A. F. de Vos

Polarized secretion of IL-6 and IL-8 by human retinal pigment epithelial cells

A number of cell types situated along interfaces of various tissues and organs such as the peritoneum and the intestine have been shown to secrete inflammatory cytokines in a polarized fashion. Retinal pigment epithelial (RPE) cells are positioned at the interface between the vascularized choroid and the avascular retina, forming part of the blood–retina barrier. These cells are potent producers of inflammatory cytokines and are therefore considered to play an important role in the pathogenesis of ocular inflammation. Whether cytokine secretion by these cells also follows a vectorial pattern is not yet known, and was therefore the subject of this study. Monolayers of human RPE cells (primary cultures and the ARPE‐19 cell line) cultured on transwell filters were stimulated to produce IL‐6 and IL‐8 by adding IL‐1β (100 U/ml) to either the upper or the lower compartment. After stimulation, the human RPE cell lines showed polarized secretion of IL‐6 and IL‐8 towards the basal side, irrespective of the side of stimulation. The ARPE‐19 cell line also secreted IL‐6 and IL‐8 in a polarized fashion towards the basal side after basal stimulation; polarized secretion was, however, not apparent after apical stimulation. The observation that human RPE cells secrete IL‐6 and IL‐8 in a polarized fashion towards the choroid may represent a mechanism to prevent damage to the adjacent fragile retinal tissue.

Effect of acute hyperglycaemia and/or hyperinsulinaemia on proinflammatory gene expression, cytokine production and neutrophil function in humans.

Aims  Type 2 diabetes is frequently associated with infectious complications. Swift activation of leucocytes is important for an adequate immune response. We determined the selective effects of hyperglycaemia and hyperinsulinaemia on lipopolysaccharide (LPS)‐induced proinflammatory gene expression and cytokine production in leucocytes and on neutrophil functions.

Analysis of the secretion pattern of monocyte chemotactic protein‐1 (MCP‐1) and transforming growth factor‐beta 2 (TGF‐β2) by human retinal pigment epithelial cells

Retinal pigment epithelial (RPE) cells, situated between the neurosensory retina and the vascularized choroid, form part of the blood–eye barrier and are important for homeostasis of the outer retina. These cells are able to produce a variety of cytokines which may play a role in the maintenance of the immunosuppressive milieu inside the eye and in intraocular inflammatory responses. In the present study, we investigated whether RPE cells secreted the anti‐inflammatory cytokine TGF‐β2 and the proinflammatory cytokine MCP‐1 in a polarized manner. Monolayers of human donor RPE cells were cultured on transwell filters. Secretion of TGF‐β2 and MCP‐1 at either the apical or basal side of the RPE cell monolayers, that were not treated or stimulated with IL‐1β (200 U/ml), was analysed by ELISA. All three cell lines examined had a different TGF‐β2 secretion pattern. In two of the three donor RPE cell lines tested, TGF‐β2 secretion was polarized, but not in the same direction. TGF‐β2 secretion was not up‐regulated by stimulation with IL‐1β. In contrast, IL‐1β strongly induced MCP‐1 secretion preferentially into the basal compartment of all RPE monolayers tested. These data indicate that human RPE cells are able to secrete TGF‐β2 and MCP‐1 in a polarized fashion. Our results suggest that MCP‐1 can be secreted by RPE cells in the direction of choroidal vessels during inflammatory responses in the posterior part of the eye, which may limit damage to the neurosensory retina.

Vagus nerve stimulation inhibits activation of coagulation and fibrinolysis during endotoxemia in rats

Summary.  Background: Sepsis and endotoxemia are associated with concurrent activation of inflammation and the hemostatic mechanism, which both contribute to organ dysfunction and death. Electrical vagus nerve stimulation (VNS) has been found to inhibit tumor necrosis factor (TNF)‐α release during endotoxemia in rodents. Objective: To determine the effect of VNS on activation of coagulation and fibrinolysis. Methods: Rats received a sublethal i.v. dose of lipopolysaccharide (LPS) after electrical VNS or sham stimulation. Activation of coagulation and fibrinolysis, as well as cytokine release, was measured before LPS injection and 2, 4 and 6 h thereafter. Results: LPS induced activation of the coagulation system (increases in the plasma concentrations of thrombin–antithrombin complexes and D‐dimer, and a decrease in antithrombin) and biphasic changes in the fibrinolytic system [early rises of plasminogen activator activity and tissue‐type plasminogen activator, followed by a delayed increase in plasminogen activator inhibitor type 1 (PAI‐1)]. VNS strongly inhibited all LPS‐induced procoagulant responses and more modestly attenuated the fibrinolytic response. In addition, VNS attenuated the LPS‐induced increases in plasma and splenic concentrations of the proinflammatory cytokines TNF‐α and interleukin‐6 (IL‐6), while not influencing the release of the anti‐inflammatory cytokine IL‐10. Conclusion: These data illustrate a thus far unrecognized effect of VNS and suggest that the cholinergic anti‐inflammatory pathway not only impacts on inflammation but also on the coagulant‐anticoagulant balance.

Toll-like receptor mRNA levels in alveolar macrophages after inhalation of endotoxin

Toll-like receptors (TLRs) are pattern-recognition receptors that have been implicated in the initiation of innate immune responses upon the first encounter with invading pathogens. The airways are frequently exposed to various types of lipopolysaccharide (LPS) from the environment or from pathogens. The current study was designed to determine the effect of LPS on TLR gene expression in human alveolar macrophages in vivo. In total, 16 healthy subjects were enrolled in a single-blinded, placebo-controlled study. Subjects inhaled 100 μg LPS or normal saline (n = 8 per group). Measurements were performed in alveolar macrophages purified from bronchoalveolar lavage fluid obtained 6 h post-challenge. Inhalation of LPS by healthy human volunteers resulted in enhanced alveolar macrophage expression of mRNAs encoding TLRs 1, 2, 7, 8 and CD14, and reduced expression of mRNAs encoding TLR4 and lymphocyte antigen 96. In conclusion, lipopolysaccharide differentially influences the toll-like receptor mRNA expression profile in human alveolar macrophages in vivo.

Osteopontin promotes host defense during Klebsiella pneumoniae-induced pneumonia.

Klebsiella pneumoniae is a common cause of nosocomial pneumonia. Osteopontin (OPN) is a phosphorylated glycoprotein involved in inflammatory processes, some of which is mediated by CD44. The aim of this study was to determine the role of OPN during K. pneumoniae-induced pneumonia. Wild-type (WT) and OPN knockout (KO) mice were intranasally infected with 104 colony forming units of K. pneumoniae, or administered Klebsiella lipopolysaccharides (LPS). In addition, recombinant OPN (rOPN) was intranasally administered to WT and CD44 KO mice. During Klebsiella pneumonia, WT mice displayed elevated pulmonary and plasma OPN levels. OPN KO and WT mice showed similar pulmonary bacterial loads 6 h after infection; thereafter, Klebsiella loads were higher in lungs of OPN KO mice and the mortality rate in this group was higher than in WT mice. Early neutrophil recruitment into the bronchoalveolar space was impaired in the absence of OPN after intrapulmonary delivery of either Klebsiella bacteria or Klebsiella LPS. Moreover, rOPN induced neutrophil migration into the bronchoalveolar space, independent from CD44. In vitro, OPN did not affect K. pneumoniae growth or neutrophil function. In conclusion, OPN levels were rapidly increased in the bronchoalveolar space during K. pneumoniae pneumonia, where OPN serves a chemotactic function towards neutrophils, thereby facilitating an effective innate immune response.

A Regional Supply and Demand Model for Inpatient Hospital Care

In this paper a regional supply and demand model for hospital admissions is developed which can be used for policymaking and planning purposes. It incorporates spatial factors such as travel-time costs into a model of market equilibrium in which waiting time acts implicitly as the equilibrating device. By distinguishing travel-time costs or distances it is shown that both supply and demand within local markets strongly influence admissions in a way which cannot be observed on aggregated levels: the tension between supply and demand is cushioned by a strong redistribution of patients. The model encompasses several well-known models for patient flows and hospital utilization originating in regional economics.

The endothelial protein C receptor and activated protein C play a limited role in host defense during experimental tuberculosis

The protein C (PC) system is an important regulator of both coagulation and inflammation. Activated PC (APC), together with its receptor the endothelial protein C receptor (EPCR), has anticoagulant and anti-inflammatory properties. During tuberculosis (TB), a devastating chronic pulmonary disease caused by Mycobacterium (M.) tuberculosis, both a local inflammatory reaction characterised by the recruitment of mainly mononuclear cells and the formation of pulmonary granulomas as well as activation of coagulation occurs as part of the host immune response. We investigated the role of EPCR and APC in a mouse model of TBusing mice overexpressing EPCR (Tie2-EPCR), mice deficient for EPCR (EPCR-/-), mice treated with APC-inhibiting antibodies and mice overexpressing APC (APChigh) and compared them with wild-type (WT) mice. Blood and organs were harvested to quantify bacterial loads, cellular influxes, cytokines, histopathology and coagulation parameters. Additionally observation studies were performed. Lung EPCR expression was upregulated during experimental TB. No significant differences in bacterial growth were seen between WT and Tie2-EPCR mice. However, Tie2-EPCR mice had decreased pulmonary coagulation activation, displayed an increased influx of macrophages 2 and 6 weeks after infection, but no increase in other proinflammatory markers. On the other hand, in EPCR-/--mice coagulation activation was decreased 6 weeks post-infection, with little impact on other inflammation markers. APC-overexpression or treatment with anti-(A)PC antibodies displayed minimal effects during experimental TB. In conclusion, EPCR and APC play a limited role in the host response during experimental pulmonary TB.

The thrombomodulin lectin-like domain does not change host responses to tuberculosis

Tuberculosis (TB), caused by Mycobacterium (M.) tuberculosis, is a devastating infectious disease causing many deaths world-wide. Thrombomodulin (TM) is a multidomain glycoprotein expressed on all vascular endothelial cells. We here studied the role of the lectin-like domain of TM, responsible for a variety of anti-inflammatory properties of TM, during TB. We compared the extent of TM-expression in human lung tissue of TB and control patients. The, the role of the lectin-like domain of TM was investigated by comparing mice lacking this domain (TMLeD/LeD mice) with wild-type (WT) mice during experimental lung TB induced by infection with M. tuberculosis via the airways. Lungs were harvested for analyses at two, six and 29 weeks after infection. Lung TM-expression was downregulated in TB patients, which was not related to changes in the amount of endothelium in infected lungs. TMLeD/LeD mice showed unaltered mycobacterial loads in lungs, liver and spleen during experimental TB. Additionally, lung histopathology and cytokine concentrations were largely similar in TMLeD/LeD and WT mice, while total leukocyte counts were increased in lungs of TMLeD/LeD mice after 29 weeks of infection. Mortality did not occur in either group. The lectin-like domain of TM does not play an important role in the host response to M. tuberculosis infection in mice.

General, but not myeloid or type II lung epithelial cell, myeloid differentiation factor 88 deficiency abrogates house dust mite induced allergic lung inflammation

Asthma is a highly prevalent chronic allergic inflammatory disease of the airways affecting people worldwide. House dust mite (HDM) is the most common allergen implicated in human allergic asthma. HDM‐induced allergic responses are thought to depend upon activation of pathways involving Toll‐like receptors and their adaptor protein myeloid differentiation factor 88 (MyD88). We sought here to determine the role of MyD88 in myeloid and type II lung epithelial cells in the development of asthma‐like allergic disease using a mouse model. Repeated exposure to HDM caused allergic responses in control mice characterized by influx of eosinophils into the bronchoalveolar space and lung tissue, lung pathology and mucus production and protein leak into bronchoalveolar lavage fluid. All these responses were abrogated in mice with a general deficiency of MyD88 but unaltered in mice with MyD88 deficiency, specifically in myeloid or type II lung epithelial cells. We conclude that cells other than myeloid or type II lung epithelial cells are responsible for MyD88‐dependent HDM‐induced allergic airway inflammation.