Lymphokine enhances oxygen-independent activity against intracellular pathogens.

To determine if mechanisms other than the generation of toxic oxygen intermediates are active against intracellular pathogens, oxidatively deficient mouse L cells and monocyte-derived macrophages from patients with chronic granulomatous disease were stimulated with soluble lymphocyte products. Despite no enhancement in oxidative activity, these cells displayed effective microbistatic activity against both T. gondii and C. psittaci. These results suggest a potential role for nonoxidative mechanisms in the mononuclear phagocyte's activity against intracellular pathogens, and indicate that lymphokines can regulate both oxygen-dependent and oxygen-independent antimicrobial responses.

Repeated and persistent infection with Chlamydia and the development of chronic inflammation and disease.

Chlamydia trachomatis is an important human pathogen that mediates disease processes capable of inflicting permanent damage. Aggressive inflammatory responses to repeated infections, and to a persistent form of this intracellular bacterium, are thought to initiate the pathogenic events that lead to the debilitating sequelae of blinding trachoma and infertility.

Interferon-induced indoleamine 2,3-dioxygenase activity in human mononuclear phagocytes.

Interferon (IFN)-induced tryptophan degradation, catalyzed by indoleamine 2,3-dioxygenase (IDO), has been shown to mediate antimicrobial activity in epithelial cells. IDO activity has also been augmented in peripheral blood mononuclear cells (PBMC) treated with IFN or interleukin-2 (IL-2). The effector cells in this population have now been further characterized. PBMCs were isolated from normal donors, separated into monocyte and lymphocyte populations by plastic adherence, treated with IFN or IL-2, and cultivated in medium supplemented with [3H]tryptophan. Culture supernatants were collected after a 48-h incubation and fractionated by high-performance liquid chromatography; radioactivity was determined in fractions corresponding to tryptophan and its metabolites. IFN-gamma and IFN-beta induced IDO activity only in monocytes (plastic-adherent, nonspecific esterase-positive PBMCs). The induction of IDO activity by IL-2 required both monocytes and lymphocytes. Interaction was required between these populations for induction of IDO by IL-2, due to production of IFN-gamma by T lymphocytes, with subsequent IFN-gamma-mediated induction of IDO in monocytes. A number of myeloid cell lines as well as monocyte-derived macrophages were also tested for their ability to be induced to degrade tryptophan in response to IFN treatment. Monocyte-derived macrophages were found to retain their capacity to be induced by IFN-gamma and IFN-beta to degrade tryptophan after differentiation, and to possess seven times more IDO activity per cell than IFN-induced monocytes. However, the presence of lipopolysaccharide (LPS) in the culture medium was required for the maximum induction of IDO activity by IFN-beta. Furthermore, higher concentrations of LPS were sufficient to induce IDO activity in macrophages in the absence of exogenous IFN.

Chlamydia pneumoniae-infected monocytes exhibit increased adherence to human aortic endothelial cells.

Interactions between monocytes and endothelial cells play an important role in the pathogenesis of atherosclerosis, and monocyte adhesion to arterial endothelium is one of the earliest events in atherogenesis. Work presented in this study examined human monocyte adherence to primary human aortic endothelial cells following monocyte infection with Chlamydia pneumoniae, an intracellular pathogen associated with atherosclerosis by a variety of sero-epidemiological, pathological and functional studies. Infected monocytes exhibited enhanced adhesion to aortic endothelial cells in a time- and dose-dependent manner. Pre-treatment of C. pneumoniae with heat did not effect the organism's capacity to enhance monocyte adhesion, suggesting that heat-stable chlamydial antigens such as chlamydial lipopolysaccharide (cLPS) mediated monocyte adherence. Indeed, treatment of monocytes with cLPS was sufficient to increase monocyte adherence to endothelial cells, and increased adherence of infected or cLPS-treated monocytes could be inhibited by the LPS antagonist lipid X. Moreover, C. pneumoniae-induced adherence could be inhibited by incubating monocytes with a mAb specific to the human beta 2-integrin chain, suggesting that enhanced adherence resulted from increased expression of these adhesion molecules. These data show that C. pneumoniae can enhance the capacity of monocytes to adhere to primary human aortic endothelial cells. The enhanced adherence exhibited by infected monocytes may increase monocyte residence time in vascular sites with reduced wall shear stress and promote entry of infected cells into lesion-prone locations.

Characterization of low-density lipoprotein uptake by murine macrophages exposed to Chlamydia pneumoniae.

Exposure to Chlamydia pneumoniae is correlated with atherosclerosis in a variety of clinical and epidemiological studies, but how the organism may initiate and promote the disease is poorly understood. One pathogenic mechanism could involve modulation of macrophage function by C. pneumoniae. We recently demonstrated that C. pneumoniae induces macrophages to accumulate excess cholesterol and develop into foam cells, the hallmark of early atherosclerotic lesions. To determine if C. pneumoniae-induced foam cell formation involved increased uptake of low-density lipoprotein (LDL), the current study examined macrophage association of a fluorescent carbocyanine (DiI)-labeled LDL following infection. C. pneumoniae enhanced the association of DiI-LDL with macrophages in a dose-dependent manner with respect to both C. pneumoniae and DiI-LDL. Interestingly, increased association was inhibited by native LDL and occurred in the absence of oxidation byproducts and in the presence of antioxidants. However, enhanced DiI-LDL association occurred without the participation of the classical Apo B/E native LDL receptor, since C. pneumoniae increased DiI-LDL association and induced foam cell formation in macrophages isolated from LDL-receptor-deficient mice. Surprisingly, DiI-LDL association was inhibited not only by unlabeled native LDL but also by high-density lipoprotein, very low density lipoprotein, and oxidized LDL. These data indicate that exposure of macrophages to C. pneumoniae increases the uptake of LDL and foam cell formation by an LDL-receptor-independent mechanism.

Effect of interferon on a primary conjunctival epithelial cell model of trachoma.

An in vitro human primary conjunctival epithelial system was adapted to determine if the effects of interferon-gamma (IFN-gamma), as described in cultured cell lines, were applicable to human ocular infection with Chlamydia trachomatis, Primary human epithelial cell cultures were exposed to varying concentrations of IFN-gamma. The treatment resulted in the induction of the tryptophan decyclizing enzyme indolamine 2,3-deoxygenase (IDO) in a dose-dependent manner as determined by assaying the conversion of tryptophan to its metabolites using reversed-phase high-performance liquid chromatography. Little IDO induction occurred in the presence of IFN-alpha or IFN-beta. Catabolism of up to 38% of available tryptophan occurred in IFN-gamma-treated cells in contrast to controls that showed only baseline activity. Cells cultured with IFN-gamma and then infected with an ocular isolate of C. trachomatis (TW-5), had a reduction in the percentage of inclusion-containing cells by over 80% in a dose-dependent manner. Reversal by the addition of exogenous tryptophan substantiated that IFN-gamma-mediated induction of IDO and catabolism of tryptophan were responsible for inhibition of intracellular growth of C. trachomatis.

Immunomodulation and Chlamydia: immunosuppression and the protective immune response to C. psittaci in mice.

Mice immunized intramuscularly with a low dose, viable inoculum of C. psittaci survived an otherwise lethal intraperitoneal challenge with the homologous chlamydial strain. Immunized animals were not protected from intraperitoneal challenge by the unrelated pathogen, Listeria monocytogenes. Spleen cells from animals that exhibited protective immunity were suppressed in their proliferative responses to mitogens or chlamydial antigen in an in vitro blastogenic assay. This suppression was transferable to normal spleen cells by adding irradiated cells from immunized animals to normal cell populations. The degree of normal cell blastogenic suppression was dependent on the ratio of irradiated immune to normal cells present in the assay medium. Suppression of humoral responses was demonstrated in vivo. Immunized animals were incapable of producing antibody secreting cells to sheep red blood cells after an intraperitoneal inoculation of SRBC. Unimmunized animals produced a significant number of plaque forming cells as measured by a direct plaque forming cell assay. Lymphokine activity was not impaired in spleen cells from mice that exhibited other manifestations of suppression. Taken together, these data provide evidence to indicate that the induction of suppression may not correlate with increased pathogenesis, but rather be closely associated with protective immunity. Data also provide circumstantial evidence to indicate that lymphokine induction may be important in the development of protective immunity to C. psittaci in the mouse.

Requirements for ingestion of Chlamydia psittaci by mouse fibroblasts (L cells).

Ingestion of 14C-amino acid-labeled Chlamydia psittaci (6BC) by mouse fibroblasts (L cells) was inhibited when the host cells were incubated for 30 min at 37 degrees C in Earle salts containing 10 mug of crystalline trypsin per ml. Tryptic digestion also inhibited the ingestion of 1-mum polystrene latex beads. Trypsin-treated L cells almost completely recovered their ability to ingest chlamydiae after 4 h at 37 degrees C in medium 199 with 5% fetal calf serum. Cycloheximide (10 mug/ml) blocked this recovery. Heating 14C-amino acid-labeled C. psittaci for 3 min at 60 degrees C inhibited its ingestion by L cells, whereas inactivating it with ultraviolet light was without effect on the ingestion rate. These results show that efficient ingestion of C. psittaci by L cells involves trypsin-labile sites on the host and heat-sensitive sites on the parasite. The failure of excess unlabeled infectious C. psittaci to promote the ingestion of 14C-labeled heat-inactivated chlamydiae suggests that direct interaction between these two sites must occur for uptake to proceed normally.

Kinetics of phagocytosis of Chlamydia psittaci by mouse fibroblasts (L cells): separation of the attachment and ingestion stages.

The kinetics of phagocytosis of Chlamydia psittaci (6BC) by monolayers of mouse fibroblasts (L cells) was studied with an assay that distinguished between the attachment and ingestion phases of phagocytosis. At multiplicities of 10 and 100 50% infectious doses (ID50) per L cell, virtually all of the inoculated C. psittaci had been attached and ingested after 60 min at 37 degrees C. At multiplicities of 500 to 5,000 ID50 per L cell, the initial rates of attachment and ingestion of C. psittaci to L cells increased with the multiplicity of infection, but phagocytosis stopped even though many chlamydial cells remained free in suspension and readily available for attachment to the host-cell monolayers. Phagocytosis probably ceased because the L cells were injured when they took up large numbers of chlamydial cells. This injury prevented direct determination of the number of potential binding sites for C. psittaci on each L cell. However, this number is large enough to make the rates of chlamydial attachment and ingestion predominantly dependent on the multiplicity of infection.

In vivo-activated mononuclear phagocytes and protective immunity to chlamydiae in mice.

Peritoneal macrophages (M phi s) collected from Chlamydia psittaci 6BC-immune mice after intraperitoneal challenge with 10(6) 6BC (immune-boosted [IB] M phi s) were compared by various functional criteria with other in vivo- and in vitro-activated M phi populations. While casein-, protease peptone-, and thioglycolate (Thio)-elicited M phi s were equally susceptible to in vitro infection with 6BC, IB M phi s did not support chlamydial growth and M phi s from Mycobacterium tuberculosis BCG- or Listeria monocytogenes-sensitized mice exhibited intermediate susceptibility to infection. The resistance of IB M phi s was not due to the ingestion of fewer 6BC organisms, nor were these cells persistently infected, since chlamydiae could not be recovered from infected IB M phi s after in vitro infection, even after extended incubation times. In contrast, Thio M phi s stimulated in vitro with gamma interferon (IFN-gamma), with or without lipopolysaccharide, resulted in cells that exhibited chlamydiastatic activity which was lost shortly after IFN-gamma was removed from the culture medium. Conversely, the antichlamydial activity of IB M phi s was stable over time but not through the production of autostimulatory cytokines, as evidenced by the lack of stimulation of Thio M phi s to restrict 6BC replication in coculture experiments. IB M phi s exhibited enhanced oxidative activity, but anti-IFN-gamma antibody did not abrogate this response. IB M phi s were recovered only from immunized mice that survived an otherwise lethal 6BC intraperitoneal challenge. These cells appear to be important for development of protective immunity to chlamydiae, and evidence suggests that stimulation by cytokines other than IFN-gamma (with or without lipopolysaccharide) is required for the observed heightened in vivo activation.

Differential susceptibility of chlamydiae to exogenous fibroblast interferon.

Mouse fibroblasts (L cells) were incubated for 5 h with 1,000 U of murine fibroblast interferon (MuIFN alpha+ beta) per ml and then were infected with either Chlamydia trachomatis (LGV 440), C. psittaci (6BC), or C. psittaci (Cal 10). Intracellular development of C. trachomatis was reduced 90% in interferon-treated cells 24 h after infection when compared with controls, whereas C. psittaci growth was not affected in interferon-treated cells.

Inhibition of Chlamydia psittaci in oxidatively active thioglycolate-elicited macrophages: distinction between lymphokine-mediated oxygen-dependent and oxygen-independent macrophage activation.

Immune sensitization of spleen cells was required to generate lymphokines (LK) that activated thioglycolate-elicited peritoneal macrophages (thio MACs) to respond via both oxygen-dependent and oxygen-independent systems. LK produced by incubating spleen cells from immunized A/J and LAF mice with concanavalin A stimulated a response by thio MACs to phorbol-12-myristate-13-acetate (PMA)-induced chemiluminescence and activated these cells to inhibit intracellular Chlamydia psittaci replication. Concanavalin A-incubated spleen cell preparations from unimmunized animals stimulated neither PMA-induced chemiluminescence nor antichlamydial activity. Activated thio MACs demonstrated a rapid chemiluminescence response to the intracellular protozoan Toxoplasma gondii, but C. psittaci did not induce chemiluminescence in LK-activated thio MACs, although cells exposed to C. psittaci retained their responsiveness to PMA-induced chemiluminescence. The PMA-induced response was inhibited by the addition of exogenous superoxide dismutase and catalase and was therefore related to the production of superoxide anion (O2 . -) and H2O2 by these cells. LK preparations incubated at 56 degrees C before macrophage treatment retained antichlamydial activity, but heated preparations no longer stimulated thio MACs to respond in the chemiluminescence assay. These data provide evidence that macrophage oxygen-dependent and oxygen-independent systems are simultaneously activated by LK, and these preparations comprise at least two distinct activities. The portion responsible for activating oxygen-dependent systems (PMA-induced chemiluminescence) is heat labile, whereas the portion responsible for activating oxygen-independent systems is heat stable. It is the latter system that results in restriction of chlamydial growth and in vitro parasite persistence.

Lymphokine-mediated inhibition of Chlamydia replication in mouse fibroblasts is neutralized by anti-gamma interferon immunoglobulin.

Experiments were carried out to characterize immunologically mediated chlamydial persistence in cell culture. Mouse fibroblasts were activated to restrict Chlamydia psittaci 6BC replication by including mitogen (concanavalin A)-induced spleen cell supernatant fluids from immunized animals in the growth medium. When mouse fibroblasts were incubated with lymphokine for 24 h before infection and then with growth medium after infection (preinfection treatment), chlamydial replication was delayed but eventually detected. No substantial chlamydial growth occurred, even with extended incubation times when mouse fibroblasts were continuously exposed to lymphokine before and after infection. Low levels of infectious chlamydiae were produced in preinfection-treated mouse fibroblasts but not in mouse fibroblasts subjected to continuous lymphokine exposure. Incubation of lymphokine with anti-murine gamma interferon immunoglobulin neutralized the observed lymphokine-mediated activity, but incubation in the presence of anti-murine alpha plus beta interferon serum did not alter lymphokine activity. We conclude that the lymphokine components responsible for activating fibroblasts to restrict C. psittaci replication exhibits properties similar to gamma interferon.

Induction of macrophage foam cell formation by Chlamydia pneumoniae.

Foam cell formation is the hallmark of early atherosclerosis. It was found that the intracellular bacterium Chlamydia pneumoniae induces foam cell formation by human monocyte-derived macrophages. Exposure of macrophages to C. pneumoniae followed by low-density lipoprotein (LDL) caused a marked increase in the number of foam cells and accumulation of cholesteryl esters. Foam cell formation was not inhibited by the antioxidant butylated hydroxytoluene nor fucoidan, suggesting that lipid accumulation did not involve scavenger receptors. In contrast, addition of heparin, which blocks binding of LDL to the LDL receptor, inhibited C. pneumoniae-induced foam cell formation, suggesting that the pathogen induced lipid accumulation by dysregulating native LDL uptake or metabolism (or both). These data demonstrate that an infectious agent can induce macrophage foam cell formation and implicate C. pneumoniae as a causative factor in atherosclerosis.

Parasite-specified phagocytosis of Chlamydia psittaci and Chlamydia trachomatis by L and HeLa cells.

Phagocytosis of the 6BC strain of Chlamydia psittaci and the lymphogranuloma venereum 440L strain of Chlamydia trachomatis by L cells and HeLa 229 cells occurred at rates and to extents that were 10 to 100 times greater than those observed for the phagocytosis of Escherichia coli and polystyrene latex spheres. Both species of Chlamydia were efficiently taken up by host cells of a type they had not previously encountered. Phagocytosis of chlamydiae was brought about by the interaction of parasite surface ligands with elements of the host cell surface. The chlamydial ligands were readily denatured by heat, were masked by antibody, and were resistant to proteases and detergents. The host cell components were reversibly removed by proteases. Chlamydial phagocytosis was inhibited when host cells were incubated for many hours with cycloheximide. It was suggested that the presence on the chlamydial cell surface of ligands with high affinity for normal, ubiquitously occurring structures on the surface of host cells is an evolutionary adaptation to intracellular existence. The term parasite-specified phagocytosis was used to describe the efficient phagocytosis of chlamydiae by nonprofessional phagocytes and to distinguish it from the host-specified immunological and non-immunological phagocytosis carried out by professional phagocytes.

Lymphokine-mediated microbistatic mechanisms restrict Chlamydia psittaci growth in macrophages.

The obligate intracellular procaryote Chlamydia psittaci replicated in cultures of macrophages taken from the peritoneal cavities of unstimulated or thioglycollate-elicited A/J mice. When treated with supernatant fluids (lymphokines) from C. psittaci-immune mice spleen cells that were stimulated for 24 hr in vitro by the mitogen concanavalin A, resident macrophages supplemented with heart infusion broth (8 mg/ml) and elicited macrophages markedly suppressed intracellular chlamydial development. Uptake of the parasites was unaffected by LK-activated macrophages. The LK-induced inhibition was a static rather than a cidal effect. Chlamydial replication was detected 24 to 41 hr after removal of LK and infection with C. psittaci. Evidence that intermediates of oxygen metabolism did not contribute to the microbistatic process in either resident or elicited macrophages was provided by the inability of exogenous catalase, deprivation of glucose, or pretreatment with phorbol myristate acetate (PMA) to reverse LK-induced inhibition of parasite replication.

A Chlamydia pneumoniae component that induces macrophage foam cell formation is chlamydial lipopolysaccharide.

Chlamydia pneumoniae infection is associated with atherosclerotic heart and vessel disease, but a causal relationship between this pathogen and the disease process has not been established. Recently, it was reported that C. pneumoniae induces human macrophage foam cell formation, a key event in early atheroma development, suggesting a role for the organism in atherogenesis. This study further examines C. pneumoniae-induced foam cell formation in the murine macrophage cell line RAW-264.7. Infected RAW cells accumulated cholesteryl esters when cultured in the presence of low-density lipoprotein in a manner similar to that described for human macrophages. Exposure of C. pneumoniae elementary bodies to periodate, but not elevated temperatures, inhibited cholesteryl ester accumulation, suggesting a role for chlamydial lipopolysaccharide (cLPS) in macrophage foam cell formation. Purified cLPS was found to be sufficient to induce cholesteryl ester accumulation and foam cell formation. Furthermore, the LPS antagonist lipid X inhibited C. pneumoniae and cLPS-induced lipid uptake. These data indicate that cLPS is a C. pneumoniae component that induces macrophage foam cell formation and suggest that infected macrophages chronically exposed to cLPS may accumulate excess cholesterol to contribute to atheroma development.