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Recent data have shown that the respiratory pathogen Chlamydia pneumoniae expresses an altered gene transcription profile during gamma interferon (IFN-γ)-induced persistent infection in vitro. In the present study, we examined, by proteomics, expression of C. pneumoniae proteins labeled intracellularly with [ 35 S]methionine/cysteine under normal conditions or IFN-γ-mediated persistence. The identity of differentially expressed proteins during persistent infection was determined by matching spots to those of proteins identified in C. pneumoniae elementary bodies by matrix-assisted laser desorption ionization mass spectrometry. Upon treatment with 50 U of IFN-γ per ml, a marked upregulation of major outer membrane protein (MOMP), heat shock protein 60 (Hsp-60/GroEL), and proteins with functions in DNA replication (GyrA), transcription (RpoA, PnP), translation (Rrf), glycolysis (PgK, GlgP), and type III secretion (SctN) was observed at 24 h of infection. In contrast, no significant decreases in bacterial protein expression were found in C. pneumoniae -infected cells due to IFN-γ treatment. Upregulation of C. pneumoniae proteins involved in diverse functions during persistent infection may allow the organism to resist the inhibitory effects of IFN-γ while retaining basic functions. Future studies should examine the differential expression of chlamydial proteins during the developmental cycle under IFN-γ pressure to obtain a finer representation of the gene products involved in establishing persistence.

Infection with Chlamydia pneumoniae , a human respiratory pathogen, has been implicated as a potential risk factor in atherosclerosis, possibly because the pathogen can exist in a persistent form similar to that described for Chlamydia trachomatis . The present study investigated whether gamma interferon (IFN-γ) can induce indoleamine 2,3-dioxygenase (IDO) activity in aortic smooth muscle cells, leading to a marked inhibition of C. pneumoniae growth. Our data indicate a stimulation of IDO mRNA expression and dose-dependent enzymatic activity following IFN-γ treatment. IDO-mediated increase in tryptophan catabolism resulted in a dose-dependent marked inhibition of C. pneumoniae replication.

We have previously shown that different isolates of Chlamydia pneumoniae display heterogeneity in the in vitro stimulation of chemokines and adhesion molecules from infected human endothelial cells. In the present study, we examined the ability of different isolates of C. pneumoniae to promote transendothelial migration of neutrophils and monocytes. Human umbilical vein endothelial cells (HUVEC) were infected with low (C. pneumoniae isolates A-03, PS-32, and BR-393 and high (>40)-passage isolates BAL-16, TW-183, and T-2634, and levels of neutrophil and monocyte transendothelial migration were determined following 24 h of infection. Compared to mock-infected controls, significant increases in neutrophil migration were observed in response to most C. pneumoniae isolates examined ( P < 0.001). Levels of monocyte migration were significantly increased in response to TW-183 and T-2634 ( P < 0.001). Serial passage (>40 times) of the three low-passage isolates in HEp-2 cell cultures prior to infection of HUVEC generally resulted in the promotion of higher levels of neutrophil and monocyte transendothelial migration. These findings were compatible with differences observed in the extent of interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) stimulation between low- and high-passage A-03, PS-32, and BR-393. As opposed to C. pneumoniae , infection with C. trachomatis L2 caused only a slight increase in neutrophil transendothelial migration, which correlated with the lack of measurable IL-8 levels by this species. However, significant levels of monocyte migration were induced in response to C. trachomatis L2 despite a lack of measurable MCP-1 stimulation. C. trachomatis serovars A and E also failed to induce IL-8 and MCP-1 production in HUVEC. Results from this study indicate that the passage history of C. pneumoniae may play a role in the divergence of stimulatory activities observed among isolates in human endothelial cells. In addition, the differences observed between this organism and C. trachomatis suggest that the upregulation of IL-8 and MCP-1 in endothelial cells may be unique to C. pneumoniae .

Chlamydia pneumoniae is a respiratory pathogen that has been associated with chronic inflammatory diseases such as asthma and atherosclerosis. Recent isolation of C. pneumoniae from human carotid and coronary atheromas provides additional support for a role of this organism in atherogenesis. We characterized the coronary strain C. pneumoniae A-03 by sequence analysis of the major outer membrane protein gene ( omp1 ). In addition, the in vitro activities of A-03 and three respiratory strains of C. pneumoniae (BAL-16, TW-183, and T-2634) were examined in infected human umbilical vein endothelial cells (HUVEC) by analysis of the production of interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1), and soluble intercellular cell adhesion molecule 1 (sICAM-1). Sequence analysis of omp1 of C. pneumoniae A-03, compared to prototype strains TW-183 and AR-39, revealed five nucleotide changes resulting in nonsynonymous codons. Of interest was a nonconservative amino acid substitution (Ser to Pro) in position 61 of variable segment 1. In vitro, the extent of MCP-1, IL-8, and sICAM-1 production was dependent on the C. pneumoniae strain examined at low multiplicities of infection following 24 h of incubation. Strain A-03 displayed the lowest stimulatory activity in infected HUVEC, while T-2634 induced the highest levels of MCP-1, IL-8, and sICAM-1 among all strains examined. Heat-inactivated C. pneumoniae failed to stimulate production of these proteins by all strains tested. In contrast, only partial inhibition was observed by UV-inactivated organisms. Results from this study demonstrate that unlike prototype respiratory strains of C. pneumoniae , the coronary strain A-03 displays divergence in the omp1 gene. In addition, the stimulation of chemokines and adhesion molecules involved in the recruitment of leukocytes to sites of inflammation by C. pneumoniae may be important in the pathogenesis of diseases associated with this organism, including atherosclerosis.

Chlamydia pneumoniae has recently been associated with atherosclerotic lesions in coronary arteries. To investigate the biological basis for the dissemination and proliferation of this organism in such lesions, the in vitro growth of C. pneumoniae was studied in two macrophage cell lines, peripheral blood monocyte-derived macrophages, human bronchoalveolar lavage macrophages, several endothelial cell lines, and aortic smooth muscle cells. Five strains of C. pneumoniae were capable of three passages in human U937 macrophages and in murine RAW 246.7 macrophages. Titers were suppressed in both macrophage types with each passage, as compared with growth titers in HEp-2 cells. Both human bronchoalveolar lavage macrophages and peripheral blood monocyte-derived macrophages were able to inhibit C. pneumoniae after 96 h of growth. Eleven C. pneumoniae strains were capable of replicating in normal human aortic artery-derived endothelial cells, umbilical vein-derived endothelial cells, and pulmonary artery endothelial cells. Infection in human aortic artery smooth muscle cells was also established for 13 strains of C. pneumoniae. The in vitro ability of C. pneumoniae to maintain infections in macrophages, endothelial cells, and aortic smooth muscle cells may provide support for the hypothesis that C. pneumoniae can infect such cells and, when infection is followed by an immune response, may contribute to atheroma formation in vivo. More studies are needed to investigate the complex relationship between lytic infection and persistence and the potential for C. pneumoniae to influence the generation of atheromatous lesions.

An in vitro culture system was used to study the effects of increasing concentrations of human cytokines on the intracellular replication of Chlamydia pneumoniae. HEp-2 cell monolayers, pretreated for 24 h with 200 U of human recombinant gamma interferon (IFN-gamma) per ml restricted the intracellular replication of C. pneumoniae. Tumor necrosis factor alpha (TNF-alpha; 25 ng/ml) exhibited a synergistic effect with IFN-gamma by reducing the concentration of IFN-gamma necessary to restrict intracellular growth to 100 U/ml. The addition of 200 micrograms of tryptophan per ml significantly reversed the inhibitory effects of IFN-gamma and TNF-alpha, suggesting involvement of the indoleamine-2,3-dioxygenase pathway in the restriction process.

Legionella pneumophila causes Legionnaires’ disease by replication in alveolar macrophages and monocytes. The bacteria are internalized most efficiently by opsonin-dependent, CR3-mediated phagocytosis. This investigation focused on determining the role of actin polymerization and phosphorylation signals in this uptake mechanism. Uptake inhibition assays and confocal microscopic analysis indicated that entry of L. pneumophila activated tyrosine kinase (TK) and protein kinase C (PKC) and induced actin polymerization at the site of bacterial entry. Upon L. pneumophila entry, six major cellular proteins (75, 71, 59, 56, 53, and 52 kDa) were TK phosphorylated in soluble fractions of monocytes, and three of these proteins (52, 53, and 56 kDa) were consistently found in insoluble (i.e., cytoskeletal) fractions of monocytes as well. Tyrosine phosphorylation was suppressed when cells were pretreated with the kinase inhibitor genistein, tyrphostin, or staurosporine. A similar tyrosine-phosphorylated protein pattern was observed with CR3-mediated entry of avirulent L. pneumophila , Escherichia coli , or zymosan into monocytes. This study has shown that PKC and TK signals which activate actin polymerization during the process of phagocytosis are induced upon L. pneumophila entry. In addition, CR3 receptor-mediated phagocytosis into monocytes may involve tyrosine phosphorylation of similar proteins, regardless of the particle being phagocytosed. Therefore, the tyrosine-induced phosphorylation observed during opsonized L. pneumophila entry is not a virulence-associated event.