Interleukin-1ß regulates CXCL8 release and influences disease outcome in response to Streptococcus pneumoniae, defining intercellular cooperation between pulmonary epithelial cells and macrophages.

The success of Streptococcus pneumoniae (the pneumococcus) as a pulmonary pathogen is related to its restriction of innate immune responses by respiratory epithelial cells. The mechanisms used to overcome this restriction are incompletely elucidated. Pulmonary chemokine expression involves complex cellular and molecular networks, involving the pulmonary epithelium, but the specific cellular interactions and the cytokines that control them are incompletely defined. We show that serotype 2 or 4 pneumococci induce only modest levels of CXCL8 expression from epithelial cell lines, even in the absence of a polysaccharide capsule. In contrast, coculture of A549 cells with the macrophage-like THP-1 cell line, differentiated with vitamin D, or monocyte-derived macrophages enhanced CXCL8 release. Supernatants from the THP-1 cell line prime A549 cells to release CXCL8 at levels similar to cocultures. Interleukin-1Ra (IL-1Ra) inhibits CXCL8 release from cocultures and reduces the activity of macrophage-conditioned media, but inhibition of tumor necrosis factor alpha (TNF-α) had only a minimal effect on CXCL8 release. Release of IL-1ß but not TNF-α was upregulated in cocultures. IL-1 type 1 receptor knockout C57BL/6 and BALB/c mice confirmed the importance of IL-1 signaling in CXC chemokine expression and neutrophil recruitment in vivo. In fulminant disease, increased IL-1 signaling resulted in increased neutrophils in the airway and more invasive disease. These results demonstrate that IL-1 is an important component of the cellular network involving macrophages and epithelial cells, which facilitates CXC chemokine expression and aids neutrophil recruitment during pneumococcal pneumonia. They also highlight a potential clinical role for anti-IL-1 treatment to limit excessive neutrophilic inflammation in the lung.

Role of activins and inducible nitric oxide in the pathogenesis of ectopic pregnancy in patients with or without Chlamydia trachomatis infection.

Chlamydia trachomatis infection can lead to pelvic inflammatory disease, ectopic pregnancy (EP), infertility, and chronic pelvic pain in women. Activins and inducible nitric oxide synthase (iNOS) are produced by the human fallopian tube, and we speculate that tubal activins and iNOS may be involved in the immune response to C. trachomatis in humans and their pathological alteration may result in tubal pathology and the development of EP. Blood and fallopian tubes were collected from 14 women with EP. Sera were analyzed by enzyme-linked immunosorbent assay to detect antibodies against chlamydial heat shock protein 60 (chsp60) and the major outer membrane protein of C. trachomatis. Confirmation of C. trachomatis serology was made using the microimmunofluorescence test. The patients were classified into three groups according to their serological results, and immunohistochemistry and quantitative reverse transcription-PCR were performed to investigate the expression of candidate molecules by tubal epithelial cells among the three groups. This is the first study to show an increase in the expression of activin betaA subunit, type II receptors, follistatin, and iNOS within the human fallopian tube of EP patients who were serologically positive for C. trachomatis. A similar expression profile was observed in the fallopian tubes with detectable antibodies only against chsp60. These results were shown at the mRNA and protein levels. We suggest that tubal activin A, its type II receptors, follistatin, and NO could be involved in the microbial-mediated immune response within the fallopian tube, and their pathological expression may lead to tubal damage and the development of EP.

Spermicidal activity of bacterial lipopolysaccharide is only partly due to lipid A.

We have previously shown that co-incubation of Chlamydia trachomatis lipopolysaccharide (LPS) leads to premature sperm death by an apoptosis-like mechanism. It was always assumed that lipid A is the toxic component of LPS. Here we investigate the possible involvement of 3-deoxy-D-manno-octulosonic acid (Kdo), which is an additional component of the LPS in C. trachomatis. Highly motile preparations of sperm from normozoospermic patients were incubated for 6 hours with commercial sources of lipid A and Kdo. Conventional lipid A inhibitors, polymyxin B (PMB) and anti-CD14 monoclonal antibody (mAb) were used to test the ability of both lipid A and Kdo to induce an apoptotic-like response in mature sperm. Flow cytometry was used to determine apoptosis by the expression of annexin V. Caspase activity was also measured by fluorometry and by the use of a pan-caspase inhibitor and caspase-3 inhibitor. Both lipid A and Kdo at 50 micro g/mL caused significant mortality of sperm. However, although PMB and anti-CD14 mAb were inhibitory to the activity of lipid A on sperm, no such effect was seen against Kdo. In the presence of either lipid A or Kdo, sperm death was caused by an apoptotic-like effect that was caspase mediated. We conclude that Kdo shares its spermicidal properties with lipid A and seems to kill sperm in a similar manner. These results provide an explanation for higher than expected levels of spermicidal activity of LPS that are not caused by lipid A.

Effect of storage temperature on survival of Chlamydia trachomatis after lyophilization.

Lyophilized preparations of Chlamydia trachomatis were made to investigate how well they would survive storage at four relevant incubation temperatures for 1 week and 1 month. Good viability was maintained by storage at either 4 degrees C or 20 degrees C for 1 week. If the ambient temperature is not too high, short-term transportation of C. trachomatis is achievable through lyophilization.

Induction of neutrophil apoptosis by the Pseudomonas aeruginosa exotoxin pyocyanin: a potential mechanism of persistent infection.

Pseudomonas aeruginosa colonizes and infects human tissues, although the mechanisms by which the organism evades the normal, predominantly neutrophilic, host defenses are unclear. Phenazine products of P. aeruginosa can induce death in Caenorhabditis elegans. We hypothesized that phenazines induce death of human neutrophils, and thus impair neutrophil-mediated bacterial killing. We investigated the effects of two phenazines, pyocyanin and 1-hydroxyphenazine, upon apoptosis of neutrophils in vitro. Pyocyanin induced a concentration- and time-dependent acceleration of neutrophil apoptosis, with 50 microM pyocyanin causing a 10-fold induction of apoptosis at 5 h (p < 0.001), a concentration that has been documented in sputum from patients colonized with P. aeruginosa. 1-hydroxyphenazine was without effect. In contrast to its rapid induction of neutrophil apoptosis, pyocyanin did not induce significant apoptosis of monocyte-derived macrophages or airway epithelial cells at time points up to 24 h. Comparison of wild-type and phenazine-deleted strains of P. aeruginosa showed a highly significant reduction in neutrophil killing by the phenazine-deleted strain. In clinical isolates of P. aeruginosa pyocyanin production was associated with a proapoptotic effect upon neutrophils in culture. Pyocyanin-induced neutrophil apoptosis was not delayed either by treatment with LPS, a powerfully antiapoptotic bacterial product, or in neutrophils from cystic fibrosis patients. Pyocyanin-induced apoptosis was associated with rapid and sustained generation of reactive oxygen intermediates and subsequent reduction of intracellular cAMP. Treatment of neutrophils with either antioxidants or synthetic cAMP analogues significantly abrogated pyocyanin-induced apoptosis. We conclude that pyocyanin-induced neutrophil apoptosis may be a clinically important mechanism of persistence of P. aeruginosa in human tissue.