Johan Kuiper

Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock

SUMMARY Bacterial sepsis and septic shock result from the overproduction of inflammatory mediators as a consequence of the interaction of the immune system with bacteria and bacterial wall constituents in the body. Bacterial cell wall constituents such as lipopolysaccharide, peptidoglycans, and lipoteichoic acid are particularly responsible for the deleterious effects of bacteria. These constituents interact in the body with a large number of proteins and receptors, and this interaction determines the eventual inflammatory effect of the compounds. Within the circulation bacterial constituents interact with proteins such as plasma lipoproteins and lipopolysaccharide binding protein. The interaction of the bacterial constituents with receptors on the surface of mononuclear cells is mainly responsible for the induction of proinflammatory mediators by the bacterial constituents. The role of individual receptors such as the toll-like receptors and CD14 in the induction of proinflammatory cytokines and adhesion molecules is discussed in detail. In addition, the roles of a number of other receptors that bind bacterial compounds such as scavenger receptors and their modulating role in inflammation are described. Finally, the therapies for the treatment of bacterial sepsis and septic shock are discussed in relation to the action of the aforementioned receptors and proteins.

The influence of cytokines on the integrity of the blood-brain barrier in vitro

The effects of the cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta and IL-6 on the permeability of monolayers of rat cerebral endothelial cells (RCEC) were investigated to assess potential changes in the integrity of the blood-brain barrier (BBB). RCEC were cultured to tight monolayers with a trans endothelial electrical resistance (TEER) of 100-150 ohm . cm2 on polycarbonate filters. Exposure of the RCEC to TNF-alpha, IL-1 beta and IL-6 induced a decline in the TEER, which could be completely abolished by 1 muM of indomethacin, a cyclooxygenase inhibitor. In addition, the effect of IL-1 beta on TEER across monolayers of RCEC could be completely inhibited by IL-1 receptor antagonist. In conclusion, cytokines induce a disruption of the BBB in vitro. In this process, cyclooxygenase activation within the endothelial cells seems to play a key role.

FcαRI-positive liver Kupffer cells: Reappraisal of the function of immunoglobulin A in immunity

Despite the well-recognized involvement of immunoglobulin (Ig) A in mucosal immunity, the function of its receptor, FcαRI (CD89), is poorly understood. The ability of FcαRI to activate leukocytes seems to conflict with the proposed anti-inflammatory activity of secretory IgA. We show here that in a transgenic mouse model, inflammatory mediators induced expression of FcαRI on Kupffer cells, which enabled efficient phagocytosis in vivo of bacteria coated with serum IgA. Secretory IgA did not initiate phagocytosis. Therefore, interactions between serum IgA and FcαRI on Kupffer cells may provide a ‘second line of defense’ in mucosal immunity, by eliminating invasive bacteria entering through the portal circulation and thus preventing disease.

Interleukins in Atherosclerosis: Molecular Pathways and Therapeutic Potential

Interleukins are considered to be key players in the chronic vascular inflammatory response that is typical of atherosclerosis. Thus, the expression of proinflammatory interleukins and their receptors has been demonstrated in atheromatous tissue, and the serum levels of several of these cytokines have been found to be positively correlated with (coronary) arterial disease and its sequelae. In vitro studies have confirmed the involvement of various interleukins in pro-atherogenic processes, such as the up-regulation of adhesion molecules on endothelial cells, the activation of macrophages, and smooth muscle cell proliferation. Furthermore, studies in mice deficient or transgenic for specific interleukins have demonstrated that, whereas some interleukins are indeed intrinsically pro-atherogenic, others may have anti-atherogenic qualities. As the roles of individual interleukins in atherosclerosis are being uncovered, novel anti-atherogenic therapies, aimed at the modulation of interleukin function, are being explored. Several approaches have produced promising results in this respect, including the transfer of anti-inflammatory interleukins and the administration of decoys and antibodies directed against proinflammatory interleukins. The chronic nature of the disease and the generally pleiotropic effects of interleukins, however, will demand high specificity of action and/or effective targeting to prevent the emergence of adverse side effects with such treatments. This may prove to be the real challenge for the development of interleukin-based anti-atherosclerotic therapies, once the mediators and their targets have been delineated.

Recombinant lipoproteins: lipoprotein-like lipid particles for drug targeting

Lipoproteins are endogenous particles that transport lipids through the blood to various cell types, where they are recognised and taken up via specific receptors. These particles are, therefore, excellent candidates for the targeted delivery of drugs to various tissues. For example, the remnant receptor and the asialoglycoprotein receptor (ASGPr), which are uniquely localised on hepatocytes, recognise chylomicrons and lactosylated high density lipopoteins (HDL), respectively. In addition, tumour cells of various origins overexpress the low density lipoprotein (LDL) receptor that recognises apolipoprotein E (apoE) on small triglyceride-rich particles and apoB-100 on LDL. Being endogenous, lipoproteins are biodegradable, do not trigger immune reactions, and are not recognised by the reticuloendothelial system (RES). However, their endogenous nature also hampers large-scale pharmaceutical application. In the past two decades, various research groups have successfully synthesised recombinant lipoproteins from commercially available natural and synthetic lipids and serum-derived or recombinant apolipoproteins, which closely mimic the metabolic behaviour of their native counterparts in animal models as well as humans. In this paper, we will summarise the studies that led to the development of these recombinant lipoproteins, and we will address the possibility of using these lipidic particles to selectively deliver a wide range of lipophilic, amphiphilic, and polyanionic compounds to hepatocytes and tumour cells. In addition, the intrinsic therapeutic activities of recombinant chylomicrons and HDL in sepsis and atherosclerosis will be discussed.

The peripheral blood mononuclear cell microRNA signature of coronary artery disease.

BACKGROUND MicroRNAs are being used in the oncology field to characterize tumors and predict the survival of cancer patients. Here, we explored the potential of microRNAs as biomarkers for coronary artery disease (CAD) and acute coronary syndromes. METHODS AND RESULTS Using real-time PCR-based profiling, we determined the microRNA signature of peripheral blood mononuclear cells (PBMCs) from stable and unstable CAD patients and unaffected controls. 129 of 157 microRNAs measured were expressed by PBMCs and low variability between separate PBMC pools was observed. The presence of CAD in general coincided with a marked 5-fold increase (P<0.001) in the relative expression level of miR-135a, while the expression of miR-147 was 4-fold decreased (P<0.05) in PBMCs from CAD patients as compared to controls, resulting in a 19-fold higher miR-135a/miR-147 ratio (P<0.001) in CAD. MicroRNA/target gene/biological function linkage analysis suggested that the change in PBMC microRNA signature in CAD patients is probably associated with a change in intracellular cadherin/Wnt signaling. Interestingly, unstable angina pectoris patients could be discriminated from stable patients based upon their relatively high expression level of a cluster of three microRNAs including miR-134, miR-198, and miR-370, suggesting that the microRNA signatures can be used to identify patients at risk for acute coronary syndromes. CONCLUSIONS The present study is the first to show that microRNA signatures can possibly be utilized to identify patients exhibiting atherosclerotic CAD in general and those at risk for acute coronary syndromes. Our findings highlight the importance of microRNAs signatures as novel tool to predict clinical disease outcomes.

Attenuation of atherogenesis by systemic and local adenovirus-mediated gene transfer of interleukin-10 in LDLr−/− mice

In view of its multifaceted anti‐inflammatory properties, interleukin‐10 (IL‐10) has been deemed to be potentially anti‐atherogenic. We have evaluated the capacity of adenoviral gene transfer of IL‐10 for the modulation of de novo atherosclerotic lesion formation by systemic and by local overexpression. Atherogenesis was initiated in the carotid arteries of low‐density lipoprotein receptor deficient mice by perivascular placement of silastic collars. One week after collar placement, mice were injected intravenously with 1 × 109 plaque‐forming units (pfus) of IL‐10 (Ad.IL‐10) or control adenovirus (Ad.empty). Administration of Ad.IL‐10 resulted in extended systemic expression of IL‐10 (peak serum level 3.0 ± 1.1 ng/ml) and a reduction in atherosclerotic lumen stenosis by 62.2% (P < 0.02). This finding was accompanied by monocyte deactivation and lowering of serum cholesterol levels (maximum decrease 44%). In a second experiment, collared arteries were transfected locally by transluminal instillation of adenovirus (titer 1.5×1010 pfu/ml). Systemic parameters remained unchanged following local transfection, but the degree of stenosis was, nonetheless, decreased by 44.9% (P < 0.05). We conclude that a marked inhibition of atherogenesis can be achieved by systemic overexpression of Ad.IL‐10, owing to its metabolic and immunomodulatory effects. Local IL‐10 transfer is virtually equipotent, however, and it may represent a valuable addition to the armory of anti‐atherosclerotic therapies.

Apolipoprotein E Protects Against Bacterial Lipopolysaccharide-induced Lethality A NEW THERAPEUTIC APPROACH TO TREAT GRAM-NEGATIVE SEPSIS

Septic shock is the most common cause of death in intensive care units and no effective treatment is available at present. Lipopolysaccharide (LPS) is the primary mediator of Gram-negative sepsis by inducing the production of macrophage-derived cytokines. Previously, we showed that apolipoprotein E (apoE), an established modulator of lipid metabolism, can bind LPS, thereby redirecting LPS from macrophages to hepatocytes in vivo. We now report that intravenously administered LPS strongly increases the serum levels of apoE. In addition, apoE can prevent the LPS-induced production of cytokines and subsequent death in rodents. Finally, apoE-deficient mice show a significantly higher sensitivity toward LPS than control wild-type mice. These findings indicate that apoE may have a physiological role in the protection against sepsis, and recombinant apoE may be used therapeutically to protect against LPS-induced endotoxemia.

Combined Deletion of Macrophage ABCA1 and ABCG1 Leads to Massive Lipid Accumulation in Tissue Macrophages and Distinct Atherosclerosis at Relatively Low Plasma Cholesterol Levels

Objective—The purpose of this study was to evaluate the effect of the combined deletion of ABCA1 and ABCG1 expression in macrophages on foam cell formation and atherosclerosis. Methods and Results—LDL receptor knockout (KO) mice were transplanted with bone marrow from ABCA1/ABCG1 double KO (dKO) mice. Plasma cholesterol levels after 6 weeks of Western-type diet (WTD) feeding were significantly lower in dKO transplanted mice than ABCA1 KO, ABCG1 KO, and control transplanted animals. Extreme foam cell formation was present in macrophages of various tissues and the peritoneal cavity of dKO transplanted animals. Furthermore, severe hypoplasia of the thymus and a significant decrease in CD4-positive T cells in blood was observed. Despite relatively low plasma cholesterol levels dKO transplanted animals developed lesion sizes of 156±19×103 &mgr;m2 after only 6 weeks of WTD feeding. Lesions, however, were smaller than single ABCA1 KO transplanted animals (226±30×103 &mgr;m2; P<0.05) and not significantly different from single ABCG1 KO (117±22×103 &mgr;m2) and WT transplanted mice (112±15×103 &mgr;m2). Conclusions—Macrophage ABCA1 and ABCG1 play a crucial role in the prevention of macrophage foam cell formation, whereas combined deletion only modestly influences atherosclerosis which is associated with an attenuated increase in WTD-induced plasma cholesterol and decreased proinflammatory CD4-positive T cell counts.

Blockade of Interleukin-12 Function by Protein Vaccination Attenuates Atherosclerosis

Background—Interleukin-12 (IL-12) has been identified as a key inducer of a type 1 T-helper cell cytokine pattern, which is thought to contribute to the development of atherosclerosis. We sought to study the role of IL-12 in atherosclerosis by inhibition of IL-12 using a newly developed vaccination technique that fully blocks the action of IL-12. Methods and Results—LDL receptor–deficient (LDLr−/−) mice were vaccinated against IL-12 by 5 intramuscular injections of IL-12–PADRE complex in combination with adjuvant oil-in-water emulsion (low dose)/MPL/QS21 every 2 weeks. Two weeks thereafter, atherogenesis was initiated in the carotid artery by perivascular placement of silicone elastomer collars. IL-12 vaccination resulted in the induction of anti–IL-12 antibodies that functionally blocked the action of IL-12 as determined in an IL-12 bioassay. Blockade of IL-12 by vaccination of LDLr−/− mice resulted in significantly reduced (68.5%; P<0.01) atherogenesis compared with control mice without a change in serum cholesterol levels. IL-12 vaccination also resulted in a significant decrease in intima/media ratios (66.7%; P<0.01) and in the degree of stenosis (57.8%; P<0.01). On IL-12 vaccination, smooth muscle cell and collagen content in the neointima increased 2.8-fold (P<0.01) and 4.2-fold (P<0.01), respectively. Conclusions—Functional blockade of endogenous IL-12 by vaccination resulted in a significant 68.5% reduction in atherogenesis in LDLr−/− mice. Vaccination against IL-12 also improved plaque stability, from which we conclude that the blockade of IL-12 by vaccination may be considered a promising new strategy in the treatment of atherosclerosis.