Peter G. Tipping

The Role of Interleukin-4 and Interleukin-12 in the Progression of Atherosclerosis in Apolipoprotein E-Deficient Mice

Accumulation of T cells and macrophages in atherosclerotic plaques and the formation of antibodies directed against plaque proteins suggests that adaptive immunity contributes to the development of atherosclerosis. The contribution of Th1 and Th2 helper cell subsets to atherogenesis was studied in a murine model by interbreeding apolipoprotein E-deficient (apoE(-/-)) mice with mice deficient in key cytokines that drive either Th1 responses [interleukin (IL)-12] or Th2 responses (IL-4). Compared to apoE(-/-) mice, apoE(-/-)/IL-12(-/-) mice had a 52% reduction in plaque area in the aortic root at 30 weeks of age (P < 0.001). ApoE(-/-)/IL-4(-/-) mice had a 27% reduction in plaque area compared to apoE(-/-) mice (P < 0.05) at 30 weeks of age, but their plaques were significantly larger than in apoE(-/-)/IL-12(-/-) mice at this stage (P < 0.05). By 45 weeks of age, there were no significant differences in lesion sizes in the aortic root between the strains, however apoE(-/-)/IL-4(-/-) mice showed a 58% and 64% decrease in disease in their aortic arch compared to apoE(-/-) (P < 0.05) and apoE(-/-)/IL-12(-/-) (P < 0.05) mice, respectively, and a 78% decrease in thoracic lesions compared to apoE(-/-)/IL-12(-/-) (P < 0.05). This suggests that both Th1 and Th2 cytokines play roles throughout the development of atherosclerosis in various vascular sites in apoE(-/-) mice.

A phase II randomized, controlled trial of continuous hemofiltration in sepsis.

Objective To study the effect of early and continuous venovenous hemofiltration (CVVH) on the plasma concentrations of several humoral mediators of inflammation and subsequent organ dysfunction in septic patients. Design Randomized, controlled trial. Setting Intensive care unit of a tertiary hospital. Patients Twenty-four patients with early septic shock or septic organ dysfunction. Interventions Random allocation to receive 48 hrs of isovolemic CVVH at 2 L/hr of fluid exchange or no hemofiltration. Measurements and Main Results We measured the plasma concentrations of complement fractions C3a and C5a, interleukins 6, 8, and 10, and tumor necrosis factor &agr; at baseline and 2, 24, 26, 48, and 72 hrs. A multiple organ dysfunction score (MODS) was calculated daily for each patient until death or discharge from the intensive care unit. The concentrations of most mediators decreased between baseline and 72 hrs. Some significant falls in concentration could be identified between specific time points, but CVVH was not associated with an overall reduction in any plasma cytokine concentrations. There was also no difference between the mean cumulative MODS for control survivors (43.3 ± 19.7) and CVVH survivors (33.2 ± 19.0;p = .30), and no difference between the average MODS calculated for all controls (4.1 ± 1.9) and all CVVH subjects (3.3 ± 1.7;p = .26). CVVH did not improve oxygenation, lower the platelet count, or reduce the duration of vasopressor support and mechanical ventilation. Conclusions Early use of CVVH at 2 L/hr did not reduce the circulating concentrations of several cytokines and anaphylatoxins associated with septic shock, or the organ dysfunction that followed severe sepsis. CVVH using current technology cannot be recommended as an adjunct to the treatment of septic shock unless severe acute renal failure is present.

Conditional Ablation of Macrophages Halts Progression of Crescentic Glomerulonephritis

The presence of macrophages in inflamed glomeruli of rat kidney correlates with proliferation and apoptosis of resident glomerular mesangial cells. We assessed the contribution of inflammatory macrophages to progressive renal injury in murine crescentic glomerulonephritis (GN). Using a novel transgenic mouse (CD11b-DTR) in which tissue macrophages can be specifically and selectively ablated by minute injections of diphtheria toxin, we depleted renal inflammatory macrophages through days 15 and 20 of progressive crescentic GN. Macrophage depletion reduced the number of glomerular crescents, improved renal function, and reduced proteinuria. Morphometric analysis of renal tubules and interstitium revealed a marked attenuation of tubular injury that was associated with reduced proliferation and apoptosis of tubular cells. The population of interstitial myofibroblasts decreased after macrophage depletion and interstitial fibrosis also decreased. In the presence of macrophages, interstitial myofibroblasts exhibited increased levels of both proliferation and apoptosis, suggesting that macrophages act to support a population of renal myofibroblasts in a high turnover state and in matrix deposition. Finally, deletion of macrophages reduced CD4 T cells in the diseased kidney. This study demonstrates that macrophages are key effectors of disease progression in crescentic GN, acting to regulate parenchymal cell populations by modulating both cell proliferation and apoptosis.

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 × 106 or 5 × 107 conventional B2 B cells but not 5 × 106 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 × 106 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.

Interleukin-8 Production by Macrophages From Atheromatous Plaques

Interleukin-8 (IL-8) is a chemotactic peptide produced by macrophages that may be involved in the recruitment of inflammatory cells into atherosclerotic plaques. In vitro, IL-8 production by macrophages isolated from carotid plaques (1240 +/- 510 pg.10(5) cells-1.24h-1, mean +/- SEM, n = 6) and noncarotid plaques (4312 +/- 1588 pg.10(5) cells-1.24 h-1, n = 9) was significantly greater than IL-8 production by blood monocytes isolated from the same patients (526 +/- 278 pg.10(5) cells-1.24 h-1, n = 6, P < .05 and 726 +/- 384 pg.10(5) cells-1.24 h-1, n = 9, P < .01, respectively). IL-8 produced by atherosclerotic macrophages was demonstrated to be biologically active in a neutrophil chemotaxis assay. IL-8 mRNA was detectable in plaque macrophages and blood monocytes from these patients, but blood monocytes from normal donors did not exhibit detectable IL-8 mRNA. IL-8 mRNA was localized in macrophage-rich areas of atherosclerotic plaques by in situ hybridization. These studies demonstrate that macrophages from atherosclerotic plaques show an enhanced capacity to produce IL-8 compared with normal and patient blood monocytes and that macrophages are a major site of IL-8 mRNA production in atherosclerotic plaques. These results provide further evidence for a proinflammatory role for macrophages in atherosclerosis.

Depletion of B2 but Not B1a B Cells in BAFF Receptor-Deficient ApoE−/− Mice Attenuates Atherosclerosis by Potently Ameliorating Arterial Inflammation

We have recently identified conventional B2 cells as atherogenic and B1a cells as atheroprotective in hypercholesterolemic ApoE−/− mice. Here, we examined the development of atherosclerosis in BAFF-R deficient ApoE−/− mice because B2 cells but not B1a cells are selectively depleted in BAFF-R deficient mice. We fed BAFF-R−/− ApoE−/− (BaffR.ApoE DKO) and BAFF-R+/+ApoE−/− (ApoE KO) mice a high fat diet (HFD) for 8-weeks. B2 cells were significantly reduced by 82%, 81%, 94%, 72% in blood, peritoneal fluid, spleen and peripheral lymph nodes respectively; while B1a cells and non-B lymphocytes were unaffected. Aortic atherosclerotic lesions assessed by oil red-O stained-lipid accumulation and CD68+ macrophage accumulation were decreased by 44% and 50% respectively. B cells were absent in atherosclerotic lesions of BaffR.ApoE DKO mice as were IgG1 and IgG2a immunoglobulins produced by B2 cells, despite low but measurable numbers of B2 cells and IgG1 and IgG2a immunoglobulin concentrations in plasma. Plasma IgM and IgM deposits in atherosclerotic lesions were also reduced. BAFF-R deficiency in ApoE−/− mice was also associated with a reduced expression of VCAM-1 and fewer macrophages, dendritic cells, CD4+ and CD8+ T cell infiltrates and PCNA+ cells in lesions. The expression of proinflammatory cytokines, TNF-α, IL1-β and proinflammatory chemokine MCP-1 was also reduced. Body weight and plasma cholesterols were unaffected in BaffR.ApoE DKO mice. Our data indicate that B2 cells are important contributors to the development of atherosclerosis and that targeting the BAFF-R to specifically reduce atherogenic B2 cell numbers while preserving atheroprotective B1a cell numbers may be a potential therapeutic strategy to reduce atherosclerosis by potently reducing arterial inflammation.

Mechanisms of T cell‐induced glomerular injury in anti‐glomeruler basement membrane (GBM) glomerulonephritis in rats

The effector mechanisms of T cell‐dependent acute glomerular injury were studied in autologous phase anti‐GBM glomerulonephritis (GN) in rats. Acute proliferative GN was induced in sensitized rats by a subnephritogenic dose of sheep anti‐rat GBM antibody. Injury was manifested by proteinuria and glomerular leucocyte infiltration composed predominantly of macrophages but also CD4+ and CD8+ T cells. T cell depletion, using an anti‐CD5 MoAb, demonstrated that glomerular leucocyte infiltration and proteinuria were T cell‐dependent. Inhibition of T helper cell function using an anti‐CD4 MoAb prevented proteinuria and glomerular macrophage and CD4+ T cell influx, but not accumulation of CD8+ T cells. Depletion of CD8+ T cells also prevented proteinuria and the influx of macrophages and CD8+ T cells, but not accumulation of CD4+ T cells. Macrophage depletion, using micro‐encapsulated clodronate, prevented proteinuria and glomerular macrophage infiltration, but not the accumulation of CD4+ or CD8+ T cells, indicating that macrophages are the common cellular effectors for both CD4 and CD8 T cell‐dependent injury. Evidence for cytotoxic mechanisms of injury (increased numbers of apoptotic cells or accumulation of natural killer (NK) cells in glomeruli) could not be demonstrated. These data suggest that acute glomerular injury in anti‐GBM GN is the result of macrophage recruitment, which is dependent on both CD4 and CD8 T cells, and that direct T cell‐mediated injury (cellular cytotoxicity) is not involved.

Intrinsic Renal Cells Are the Major Source of Tumor Necrosis Factor Contributing to Renal Injury in Murine Crescentic Glomerulonephritis

Macrophages are prominent participants in crescentic glomerulonephritis (GN) and have been suggested to be the major source of TNF in this cell-mediated form of glomerular inflammation. Intrinsic renal cells also have the capacity to produce TNF. For dissecting the contribution of local versus bone marrow (BM)-derived TNF in inflammatory renal injury, TNF chimeric mice were created by transplanting normal wild-type (WT) BM into irradiated TNF-deficient recipients (WT-->TNF-/- chimeras) and vice versa (TNF-/- -->WT chimeras). A model of crescentic GN induced by an intravenous injection of sheep anti-murine glomerular basement membrane antibody was studied in WT mice, mice with complete TNF deficiency (TNF-/-), and chimeric mice. Crescentic GN was attenuated in TNF-/- mice with fewer crescents (crescents, 13.7 +/- 1.7% of glomeruli) and reduced functional indices of renal injury (serum creatinine, 15.2 +/- 0.8 micromol/L). Similar protection (crescents, 14.3 +/- 1.9% of glomeruli; serum creatinine, 18.9 +/- 1.1 micromol/L) was observed in chimeric mice with intact BM but absent renal-derived TNF (WT-->TNF-/- chimeras), suggesting a minor contribution of infiltrating leukocytes to TNF-mediated renal injury. Chimeric mice with TNF-deficient leukocytes but intact intrinsic renal cell-derived TNF (crescents, 20.5 +/- 2.0% of glomeruli; serum creatinine, 21.6 +/- 1.4 micromol/L) developed similar crescentic GN to WT mice (crescents, 22.3 +/- 1.4% of glomeruli; serum creatinine, 24.8 +/- 1.9 micromol/L). Cutaneous delayed-type hypersensitivity after subdermal challenge with the nephritogenic antigen was attenuated in the absence of BM cell-derived TNF but unaffected in WT-->TNF-/- chimeric mice. These studies suggest that intrinsic renal cells are the major cellular source of TNF contributing to inflammatory injury in crescentic GN.

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 CD8&agr; or CD8&bgr; 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 1&bgr;, interferon &ggr;, 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 1&bgr; in atherosclerotic lesions. Transfer of CD8+ T cells deficient in perforin, granzyme B, or tumor necrosis factor &agr; but not interferon &ggr; 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 &agr; secretion.