basis for atherosclerosis susceptibility in these regions is not completely understood. In the intima of atherosclerosis-predisposed regions of the wild-type C57BL/6 mouse aorta, we quantified increased expression of several proinflammatory genes that have been implicated in atherogenesis, including vascular cell adhesion molecule–1 (VCAM-1) and a relative abundance of dendritic cells, but only occasional T cells. In contrast, very few intimal leukocytes were detected in regions resistant to atherosclerosis; however, abundant macrophages, including T cells, were found throughout the adventitia (Adv). Considerably lower numbers of intimal CD68+ leukocytes were found in inbred atherosclerosis-resistant C3H and BALB/c mouse strains relative to C57BL/6 and 129; however, leukocyte distribution throughout the Adv of all strains was similar. The predominant mechanism for the accumulation of intimal CD68+ cells was continued recruitment of bone marrow–derived blood monocytes, suggestive of low-grade chronic inflammation. Local proliferation of intimal leukocytes was low. Intimal CD68+ leukocytes were reduced in VCAM-1–deficient mice, suggesting that mechanisms of leukocyte accumulation in the intima of normal aorta are analogous to those in atherosclerosis.

Foam cells are derived primarily from monocytes/ macrophages, 2,3 but the majority of research into the nature of foam cells has focused on relatively advanced lesions and has generally used a single monocyte/macrophage marker, such as CD68, MOMA-2, Mac-2, or Mac-3. 4 -6 Some of these markers (eg, CD68) can be expressed by both macrophages and dendritic cells (DCs) and thus cannot differentiate between these cell types. There are, however, several markers expressed preferentially by DCs, including CD11c, major histocompatibility complex (MHC) class II and 33D1 antigen. 7 An in vitro study showed that macrophages, when treated with oxidatively modified lipids, differentiate into foam cells and in the process acquire expression of DC markers. 8 The identity of foam cells is of great significance, because DCs possess distinct functions from macrophages and DC-derived foam cells may retain some or all of their properties.DCs are found in the intima of human arteries. 9 We and others have shown that DCs reside in the normal murine aortic intima in areas predisposed to atherosclerotic lesion formation and are absent in areas protected from atherosclerosis. 10,11 We refer to these cells as resident intimal DCs. Macrophages are rare in the normal aortic intima but are abundant throughout the adventitia. 10 DCs are also found in atherosclerotic lesions, in both humans 12 and animal models. 13,14 The role of DCs in atherosclerosis is not well understood. DCs isolated from the normal aorta are capable of cross-presenting antigen 15 ; however, it is not likely that antigen presentation occurs in the normal intima, because T lymphocytes are very scarce in this location. 10 DCs may migrate to secondary lymphoid organs, where they encounter a wide repertoire of T cells. This phenomenon may decrease during atherogenesis, because hypercholesterolemia impairs migration of DCs to lymph nodes. 16 DCs can also regulate inflammation by producing either proinflammatory or tolerogenic factors in different contexts. 17,18 Recent studies suggested that lesion Original received May 29, 2009; resubmission received October 7, 2009; accepted October 28, 2009. From the Toronto General Research Institute (K.E.P., S.-N.Z., M.C., S.N., J.J.-B., M.I.C.), University Health Network; and Departments of Immunology (K.E.P., J.J.-B.) and Laboratory Medicine and Pathobiology (J.J.-B., M.I.C.), University of Toronto, Canada.Correspondence 19 We hypothesize that resident intimal DCs may play a key role in the initiation of atherosclerosis by engulfing lipid and differentiating into foam cells.Conditional depletion of specific cell types can be accomplished using transgenic murine models in which the simian diphtheria toxin receptor (DTR) is placed under the transcriptional control of a cell-specific promoter. A single injection of diphtheria toxin (DT) into mice bearing a murine CD11c promoter-DTR transgene (CD11c-DTR mice) induces apoptosis and depletes DCs, which express high levels of CD11c. 24 The murine homolog of DTR binds DT with very low affinit...

The contribution of intimal cell proliferation to the formation of early atherosclerotic lesions is poorly understood. We combined 5-bromo-2′-deoxyuridine pulse labeling with sensitive en face immunoconfocal microscopy analysis, and quantified intimal cell proliferation and Ly-6Chigh monocyte recruitment in low density lipoprotein receptor–null mice. Cell proliferation begins in nascent lesions preferentially at their periphery, and proliferating cells accumulate in lesions over time. Although intimal cell proliferation increases in parallel to monocyte recruitment as lesions grow, proliferation continues when monocyte recruitment is inhibited. The majority of proliferating intimal cells are dendritic cells expressing CD11c and major histocompatibility complex class II and 33D1, but not CD11b. Systemic injection of granulocyte/macrophage colony-stimulating factor (GM-CSF) markedly increased cell proliferation in early lesions, whereas function-blocking anti–GM-CSF antibody inhibited proliferation. These findings establish GM-CSF as a key regulator of intimal cell proliferation in lesions, and demonstrate that both proliferation and monocyte recruitment contribute to the inception of atherosclerosis.

Actin polymerization drives the extension of pseudopods required for phagocytosis. Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is thought to play a central role in this process, because it interacts with several actin-regulatory proteins and undergoes acute and localized changes at sites of phagocytosis. We therefore studied whether phosphatidylinositol-4-phosphate 5-kinase (PIPK), the enzyme responsible for the generation of PIP 2 from phosphatidylinositol 4-phosphate, is involved in the control of phagocytosis. PIPKI␣ was found to accumulate transiently on forming phagosomes. To test the functional involvement of PIPKI␣ in particle engulfment, we generated a double mutant (D309N/ R427Q) that lacks kinase activity. When ectopically expressed in cultured cells, this mutant is targeted to the plasma membrane and accumulates at the phagosomal cup during particle engulfment. Expression of PIP5KI␣ D309N/R427Q impaired phagocytosis in RAW264.7 macrophages and in engineered phagocytes generated by transfection of Fc receptors in Chinese hamster ovary cells. Inhibition of phagocytosis could not be attributed to defects in particle binding or receptor clustering, which was monitored using green fluorescent protein-tagged Fc␥ receptors. Instead, expression of the inactive kinase diminished the accumulation of PIP 2 and of F-actin in the phagosomal cup. These data suggest that PIPKI␣ activity is involved in the actin remodeling that is a prerequisite for efficient phagocytosis. PIPKI␣ appears to contribute to the transient changes in PIP 2 levels that are associated with, and likely required for, the recruitment and regulation of actinmodulating proteins.

Leukocyte-specific protein 1 (LSP1), an F-actin binding protein and a major downstream substrate of p38 mitogen-activated protein kinase as well as protein kinase C, has been reported to be important in leukocyte chemotaxis. Although its distribution has been thought to be restricted to leukocytes, herein we report that LSP1 is expressed in endothelium and is essential to permit neutrophil emigration. Using intravital microscopy to directly visualize leukocyte rolling, adhesion, and emigration in postcapillary venules in LSP1-deficient (Lsp1 −/−) mice, we found that LSP1 deficiency inhibits neutrophil extravasation in response to various cytokines (tumor necrosis factor-α and interleukin-1β) and to neutrophil chemokine keratinocyte-derived chemokine in vivo. LSP1 deficiency did not affect leukocyte rolling or adhesion. Generation of Lsp1 −/− chimeric mice using bone marrow transplantation revealed that in mice with Lsp1 −/− endothelial cells and wild-type leukocytes, neutrophil transendothelial migration out of postcapillary venules is markedly restricted. In contrast, Lsp1 −/− neutrophils in wild-type mice were able to extravasate normally. Consistent with altered endothelial function was a reduction in vascular permeability to histamine in Lsp1 −/− animals. Western blot analysis and immunofluorescence microscopy examination confirmed the presence of LSP1 in wild-type but not in Lsp1 −/− mouse microvascular endothelial cells. Cultured human endothelial cells also stained positive for LSP1. Our results suggest that LSP1 expressed in endothelium regulates neutrophil transendothelial migration.

Functional interactions betweenMac-1, a heterodimeric receptor primarily expressed in neutrophils and monocytes/macrophages, is composed of a specific ␣ chain (CD11b) and the ␤2 chain (CD18) which is common to the other members of the ␤ 2 integrin family (1). As is the case for other integrins, Mac-1 (also known as CD11b/CD18, ␣ M ␤ 2 , Mo-1, or CR3) activation is required for efficient binding to several ligands such as intercellular adhesion molecule 1, C3bi, or fibrinogen. Activation of the cells by specific agonists induces the receptor to undergo conformational changes, mobilization, and clustering by a process known as inside-out signaling (2, 3).

We have identified CD72 as the first clear in vivo substrate of SHP-1 in B cells. Our results suggest that tyrosine-phosphorylated CD72 may transmit signals for BCR-induced apoptosis. By dephosphorylation CD72. SHP-1 may have a positive role in B-cell signaling. These results have potentially important implications for the involvement of CD72 and SHP-1 in B-cell development and autoimmunity.