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Abstract: Chlamydia trachomatis infections manifest as unique, chronic inflammatory diseases, indicating a relative compromise in the capacity of early immune responders such as macrophages to resolve the infection. We decided to investigate whether or not the chronic inflammatory manifestations are influenced by a disturbance in the pattern of inflammatory:anti-inflammatory cytokine elaboration early in the infection cycle in macrophages and assess the possible modulatory role of Ca2+ signals in the process. Although the basal and functional levels of IL-12 and IL-10 are not identical in concentration, chlamydia initiated a significant decline in IL-12. This led to a difference in the ratio of time-course decline in IL-12 compared with IL-10 in a Ca2+-poor medium, while there was significant increase in IL-10 in a Ca2+-rich medium. Also, when macrophages were infected after treatment with drugs that either facilitated Ca2+ influx into cells or inhibited efflux from intracellular stores into cytosol, there was a significant enhancement of the elaboration of IL-10 compared with IL-12. The immobilization of cytosolic Ca2+ by BAPTA-AM resulted in the decline of macrophage IL-12 and IL-10 in both infected and uninfected cases. There was evidence that infectivity and status of chlamydial elementary bodies harvested from macrophages during these experiments were consistent with chronic forms as assessed by HSP-60:MOMP ratio. The implication of these findings is that chlamydia infection of macrophages, together with its capacity to moderate macrophage intracellular Ca2+ levels, may evoke a net anti-inflammatory response that presumably favors chronic chlamydia infections. [ABSTRACT FROM AUTHOR]

Abstract: Inducible nitric oxide synthase (iNOS) reaction is of enormous significance in innate immune protective response of host to microbial challenges. We speculate that microbial challenges, by virtue of their lipopolysaccharide (LPS) content, exert an effect on intracellular free Ca2+ levels ([Ca2+]i), which evokes cellular signaling in a manner that depends on LPS type. In this study, we investigated the effect of LPS from heterogeneous sources (Chlamydia pneumoniae [cLPS], Pseudomonas aeruginosa [pLPS], and Salmonella typhimurium [sLPS]) on nitric oxide (NO) release and iNOS expression in THP-1- and U937-derived macrophages, and determined the role of free [Ca2+]i on LPS-mediated generation of NO and iNOS expression in these cell types. The role of free [Ca2+]i oscillation and the probable input of extracellular Ca2+ influx on Ca2+-signals was monitored in relation to iNOS expression in LPS-stimulated macrophages. There was a significant difference in the time course release of NO in both macrophage types when stimulated with cLPS, or pLPS, or sLPS. In all instances, LPS induced a significant release of NO compared with interferon-γ. There was a difference in the pattern of basal iNOS reaction leading to NO release compared to iNOS expression observed at 24h post-stimulation. While a biphasic pattern was observed in the manner in which free [Ca2+]i affected iNOS expression, inhibition of extracellular Ca2+ influx resulted in a steady increase in iNOS expression. The implications of these findings are: moderate free [Ca2+]i affects macrophage release of NO and iNOS expression during LPS stimulation; also, LPS of heterogeneous sources differentially evokes macrophage iNOS reactions irrespective of macrophage types. [Copyright &y& Elsevier]

The steroid 17β-estradiol has emerged as having both beneficial and detrimental effects in relation to cardiovascular physiology. To provide clarity on the effect of 17β-estradiol on atherogenic process, we examined its regulatory role in LDL-receptor expression in macrophages. We determined if the previously reported 17β-estradiol-mediated reactive oxygen species (ROS) generation in macrophage has signaling consequences by examining the release of ROS, its homeostasis by antioxidant enzymes (SOD and γ-GCS) and the uptake of LDL in THP-1 derived macrophages. Also, we evaluated the antagonistic effect of progesterone on the processes. Estradiol exerted an up-regulatory effect on native LDL-receptor expression, while progesterone exhibited a reverse effect despite subsequent exposure of cells to estradiol. Also, estradiol elicited ROS release, while progesterone attenuated the process. There was evidence that ROS homeostasis was essentially controlled by SOD and inhibition of SOD down-regulates the expression of LDL-receptor. Estradiol also enhanced Ox-LDL or native LDL uptake, a process which was impaired by SOD inhibitor. These findings imply that estradiol acting alone may indeed favor atherogenic process by up-regulating LDL-receptor via induction of ROS signaling. However, in physiologic circumstances progesterone is present; therefore, its inhibitory effect may annul the process. [ABSTRACT FROM AUTHOR]

Chlamydia pneumoniae, a respiratory pathogen implicated in the development and progress of atherosclerosis, is known to infect and survive in macrophages, despite macrophage producing reactive oxygen species (ROS). To gain insight into ROS generation in macrophages infected with C. pneumoniae and to explore factors accounting for their final levels and effect, we investigated the role of NADPH oxidase and cytochrome oxidase pathways in the production and modulation of ROS. We also determined the operational role of Ca2+ signaling in the process. Macrophages stimulated with C. pneumoniae exhibit early release of ROS via up-regulation of NADPH oxidase and cytochrome c oxidase activities. Increasing the dose of C. pneumoniae led to an increase in the expression of these enzymes’ gene production, which was accompanied by a significant up-regulation of their gene products, implying a probable activation of transcriptional and translational processes, respectively. The change in levels of free Ca2+, influx across plasma membrane and efflux from intracellular store into cytosol all exhibited a significant regulatory role on the ROS generation pathways in macrophages. The observed events were shown to be dependent on binding of C. pneumoniae to CD14 receptors of macrophages. The data reported here imply that macrophages infected with C. pneumoniae produce ROS through membrane-associated NADPH oxidase with oxidative phosphorylation levels depending on Ca2+ influx signals. [ABSTRACT FROM AUTHOR]

Chlamydia pneumoniae, a respiratory pathogen implicated in the development and progress of atherosclerosis, is known to infect and survive in macrophages, despite macrophage producing reactive oxygen species (ROS). To gain insight into ROS generation in macrophages infected withC. pneumoniaeand to explore factors accounting for their final levels and effect, we investigated the role of NADPH oxidase and cytochrome oxidase pathways in the production and modulation of ROS. We also determined the operational role of Ca2+ signaling in the process. Macrophages stimulated withC. pneumoniaeexhibit early release of ROS via up-regulation of NADPH oxidase and cytochrome c oxidase activities. Increasing the dose ofC. pneumoniaeled to an increase in the expression of these enzymes’ gene production, which was accompanied by a significant up-regulation of their gene products, implying a probable activation of transcriptional and translational processes, respectively. The change in levels of free Ca2+, influx across plasma membrane and efflux from intracellular store into cytosol all exhibited a significant regulatory role on the ROS generation pathways in macrophages. The observed events were shown to be dependent on binding ofC. pneumoniaeto CD14 receptors of macrophages. The data reported here imply that macrophages infected withC. pneumoniaeproduce ROS through membrane-associated NADPH oxidase with oxidative phosphorylation levels depending on Ca2+ influx signals. [ABSTRACT FROM AUTHOR]

Explores the role of Chlamydia pneumoniae infection in macrophage expression of lipoprotein lipase gene. Influence of Chlamydia pneumoniae on the upregulation of lipoprotein lipase messenger RNA gene expression; Impact of calcium influx signals on lipoprotein lipase gene expression; Assessment of lipoprotein lipase activity.

Summary: Recent data have shown a unique relationship between Ca2+ signaling in macrophages through L-type channels and the outcome of C. pneumoniae infection of such cells. The present investigation seeks to provide insights into the manner in which macrophage L-type Ca2+ channel operation affects major outer membrane protein (MOMP) and heat shock protein-60 (HSP-60) mRNA gene expression (factors associated with Chlamydia chronicity), and the possible effect of this on antibiotic susceptibility. Intracellular calcium ([Ca2+]i) chelation using varying doses of 1,2-bis (o-aminophenoxy) ethane-N,N,N′N′ – tetra acetic acid (acetoxymethyl) ester (BAPTA-AM) induced an increase in MOMP and a decrease in HSP-60 mRNA gene expression. L-type Ca2+ channel antagonists produced an identical but enhanced effect. Since these findings associate specialized Ca2+ channels to Chlamydia chronicity, it was important to determine Ca2+ channel effect on the usual antibiotic refractory form of C. pneumoniae in macrophages. Inhibition of macrophage L-type Ca2+ channel operation improved C. pneumoniae antibiotic susceptibility assessed by decreased inclusion counts or down-regulated MOMP and HSP-60 mRNA gene expression. These findings provide molecular insights into how specialized Ca2+ channels influence Chlamydia chronic course in macrophages and demonstrates a role for L-type Ca2+ channel inhibitors in enhanced C. pneumoniae susceptibility to antibiotic therapy. [Copyright &y& Elsevier]

Macrophage immune capability depends on their efficient redox potential expressed in the effective release of reactive oxygen species (ROS) and nitric oxide. In this study the effect of the activation of a specialized Ca2+ channel on macrophage redox function during Chlamydia pneumoniae infection was explored. C. pneumoniae exhibited a profound and sustained Ca2+ influx capacity, with evidence of activity attributable to their lipopolysaccharide (cLPS) content. Also the organism showed an additional Ca2+ influx signal in macrophages exposed to thapsigargin, and there was evidence for the operation of a single ion channel of the L type as demonstrated by the effect of L-type channel antagonists (methoxyverapamil and nimodipine) despite exposure to Ca2+-rich medium. C. pneumoniae or cLPS induced intracellular ROS and NO generation in a manner consistent with dependence on intracellular calcium. L-type Ca2+ channel blocking significantly prompted C. pneumoniae inclusion formation. These findings suggest that Ca2+ influx signal and redox function in C. pneumoniae-infected macrophages depend on L-type Ca2+ channel activation. [ABSTRACT FROM AUTHOR]

Chlamydia pneumoniae is a recently recognized human respiratory pathogen with a unique biphasic life cycle characterized by an obligate intracellular (replicative) and an extracellular (infectious) form of the organism. C. pneumoniae is widely distributed and, via the respiratory route, infects the majority of the world's population. The majority (70%) of acute human C. pneumoniae respiratory tract infections are asymptomatic or only mildly symptomatic but a minority (30%) cause more severe respiratory illnesses including community-acquired pneumonia, bronchitis and a variety of upper airway illnesses. After acute infection the C. pneumoniae intracellular life cycle is characterized by the development of metabolically inert (and thus antibiotic resistant) atypical "persistent" inclusions; this biologic behavior correlates with a clinical course following acute symptomatic illness that is characterized by persistence of symptoms that are difficult to treat with antibiotics. A role for C. pneumoniae in chronic respiratory illness is currently under investigation: "persistent" intracellular inclusions contain increased quantities of chlamydial heat shock protein 60 (hsp 60), a highly immunogenic protein that has been implicated in the pathogenesis of established chronic inflammatory chlamydial diseases (blinding trachoma, pelvic inflammatory disease and tubal infertility). An emerging body of evidence, including host immune response to chlamydial hsp 60, links C. pneumoniae infection with a spectrum of chronic inflammatory lung diseases of currently unknown etiology (asthma, chronic bronchitis and chronic obstructive pulmonary disease (COPD)). Further laboratory developments, including reliable and practical diagnostic methods and antibiotics effective against persistent infection, will be required to recognize and treat acute C. pneumoniae infection, and to advance our knowledge and understanding of the role of chronic infection in asthma, chronic bronchitis and COPD.