Where the Action Is—Leukocyte Recruitment in Atherosclerosis

Atherosclerosis is the leading cause of death worldwide and leukocyte recruitment is a key element of this phenomenon, thus allowing immune cells to enter the arterial wall. There, in concert with accumulating lipids, the invading leukocytes trigger a plethora of inflammatory responses which promote the influx of additional leukocytes and lead to the continued growth of atherosclerotic plaques. The recruitment process follows a precise scheme of tethering, rolling, firm arrest, crawling and transmigration and involves multiple cellular and subcellular players. This review aims to provide a comprehensive up-to-date insight into the process of leukocyte recruitment relevant to atherosclerosis, each from the perspective of endothelial cells, monocytes and macrophages, neutrophils, T lymphocytes and platelets. In addition, therapeutic options targeting leukocyte recruitment into atherosclerotic lesions—or potentially arising from the growing body of insights into its precise mechanisms—are highlighted.

Neutralization of Inflammasome-Processed Cytokines Reduces Inflammatory Mechanisms and Leukocyte Recruitment in the Vasculature of TNF-α-Stimulated Sickle Cell Disease Mice

The chronic inflammatory state associated with sickle cell disease (SCD) incurs pan-cellular activation and the recruitment of leukocytes to the activated endothelium of blood vessels. The resultant rheological alterations and red cell sickling culminate in acute vaso-occlusive processes. Cytokines IL-1β and IL-18, the bio-active products of the activated inflammasome, are elevated in the plasma of SCD patients (ASH Abstract (2016) 128 (22): 854), and a previous study reported that anti-IL1β therapy alleviated reperfusion injury and flow stasis in NY1DD transgenic sickle mice exposed to hypoxia/reoxygenation (ASH Abstract (2011) 118(21): 848). The aim of this study was to determine whether antibodies that neutralize IL-1β and IL-18 could individually, or synergistically, diminish inflammatory processes and leukocyte recruitment in mice with SCD. Townes mice (5-months old; N=4-7 per group) received an i.p. administration of either saline, 200 µg/mouse anti-murine IL-1β (01BSUR), and/or 250 or 500 µg/mouse anti-murine IL-18 (SK113AE-4), or an IgG1 control antibody (iProt105125; 200 or 500 µg/mouse). At 21h after treatments, vaso-occlusive-like processes were induced in mice by the injection of tumor necrosis factor-α (TNF; 0.5μg, i.p.). At 3h after TNF, the cremaster muscles of anesthetized mice were surgically exposed, and leukocyte TNF recruitment and extravasation in venules of the microcirculation were observed using intravital microscopy. Another set of Townes mice (N=4-12 per group) was submitted to the same procedures, with blood sampling for ELISA at 3h post TNF. Optimal concentrations of antibodies were determined by observing leukocyte recruitment by intravital microscopy (doses; 100, 200 µg/mouse anti-IL-1β [N=2; 3, respectively] and 250, 500 µg/mouse anti-IL-18, [N=2 each]) in TNF-stimulated C57BL/6J mice (data not shown). Figure 1 (A-C) demonstrates the extensive recruitment and extravasation of leukocytes that occurs in the microvasculature of Townes mice at 3h post-TNF (saline group). Pre-treatment of mice with either anti-IL-1β or anti-IL-18 significantly abrogated (P<0.01) the TNF-induced adhesion (Fig. 1B) and extravasation (Fig. 1C) of leukocytes in venules, while only anti-IL-18 significantly reduced leukocyte rolling along the venule endothelium (Fig. 1A; P<0.05). In contrast, the administration of 500 µg/mouse (Fig. 1) or 200 µg/mouse (data not shown) of a non-specific IgG1 did not significantly affect TNF-induced leukocyte recruitment/extravasation (P>0.05). The combined use of the anti-IL-1β (200 µg/mouse) together with an intermediate dose of anti-IL-18 (250 µg/mouse) did not further reduce leukocyte recruitment and extravasation in this model, when compared with the effects of anti-IL-1β alone (Fig. 1A-C; P>0.05). Investigating the effects of these biological agents on inflammatory molecules production in TNF-stimulated Townes mice, we found that the administration of anti-IL-1β (200 µg) reduced IL-6 production, a pleiotropic inflammatory molecule that is upregulated by IL-1β (Fig. 1D), as did the combination of anti-IL-1β plus anti-IL-18 (200 µg and 250 µg/mouse, respectively). Pre-treatment of TNF-stimulated SCD mice with anti-IL-1β plus anti-IL-18 decreased Interferon (IFN)-γ production, a molecule that is upregulated synergistically by IL-18 and IL-12 and that mediates early host immune defenses (Fig. 1E). In contrast, anti-inflammatory IL-10 production was not significantly modulated by the pre-treatment of TNF-stimulated mice with these biological agents (Fig. 1F). As such, IL-1β and IL-18 neutralization significantly reduced inflammatory processes and leukocyte recruitment in the microvasculature of mice with SCD, indicating that biological agents that inhibit the effects of inflammasome-processed cytokines may hold potential for reducing vaso-occlusive processes in patients with this disease. Figure 1 Figure 1. Disclosures Kovarik: Novartis Institutes for Biomedical Research: Current Employment. Costa: Novartis: Consultancy. Conran: Novartis Pharma AG: Research Funding.

Leukocyte adhesion is governed by endolysosomal two pore channel 2 (TPC2)

In response to pro-inflammatory challenges including pathogenic attack and tissue damage, the endothelial cell surface is rearranged to present leukocyte-engaging cell surface receptors. The initial contact needed for leukocyte tethering and rolling is mediated via adhesion demand-driven exocytosis of Weibel-Palade bodies (WPB) that contain the leukocyte receptor P-selectin together with the stabilizing co-factor CD63. We found that diminished expression of the endolysosomal non-selective cation channel TPC2 or inhibition of TPC2-mediated Ca2+-release via trans-Ned 19 led to reduced endolysosomal Ca2+ efflux, and blocked transfer of CD63 from late endosomes/lysosomes (LEL) to WPB, and a concomitant loss of P-selectin on the endothelial cell surface. Accordingly, P-selectin-mediated leukocyte recruitment to trans-Ned 19-treated HUVEC under flow was significantly reduced without disturbing VWF exocytosis. Our findings establish the endolysosome-related TPC2 Ca2+ channel as a key element in the maintenance of proper endothelial functions and a potential pharmacological target in the control of inflammatory leukocyte recruitment.

The Absence of Extracellular Cold-Inducible RNA-Binding Protein (eCIRP) Promotes Pro-Angiogenic Microenvironmental Conditions and Angiogenesis in Muscle Tissue Ischemia

Extracellular Cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern, is released from cells upon hypoxia and cold-stress. The overall absence of extra- and intracellular CIRP is associated with increased angiogenesis, most likely induced through influencing leukocyte accumulation. The aim of the present study was to specifically characterize the role of eCIRP in ischemia-induced angiogenesis together with the associated leukocyte recruitment. For analyzing eCIRPs impact, we induced muscle ischemia via femoral artery ligation (FAL) in mice in the presence or absence of an anti-CIRP antibody and isolated the gastrocnemius muscle for immunohistological analyses. Upon eCIRP-depletion, mice showed increased capillary/muscle fiber ratio and numbers of proliferating endothelial cells (CD31+/CD45−/BrdU+). This was accompanied by a reduction of total leukocyte count (CD45+), neutrophils (MPO+), neutrophil extracellular traps (NETs) (MPO+CitH3+), apoptotic area (ascertained via TUNEL assay), and pro-inflammatory M1-like polarized macrophages (CD68+/MRC1−) in ischemic muscle tissue. Conversely, the number of regenerative M2-like polarized macrophages (CD68+/MRC1+) was elevated. Altogether, we observed that eCIRP depletion similarly affected angiogenesis and leukocyte recruitment as described for the overall absence of CIRP. Thus, we propose that eCIRP is mainly responsible for modulating angiogenesis via promoting pro-angiogenic microenvironmental conditions in muscle ischemia.

Activation of the Constitutive Androstane Receptor Inhibits Leukocyte Adhesiveness to Dysfunctional Endothelium

Leukocyte cell recruitment into the vascular subendothelium constitutes an early event in the atherogenic process. As the effect of the constitutive androstane receptor (CAR) on leukocyte recruitment and endothelial dysfunction is poorly understood, this study investigated whether the role of CAR activation can affect this response and the underlying mechanisms involved. Under physiological flow conditions, TNFα-induced endothelial adhesion of human leukocyte cells was concentration-dependently inhibited by preincubation of human umbilical arterial endothelial cells with the selective human CAR ligand CITCO. CAR agonism also prevented TNFα induced VCAM-1 expression, as well as MCP-1/CCL-2 and RANTES/CCL-5 release in endothelial cells. Suppression of CAR expression with a small interfering RNA abrogated the inhibitory effects of CITCO on these responses. Furthermore, CITCO increased interaction of CAR with Retinoid X Receptor (RXR) and reduced TNFα-induced p38-MAPK/NF-κB activation. In vivo, using intravital microscopy in the mouse cremasteric microcirculation treatment with the selective mouse CAR ligand TCPOBOP inhibited TNFα-induced leukocyte rolling flux, adhesion, and emigration and decreased VCAM-1 in endothelium. These results reveal that CAR agonists can inhibit the initial inflammatory response that precedes the atherogenic process by targeting different steps in the leukocyte recruitment cascade. Therefore, CAR agonists may constitute a new therapeutic tool in controlling cardiovascular disease-associated inflammatory processes.

The Janus-like role of neuraminidase isoenzymes in inflammation

The processes of activation, extravasation, and migration of immune cells to a site are early and essential steps in the induction of an acute inflammatory response. These events are part of the inflammatory cascade which involves multiple regulatory steps. Using a murine air-pouch model of inflammation with LPS as an inflammation inducer we demonstrate that isoenzymes of the neuraminidase family (NEU1, 3, and 4) play essential roles in this process acting as positive or negative regulators of leukocyte infiltration. Genetically knocked-out (KO) mice for different NEU genes (Neu1 KO, Neu3 KO, Neu4 KO, and Neu3/4 double KO mice) were induced with LPS, leukocytes at the site of inflammation were counted, and the inflamed tissue was analyzed using immunohistochemistry. Our data show that leukocyte recruitment was decreased in NEU1 and NEU3-deficient mice, while it was increased in NEU4-deficient animals. Consistent with these results, systemic levels of pro-inflammatory cytokines and those in pouch exudate were reduced in Neu1 and increased in Neu4 KO mice. We found that pharmacological inhibitors specific for NEU1, NEU3, and NEU4 isoforms also affected leukocyte recruitment. We conclude that NEU isoenzymes have distinct – and even opposing – effects on leukocyte recruitment, and therefore warrant further investigation to determine their mechanisms and importance as regulators of the inflammatory cascade.

Protease nexin-1 deficiency increases mouse hindlimb neovascularisation following ischemia and accelerates femoral artery perfusion

We previously identified the inhibitory serpin protease nexin-1 (PN-1) as an important player of the angiogenic balance with anti-angiogenic activity in physiological conditions. In the present study, we aimed to determine the role of PN-1 on pathological angiogenesis and particularly in response to ischemia, in the mouse model induced by femoral artery ligation. In wild-type (WT) muscle, we observed an upregulation of PN-1 mRNA and protein after ischemia. Angiography analysis showed that femoral artery perfusion was more rapidly restored in PN-1−/− mice than in WT mice. Moreover, immunohistochemistry showed that capillary density increased following ischemia to a greater extent in PN-1−/− than in WT muscles. Moreover, leukocyte recruitment and IL-6 and MCP-1 levels were also increased in PN-1−/− mice compared to WT after ischemia. This increase was accompanied by a higher overexpression of the growth factor midkine, known to promote leukocyte trafficking and to modulate expression of proinflammatory cytokines. Our results thus suggest that the higher expression of midkine observed in PN-1- deficient mice can increase leukocyte recruitment in response to higher levels of MCP-1, finally driving neoangiogenesis. Thus, PN-1 can limit neovascularisation in pathological conditions, including post-ischemic reperfusion of the lower limbs.

β2 integrin activation and signaling transduction in leukocyte recruitment

Leukocyte recruitment is a critical step in the pathogenesis of inflammatory and immunological responses. Cell adhesion molecules (CAMs) are involved in controlling cell movements and the recruitment process, and the integrin family of CAMs plays a key role. During cell movement, integrin function is dynamically and precisely regulated. However, this balance might be broken under pathological conditions. Thus, the functional regulation and molecular mechanisms of integrins related to diseases are often a focus of research. Integrin β2 is one of the most commonly expressed integrins in leukocytes that mediates leukocyte adhesion and migration, and it plays an important role in immune responses and inflammation. In this review, we focus on specific functions of integrin β2 in leukocyte recruitment, the conformational changes and signal transduction of integrin β2 activation, and how new insights into these processes can inform future therapies for inflammation and immune diseases.