Follicular B Cells Promote Atherosclerosis via T Cell-Mediated Differentiation Into Plasma Cells and Secreting Pathogenic Immunoglobulin G.

OBJECTIVE: B cells promote or protect development of atherosclerosis. In this study, we examined the role of MHCII (major histocompatibility II), CD40 (cluster of differentiation 40), and Blimp-1 (B-lymphocyte-induced maturation protein) expression by follicular B (FO B) cells in development of atherosclerosis together with the effects of IgG purified from atherosclerotic mice. APPROACH AND RESULTS: Using mixed chimeric Ldlr-/- mice whose B cells are deficient in MHCII or CD40, we demonstrate that these molecules are critical for the proatherogenic actions of FO B cells. During development of atherosclerosis, these deficiencies affected T-B cell interactions, germinal center B cells, plasma cells, and IgG. As FO B cells differentiating into plasma cells require Blimp-1, we also assessed its role in the development of atherosclerosis. Blimp-1-deficient B cells greatly attenuated atherosclerosis and immunoglobulin-including IgG production, preventing IgG accumulation in atherosclerotic lesions; Blimp-1 deletion also attenuated lesion proinflammatory cytokines, apoptotic cell numbers, and necrotic core. To determine the importance of IgG for atherosclerosis, we purified IgG from atherosclerotic mice. Their transfer but not IgG from nonatherosclerotic mice into Ldlr-/- mice whose B cells are Blimp-1-deficient increased atherosclerosis; transfer was associated with IgG accumulating in atherosclerotic lesions, increased lesion inflammatory cytokines, apoptotic cell numbers, and necrotic core size. CONCLUSIONS: The mechanism by which FO B cells promote atherosclerosis is highly dependent on their expression of MHCII, CD40, and Blimp-1. FO B cell differentiation into IgG-producing plasma cells also is critical for their proatherogenic actions. Targeting B-T cell interactions and pathogenic IgG may provide novel therapeutic strategies to prevent atherosclerosis and its adverse cardiovascular complications.

CD4+ natural killer T cells potently augment aortic root atherosclerosis by perforin- and granzyme B-dependent cytotoxicity.

RATIONALE: CD4(+) natural killer T (NKT) cells augment atherosclerosis in apolipoprotein E-deficient (ApoE)(-/-) mice but their mechanisms of action are unknown. OBJECTIVES: We investigated the roles of bystander T, B, and NK cells; NKT cell-derived interferon-γ, interleukin (IL)-4, and IL-21 cytokines; and NKT cell-derived perforin and granzyme B cytotoxins in promoting CD4(+) NKT cell atherogenicity. METHODS AND RESULTS: Transfer of CD4(+) NKT cells into T- and B-cell-deficient ApoE(-/-)Rag2(-/-) mice augmented aortic root atherosclerosis by ≈75% that was ≈30% of lesions in ApoE(-/-) mice; macrophage accumulation similarly increased. Transferred NKT cells were identified in the liver and atherosclerotic lesions of recipient mice. Transfer of CD4(+) NKT cells into T-, B-cell-deficient, and NK cell-deficient ApoE(-/-)Rag2(-/-)γC(-/-) mice also augmented atherosclerosis. These data indicate that CD4(+) NKT cells can exert proatherogenic effects independent of other lymphocytes. To investigate the role of NKT cell-derived interferon-γ, IL-4, and IL-21 cytokines and perforin and granzyme B cytotoxins, CD4(+) NKT cells from mice deficient in these molecules were transferred into NKT cell-deficient ApoE(-/-)Jα18(-/-) mice. CD4(+) NKT cells deficient in IL-4, interferon-γ, or IL-21 augmented atherosclerosis in ApoE(-/-)Jα18(-/-) mice by ≈95%, ≈80%, and ≈70%, respectively. Transfer of CD4(+) NKT cells deficient in perforin or granzyme B failed to augment atherosclerosis. Apoptotic cells, necrotic cores, and proinflammatory VCAM-1 (vascular cell adhesion molecule) and MCP-1 (monocyte chemotactic protein) were reduced in mice receiving perforin-deficient NKT cells. CD4(+) NKT cells are twice as potent as CD4(+) T cells in promoting atherosclerosis. CONCLUSIONS: CD4(+) NKT cells potently promote atherosclerosis by perforin and granzyme B-dependent apoptosis that increases postapoptotic necrosis and inflammation.

Cytotoxic and proinflammatory CD8+ T lymphocytes promote development of vulnerable atherosclerotic plaques in apoE-deficient mice.

BACKGROUND: Heart attacks and strokes, leading causes of deaths globally, arise from thrombotic occlusion of ruptured vulnerable atherosclerotic plaques characterized by abundant apoptosis, large necrotic cores derived from inefficient apoptotic cell clearance, thin fibrous caps, and focal inflammation. The genesis of apoptosis and necrotic cores in these vulnerable atherosclerotic plaques remains unknown. Cytotoxic CD8(+) T lymphocytes represent up to 50% of leukocytes in advanced human plaques and dominate early immune responses in mouse lesions, yet their role in atherosclerosis also remains unresolved. METHODS AND RESULTS: CD8(+) T-lymphocyte depletion by CD8α or CD8ß monoclonal antibody in apolipoprotein E-deficient mice fed a high-fat diet ameliorated atherosclerosis by reducing lipid and macrophage accumulation, apoptosis, necrotic cores, and monocyte chemoattractant protein 1, interleukin 1ß, interferon γ, and vascular cell adhesion molecule 1. Transfer of CD8(+) T cells into lymphocyte-deficient, apolipoprotein E-deficient mice partially reconstituted CD8(+) T cells in lymphoid compartments and was associated with CD8(+) T-cell infiltration in lesions, increased lipid and macrophage accumulation, apoptotic cells, necrotic cores, and interleukin 1ß in atherosclerotic lesions. Transfer of CD8(+) T cells deficient in perforin, granzyme B, or tumor necrosis factor α but not interferon γ failed to increase atherosclerotic lesions despite partial reconstitution in the lymphoid system and the presence in atherosclerotic lesions. Macrophages, smooth muscle cells, and endothelial cells were identified as apoptotic targets. CONCLUSIONS: We conclude that CD8(+) T lymphocytes promote the development of vulnerable atherosclerotic plaques by perforin- and granzyme B-mediated apoptosis of macrophages, smooth muscle cells, and endothelial cells that, in turn, leads to necrotic core formation and further augments inflammation by tumor necrosis factor α secretion.

Cytokine therapy with interleukin-2/anti-interleukin-2 monoclonal antibody complexes expands CD4+CD25+Foxp3+ regulatory T cells and attenuates development and progression of atherosclerosis.

BACKGROUND: CD4+CD25+Foxp3+ regulatory T cells (Tregs) attenuate atherosclerosis, but their therapeutic application by adoptive transfer is limited by the need for their expansion in vitro and limited purity. Recently, an interleukin (IL)-2/anti-IL-2 neutralizing monoclonal antibody (IL-2/anti-IL-2 mAb) complex has been shown to expand these Tregs. We examined the capacity of a modified IL-2/anti-IL-2 mAb treatment to expand Tregs and inhibit both the progression and development of developed atherosclerosis. METHODS AND RESULTS: Six-week old apolipoprotein E-deficient mice fed a high-fat diet for 8 weeks were administered IL-2/anti-IL-2 mAb commencing 2 weeks after starting the diet. Tregs in the spleen, lymph node, and liver were selectively expanded without affecting CD4+, CD8+, or natural killer cells. Tregs were increased in lesions and lesion size reduced. CD4+ T-cells, macrophages, mature dendritic cells, proliferating cell nuclear antigen+ cells, and monocyte chemoattractant protein-1 and vascular cell adhesion molecule-1 were reduced. In anti-CD3-stimulated splenocytes, proliferation and secretion of Th1, Th2, and Th17 (IL-17) cytokines and IL-1ß were reduced. To determine whether treatment attenuated progression of developed atherosclerosis, 6-week-old apolipoprotein E-deficient mice were fed a high-fat diet for 6 weeks, followed by IL-2/anti-IL-2 mAb treatment for 6 weeks while continuing the high-fat diet. Treatment also increased Tregs without affecting CD4+, CD8+, or natural killer cells, suppressed inflammation, and greatly attenuated progression of atherosclerosis. CONCLUSIONS: IL-2/anti-IL-2 mAb treatment in vivo attenuates atherosclerosis via selective Tregs expansion. The findings suggest that cytokine-based IL-2/anti-IL-2 mAb complex therapy could represent an attractive approach for treating atherosclerosis, because it markedly attenuates progression as well as development, by modulating its immunoinflammatory component.

Protease-activated receptor 2 promotes experimental liver fibrosis in mice and activates human hepatic stellate cells.

UNLABELLED: Protease-activated receptor (PAR) 2 is a G-protein-coupled receptor that is activated after proteolytic cleavage by serine proteases, including mast cell tryptase and activated coagulation factors. PAR-2 activation augments inflammatory and profibrotic pathways through the induction of genes encoding proinflammatory cytokines and extracellular matrix proteins. Thus, PAR-2 represents an important interface linking coagulation and inflammation. PAR-2 is widely expressed in cells of the gastrointestinal tract, including hepatic stellate cells (HSCs), endothelial cells, and hepatic macrophages; however, its role in liver fibrosis has not been previously examined. We studied the development of CCl(4) -induced liver fibrosis in PAR-2 knockout mice, and showed that PAR-2 deficiency reduced the progression of liver fibrosis, hepatic collagen gene expression, and hydroxyproline content. Reduced fibrosis was associated with decreased transforming growth factor beta (TGFß) gene and protein expression and decreased matrix metalloproteinase 2 and tissue inhibitor of matrix metalloproteinase 1 gene expression. In addition, PAR-2 stimulated activation, proliferation, collagen production, and TGFß protein production by human stellate cells, indicating that hepatic PAR-2 activation increases profibrogenic cytokines and collagen production both in vivo and in vitro. CONCLUSION: Our findings demonstrate the capacity of PAR-2 activation to augment TGFß production and promote hepatic fibrosis in mice and to induce a profibrogenic phenotype in human HSCs. PAR-2 antagonists have recently been developed and may represent a novel therapeutic approach in preventing fibrosis in patients with chronic liver disease.

B1a B lymphocytes are atheroprotective by secreting natural IgM that increases IgM deposits and reduces necrotic cores in atherosclerotic lesions.

RATIONALE: Aggravated atherosclerosis in B lymphocyte-deficient chimeric mice and reduced atherosclerosis after transfer of unfractionated spleen B lymphocytes into splenectomized mice have led to the widely held notion that B lymphocytes are atheroprotective. However, B lymphocytes can be pathogenic, because their depletion by anti-CD20 antibody ameliorated atherosclerosis, and transfer of B2 lymphocytes aggravated atherosclerosis. These observations raise the question of the identity of the atheroprotective B-lymphocyte population. OBJECTIVE: The purpose of the study was to identify an atheroprotective B-lymphocyte subset and mechanisms by which they confer atheroprotection. METHODS AND RESULTS: Splenectomy of apolipoprotein E-deficient mice selectively reduced peritoneal B1a lymphocytes, plasma IgM, and oxidized low-density lipoprotein IgM levels and lesion IgM deposits. These reductions were accompanied by increased oil red O-stained atherosclerotic lesions and increased necrotic cores, oxidized low-density lipoproteins, and apoptotic cells in lesions. Plasma lipids, body weight, collagen, and smooth muscle content were unaffected. Transfer of B1a lymphocytes into splenectomized mice increased peritoneal B1a lymphocytes; restored plasma IgM, oxidized low-density lipoprotein IgM levels, and lesion IgM deposits; and potently attenuated atherosclerotic lesions, with reduced lesion necrotic cores, oxidized low-density lipoprotein, and apoptotic cells. In contrast, transfer of B1a lymphocytes that cannot secrete IgM failed to protect against atherosclerosis development in splenectomized mice despite reconstitution in the peritoneum. CONCLUSIONS: B1a lymphocytes are an atheroprotective B-lymphocyte population. Our data suggest that natural IgM secreted by these lymphocytes offers protection by depositing IgM in atherosclerotic lesions, which reduces the necrotic cores of lesions.

Conventional B2 B cell depletion ameliorates whereas its adoptive transfer aggravates atherosclerosis.

Atherosclerosis is a chronic inflammatory arterial disease characterized by focal accumulation of lipid and inflammatory cells. It is the number one cause of deaths in the Western world because of its complications of heart attacks and strokes. Statins are effective in only approximately one third of patients, underscoring the urgent need for additional therapies. B cells that accumulate in atherosclerotic lesions and the aortic adventitia of humans and mice are considered to protect against atherosclerosis development. Unexpectedly, we found that selective B cell depletion in apolipoprotein E-deficient (ApoE(-/-)) mice using a well-characterized mAb to mouse CD20 reduced atherosclerosis development and progression without affecting the hyperlipidemia imposed by a high-fat diet. Adoptive transfer of 5 × 10(6) or 5 × 10(7) conventional B2 B cells but not 5 × 10(6) B1 B cells to a lymphocyte-deficient ApoE(-/-) Rag-2(-/-) common cytokine receptor γ-chain-deficient mouse that was fed a high-fat diet augmented atherosclerosis by 72%. Transfer of 5 × 10(6) B2 B cells to an ApoE(-/-) mouse deficient only in B cells aggravated atherosclerosis by >300%. Our findings provide compelling evidence for the hitherto unrecognized proatherogenic role of conventional B2 cells. The data indicate that B2 cells can potently promote atherosclerosis development entirely on their own in the total absence of all other lymphocyte populations. Additionally, these B2 cells can also significantly augment atherosclerosis development in the presence of T cells and all other lymphocyte populations. Our findings raise the prospect of B cell depletion as a therapeutic approach to inhibit atherosclerosis development and progression in humans.

Current understanding of the role of B cell subsets and intimal and adventitial B cells in atherosclerosis.

PURPOSE OF REVIEW: Inflammation, in addition to high cholesterol is a major factor contributing to atherosclerosis-associated adverse cardiovascular events. Thus, there is a pressing need for additional therapeutic strategies to reduce inflammation, by targeting immune cells and cytokines. Here we review B cell subsets and adventitial and intimal B cells in atherosclerosis development and discuss potential B cell-targeted anti-inflammatory therapies for atherosclerosis. RECENT FINDINGS: B cell subsets can have opposing proatherogenic and atheroprotective roles in atherosclerosis. CD-20-targeted B cell depletion has been shown to decrease murine atherosclerotic lesions. The accumulation of intimal and adventitial B cells associated with atherosclerotic lesions is consistent with their participation in local inflammatory responses. As B2 B cells are proatherogenic, blocking its survival factor B cell activating factor may selectively delete this proatherogenic subset. SUMMARY: Both intimal and adventitial B cells appear important in atherosclerosis. B2 B cells are proatherogenic and other subsets such as regulatory B cells are antiatherogenic. Future B cell-targeted therapy for atherosclerosis should be customized to selectively deplete damaging B2 B cells while sparing or expanding protective B cell subsets.

The cytoplasmic domain of tissue factor restricts physiological albuminuria and pathological proteinuria associated with glomerulonephritis in mice.

BACKGROUND/AIMS: Tissue factor (TF) is a transmembrane protein that is essential for coagulation. TF is expressed on podocytes and its cytoplasmic domain has cell signalling functions in epithelial cells. METHODS: Mice lacking the cytoplasmic domain of TF (TF(CT-/-) mice) were used to study its role in physiological albuminuria and pathological proteinuria following induction of glomerulonephritis (GN). RESULTS: Absence of the cytoplasmic domain of TF was associated with increased albuminuria, podocyte effacement, reduced podocyte numbers and increased spontaneous glomerular tumour necrosis factor α(TNFα) production under physiological conditions. In mice developing GN, absence of the cytoplasmic domain of TF resulted in increased proteinuria and enhanced renal TNFα production without altering other parameters of renal inflammation and injury. Studies in TF(CT-/-) chimeric mice (created by bone marrow transplantation) showed increased proteinuria and renal TNFα mRNA in GN was associated with absence of the cytoplasmic domain of TF in the kidney and was independent of the leucocyte phenotype. CONCLUSION: These studies demonstrate that the cytoplasmic domain of TF contributes to renal albumin retention and its renal expression protects against proteinuria in leucocyte-mediated renal inflammation. Increased glomerular production of TNFα in the absence of cytoplasmic domain of TF may contribute to podocyte injury resulting in albuminuria and proteinuria.

The cytoplasmic domain of tissue factor in macrophages augments cutaneous delayed-type hypersensitivity.

In addition to its procoagulant role, tissue factor (TF) has important coagulation-independent roles, including in inflammation. The cytoplasmic domain of TF has been implicated in some of these coagulation-independent roles, particularly cell signaling. To assess the contribution of the cytoplasmic domain of TF to cell-mediated adaptive immunity, the development of cutaneous delayed-type hypersensitivity (DTH) was studied in mice lacking the cytoplasmic domain of TF (TF(deltaCT/deltaCT) mice). DTH responses in sensitized mice were significantly attenuated in TF(deltaCT/deltaCT) mice, and leukocyte-endothelial cell interactions, assessed by intravital microscopy, were impaired significantly. Studies in chimeric mice, created by bone marrow transplantation, showed that the absence of the cytoplasmic domain of TF in leukocytes rather than endothelial cells was responsible for reduced DTH and leukocyte recruitment. DTH responses to OVA could be induced in wild-type mice but not in TF(deltaCT/deltaCT) mice by transfer of activated CD4(+) OVA-specific TCR transgenic T cells, demonstrating that the defective DTH response in TF(deltaCT/deltaCT) mice was independent of any defect in T cell activation. Macrophage and neutrophil accumulation and expression of TNF-alpha mRNA and phospho-p38-MAPK were reduced significantly in TF(deltaCT/deltaCT) mice, and their macrophages had reduced P-selectin-binding capacity and reduced in vivo emigration in response to MCP-1. These results indicate that leukocyte expression of the cytoplasmic domain of TF contributes to antigen-specific cellular adaptive immune responses via effects on leukocyte recruitment and activation.

Cytokines in glomerulonephritis.

Cytokines play central roles in both innate and adaptive immune responses that lead to renal inflammation. They are involved systemically in cross-talk between antigen-presenting cells, leukocytes, and regulatory cells to initiate and modulate nephritogenic immunity. Within the kidney, cytokines play a central role in signaling between infiltrating leukocytes and intrinsic renal cells and orchestrate the effector responses that lead to renal damage. Glomerulonephritis (GN) is an important cause of renal inflammation leading to renal failure that results from adaptive responses targeted at the kidney. Animal models of GN have shown that cytokines play critical roles in initiation and modulation of renal inflammatory responses through their ability to modulate the T helper 1/T helper 2 balance of nephritogenic immune responses. Evidence from clinical studies is now confirming the importance of this paradigm in directing the inflammatory mechanisms, histologic patterns, and clinical consequences of human GN. Cytokines also have critical intrarenal effector roles in the development, perpetuation, and resolution of GN. The proinflammatory role of intrarenal cytokine production by leukocytes in GN is well recognized, but, more recently, the role of intrinsic renal cell cytokine production in amplifying renal inflammation has been shown in animal models of GN. Studies showing benefits of specific anticytokine therapies directed at tumor necrosis factor in human GN are now appearing.

Killer cells in atherosclerosis.

Cytotoxic lymphocytes (killer cells) play a critical role in host defence mechanisms, protecting against infections and in tumour surveillance. They can also exert detrimental effects in chronic inflammatory disorders and in autoimmune diseases. Tissue cell death and necrosis are prominent features of advanced atherosclerotic lesions including vulnerable/unstable lesions which are largely responsible for most heart attacks and strokes. Evidence for accumulation of killer cells in both human and mouse lesions together with their cytotoxic potential strongly suggest that these cells contribute to cell death and necrosis in lesions leading to vulnerable plaque development and potentially plaque rupture. Killer cells can be divided into two groups, adaptive and innate immune cells depending on whether they require antigen presentation for activation. Activated killer cells detect damaged or stressed cells and kill by cytotoxic mechanisms that include perforin, granzymes, TRAIL or FasL and in some cases TNF-α. In this review, we examine current knowledge on killer cells in atherosclerosis, including CD8 T cells, CD28- CD4 T cells, natural killer cells and γδ-T cells, mechanisms responsible for their activation, their migration to developing lesions and effector functions. We also discuss pharmacological strategies to prevent their deleterious vascular effects by preventing/limiting their cytotoxic effects within atherosclerotic lesions as well as potential immunomodulatory therapies that might better target lesion-resident killer cells, to minimise any compromise of the immune system, which could result in increased susceptibility to infections and reductions in tumour surveillance.

Opposing roles of B lymphocyte subsets in atherosclerosis.

Atherosclerosis is initiated by cholesterol entry into arteries that triggers chronic immune-inflammatory lesions in the vessels. Early lesions are clinically insignificant but advanced complex lesions and vulnerable rupture prone lesions impact on quality of life and can be life threatening. Rupture of vulnerable atherosclerotic lesions initiates thrombotic occlusion of vital arteries precipitating heart attacks and strokes that remain major killers globally despite therapeutic use of statins to lower blood cholesterol levels. Conventional B2 cells are proatherogenic whereas peritoneal Bla cells are atheroprotective. Depletion of B2 cells by administration of mAb to CD20 or to BAFF receptor or in BAFF receptor-deficient mice ameliorates atherosclerosis. B2 cells may promote atherosclerosis by production of IgG, secretion of proinflammatory cytokine TNFα and activation of CD4 T cells. Together these B2 cell mechanisms contribute to generation of rupture-prone vulnerable atherosclerotic plaques characterised by large necrotic cores. In contrast, peritoneal Bla cells protect against atherosclerosis by secretion of natural IgM that scavenges apoptotic cells and oxidised LDL and reduces necrotic cores in atherosclerotic lesions. These atheroprotective effects can be further increased by stimulating Bla cells by administration of apoptotic cells, liposomes of phosphatidylserine abundant on surfaces of apoptotic cell, by mAb to TIM1, a phosphatidylserine receptor expressed by B1a cells and by TLR4-MyD88 activation. Experimental studies of atherosclerosis in mouse models indicate that reductions in atherogenic B2 cells and/or activation of atheroprotective B1a cells protects against atherosclerosis development, findings which have potential for clinical translation to reduce risks of deaths from heart attacks and strokes.

Cytoplasmic domain of tissue factor promotes liver fibrosis in mice.

AIM: To evaluate the role of tissue factor (TF) and protease activated receptor (PAR)-2 in liver fibrosis. METHODS: Using CCl4 administration for eight weeks, we induced hepatic fibrosis in wild-type C57BL/6 mice and in mice with deletion of the cytoplasmic signalling domain of TF (TF§CT/§CT), deletion of PAR-2 (PAR-2-/-) and combined deletion of TF signalling domain and PAR-2 (TF§CT/§CT/PAR-2-/-). Hepatic fibrosis area was assessed by quantitative imaging of picrosirius red staining. Hepatic collagen content was assessed by hydroxyproline levels. Hepatic stellate cells (αSMA positive) and hepatic macrophages (CD68 positive) were identified by immunohistochemistry. Hepatic gene expression was determined by PCR and liver TGFß1 content by ELISA. RESULTS: CCl4 treated mice with deletion of the PAR-2 gene (PAR-2-/-) and the cytoplasmic domain of TF (TF§CT/§CT) developed significantly less hepatic fibrosis, characterised by reduced liver fibrosis area and hydroxyproline content, compared to control wildtype mice treated with CCl4. The observed reduction in histological fibrosis was accompanied by a significant decrease in the hepatic content of TGFß, the prototypic fibrogenic cytokine, as well as fewer activated hepatic stellate cells and hepatic macrophages. Deletion of the TF cytoplasmic signalling domain reduced hepatic fibrosis to levels similar to those observed in mice lacking PAR-2 signalling but combined deletion provided no added protection against fibrosis indicating a lack of mutual modulating effects that have been observed in other contexts such as angiogenic responses. CONCLUSION: Tissue factor cytoplasmic domain is involved in TF-PAR-2 signalling initiating hepatic fibrosis and is a potential therapeutic target, as its deletion would not impact coagulation.

Cytotoxic lymphocytes and atherosclerosis: significance, mechanisms and therapeutic challenges.

Cytotoxic lymphocytes encompass natural killer lymphocytes (cells) and cytotoxic T cells that include CD8+ T cells, natural killer (NK) T cells, γ, δ (γδ)-T cells and human CD4 + CD28- T cells. These cells play critical roles in inflammatory diseases and in controlling cancers and infections. Cytotoxic lymphocytes can be activated via a number of mechanisms that may involve dendritic cells, macrophages, cytokines or surface proteins on stressed cells. Upon activation, they secrete pro-inflammatory cytokines as well as anti-inflammatory cytokines, chemokines and cytotoxins to promote inflammation and the development of atherosclerotic lesions including vulnerable lesions, which are strongly implicated in myocardial infarctions and strokes. Here, we review the mechanisms that activate and regulate cytotoxic lymphocyte activity, including activating and inhibitory receptors, cytokines, chemokine receptors-chemokine systems utilized to home to inflamed lesions and cytotoxins and cytokines through which they affect other cells within lesions. We also examine their roles in human and mouse models of atherosclerosis and the mechanisms by which they exert their pathogenic effects. Finally, we discuss strategies for therapeutically targeting these cells to prevent the development of atherosclerotic lesions and vulnerable plaques and the challenge of developing highly targeted therapies that only minimally affect the body's immune system, avoiding the complications, such as increased susceptibility to infections, which are currently associated with many immunotherapies for autoimmune diseases. LINKED ARTICLES: This article is part of a themed section on Targeting Inflammation to Reduce Cardiovascular Disease Risk. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.22/issuetoc and http://onlinelibrary.wiley.com/doi/10.1111/bcp.v82.4/issuetoc.

B-cell-specific depletion of tumour necrosis factor alpha inhibits atherosclerosis development and plaque vulnerability to rupture by reducing cell death and inflammation.

AIMS: B2 lymphocytes promote atherosclerosis development but their mechanisms of action are unknown. Here, we investigated the role of tumour necrosis factor alpha (TNF-α) produced by B2 cells in atherogenesis. METHODS AND RESULTS: We found that 50% of TNF-α-producing spleen lymphocytes were B2 cells and ∼20% of spleen and aortic B cells produced TNF-α in hyperlipidemic ApoE(-/-) mice. We generated mixed bone marrow (80% µMT/20% TNF-α(-/-)) chimeric LDLR(-/-) mice where only B cells did not express TNF-α. Atherosclerosis was reduced in chimeric LDLR(-/-) mice with TNF-α-deficient B cells. TNF-α expression in atherosclerotic lesions and in macrophages were also reduced accompanied by fewer apoptotic cells, reduced necrotic cores, and reduced lesion Fas, interleukin-1ß and MCP-1 in mice with TNF-α-deficient B cells compared to mice with TNF-α-sufficient B cells. To confirm that the reduced atherosclerosis is attributable to B2 cells, we transferred wild-type and TNF-α-deficient B2 cells into ApoE(-/-) mice deficient in B cells or in lymphocytes. After 8 weeks of high fat diet, we found that atherosclerosis was increased by wild-type but not TNF-α-deficient B2 cells. Lesions of mice with wild-type B2 cells but not TNF-α-deficient B2 cells also had increased apoptotic cells and necrotic cores. Transferred B2 cells were found in lesions of recipient mice, suggesting that TNF-α-producing B2 cells promote atherosclerosis within lesions. CONCLUSION: We conclude that TNF-α produced by B2 cells is a key mechanism by which B2 cells promote atherogenesis through augmenting macrophage TNF-α production to induce cell death and inflammation that promote plaque vulnerability.

Toll-Like Receptor (TLR)4 and MyD88 are Essential for Atheroprotection by Peritoneal B1a B Cells.

BACKGROUND: We previously identified peritoneal B1a cells that secrete natural IgM as a key atheroprotective B cell subset. However, the molecules that activate atheroprotective B1a cells are unknown. Here, we investigated whether Toll-like receptors (TLRs) TLR2, TLR4, and TLR9 expressed by B1a cells are required for IgM-mediated atheroprotection. METHODS AND RESULTS: We adoptively transferred B1a cells from wild-type mice or from mice deficient in TLR2, TLR4, TLR9, or myeloid differentiation primary response 88 (MyD88) into ApoE-/- mice depleted of peritoneal B1a cells by splenectomy and fed a high-fat diet for 8 weeks. Elevations in plasma total, anti-oxLDL (oxidized low-density lipoprotein), anti-leukocyte, anti-CD3, anti-CD8, and anti-CD4 IgMs in atherosclerotic mice required B1a cells expressing TLR4 and MyD88, indicating a critical role for TLR4-MyD88 signaling for IgM secretion. Suppression of atherosclerosis was also critically dependent on B1a cells expressing TLR4-MyD88. Atherosclerosis suppression was associated not only with reductions in lesion apoptotic cells, necrotic cores, and oxLDL, but also with reduced lesion CD4+ and CD8+ T cells. Transforming growth factor beta 1 (TGF-ß1) expression, including macrophages expressing TGF-ß1, was increased, consistent with increased IgM-mediated phagocytosis of apoptotic cells by macrophages. Reductions in lesion inflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin (IL) 1ß, and IL-18 were consistent with augmented TGF-ß1 expression. CONCLUSIONS: TLR4-MyD88 expression on B1a cells is critical for their IgM-dependent atheroprotection that not only reduced lesion apoptotic cells and necrotic cores, but also decreased CD4 and CD8 T-cell infiltrates and augmented TGF-ß1 expression accompanied by reduced lesion inflammatory cytokines TNF-α, IL-1ß, and IL-18.

A CD1d-dependent lipid antagonist to NKT cells ameliorates atherosclerosis in ApoE-/- mice by reducing lesion necrosis and inflammation.

AIMS: Atherosclerosis-related deaths from heart attacks and strokes remain leading causes of global mortality, despite the use of lipid-lowering statins. Thus, there is an urgent need to develop additional therapies. METHODS AND RESULTS: Reports that NKT cells promote atherosclerosis and an NKT cell CD1d-dependent lipid antagonist (DPPE-PEG350, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N[methoxy(polyethyleneglycol)-350]) reduces allergen-induced inflammation led us to investigate its therapeutic potential in preventing the development and progression of experimental atherosclerosis. DPPE-PEG350 was administered to hyperlipidaemic ApoE(-/-) mice with/without established atherosclerosis. Atherosclerosis and immune cells were assessed in the aortic sinus lesions. Lesion expression of monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion protein-1 (VCAM-1) responsible for inflammatory immune cell recruitment as well as mRNA expression of IFNγ and its plasma levels were investigated. Necrotic cores and lesion smooth muscle and collagen contents important in plaque stability were determined as were plasma lipid levels. DPPE-PEG350 reduced atherosclerosis development and delayed progression of established atherosclerosis without affecting plasma lipids. CD4 and CD8 T cells and B cells in atherosclerotic lesions were decreased in DPPE-PEG350-treated mice. Lesion MCP-1 and VCAM-1 protein expression and necrotic core size were reduced without affecting lesion smooth muscle and collagen content. IFNγ and lymphocytes were unaffected by the treatment. CONCLUSION: The attenuation of progression of established atherosclerosis together with reduced development of atherosclerosis in hyperlipidaemic mice by the NKT antagonist, without affecting NKT cell or other lymphocyte numbers, suggests that targeting lesion inflammation via CD1d-dependent activation of NKT cells using DPPE-PEG350 has a therapeutic potential in treating atherosclerosis.

Resident kidney cells and their involvement in glomerulonephritis.

Each year, worldwide, there is an increasing number of patients with chronic kidney disease that progress to end-stage renal disease. Glomerulonephritis (GN) is the commonest single cause of end-stage renal failure in the world. GN can be a manifestation of primary renal injury or may be a secondary feature of a systemic disease process, for example Systemic Lupus Erythematosus (SLE) and Anti-Neutrophilic Cytoplasmic Antibody (ANCA) associated vasculitis. Understanding of the immunopathogenesis of GN has advanced considerably over the last 25 years, particularly the immune system's role. The injurious role of infiltrating leukocytes and humoral mediators has been emphasised, however, the contribution of intrinsic renal cells has proved difficult to define. Most evidence for the pro-inflammatory capacity of intrinsic renal cells has been derived from in vitro studies. Although cytokine production by intrinsic renal cells has been demonstrated by immunohistochemistry and in situ hybridisation studies in renal tissue during the development of GN, the functional contribution of this cytokine production to renal injury was unknown. Little was known about direct and specific interactions between different glomerular cell types and infiltrating leukocytes in the pathogenesis of GN. The development of mice with genetic deficiencies of pro-inflammatory mediators and cytokines, and the technique of bone marrow transplantation into irradiated recipients to produce chimeric mice with restricted cytokine expression has allowed in vivo assessment of the functional contribution made by intrinsic renal cells. Studies have demonstrated the significant contribution of intrinsic renal cell derived cytokines (e.g. TNF) in mediating GN, whereas others (IL-1beta) have a relatively minor role.

Lack of plasminogen activator inhibitor-1 promotes growth and abnormal matrix remodeling of advanced atherosclerotic plaques in apolipoprotein E-deficient mice.

Epidemiological studies suggest that elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) predispose an individual to ischemic heart disease or promote plaque progression by inhibiting fibrinolysis. In the present study, loss of PAI-1 in apolipoprotein E (apoE)-deficient (apoE(-/-):PAI-1(-/-)) mice promoted the growth of advanced atherosclerotic plaques, which was due to enhanced extracellular matrix deposition. ApoE(-/-):PAI-1(-/-) plaques also exhibited collagen fiber disorganization and degradation. Immunostaining and bone marrow transplantation revealed that smooth muscle cells, not macrophages, primarily expressed PAI-1 in plaques. Thus, although PAI-1 may promote plaque growth because of its antifibrinolytic properties, the present study reveals a protective role for PAI-1 by limiting plaque growth and preventing abnormal matrix remodeling.