Activation of mononuclear cells from blood and lung by Streptococcus pneumoniae and Haemophilus influenzae

Abstract

The response of human peripheral blood mononuclear cells (PBMC) to stimulation with whole killed S. pneumoniae or H. influenzae is not very well characterised. The aim of the present study was to investigate the proliferative response as well as the pattern of cytokine secretion after stimulation of PBMC with S. pneumoniae and H. influenzae. In studies I and II, the cytokine secretion was measured after 48 hours of stimulation, and the proliferative response was measured after 88 hours. In study III, the kinetics of cytokine secretion was determined, as was the expression of cell surface activation markers on T cells, B cells and NK cells after PBMC stimulation with S. pneumoniae. In study IV, the antibody response in bronchoalveolar lavage and serum was measured after aerosol immunisation with two strains of whole killed S. pneumoniae. PBMC were prepared from buffy coats from healthy blood donors, and mixed with either 106 S. pneumoniae/well, or 106 or 108 H. influenzae/well. At 48 hours, culture supernatants were collected for cytokine analysis, and new medium was added to the wells. At 72 hours of stimulation, 3H-thymidine was added to the wells, and after over night incubation, the cells were harvested and the proliferation was measured. Cytokine secretion was measured using either ELISA technique or bioassays. After stimulation with either S. pneumoniae or H. influenzae, IL-1b, IL-6, IL-8, IL-10, IL-12, IFN-g and TNF-a could be measured in the culture supernatants, while IL-2, IL-4, and IL-13 were unmeasurable. In addition, GM-CSF could be found, while IL-5 and TNF-b were undetectable after S. pneumoniae stimulation. S. pneumoniae induced strikingly low levels of IL-10 and high levels of IL-12, while H. influenzae in contrast induced low levels of IL-12 and high levels of IL-10. H. influenzae actually induced 30 times more IL-10 than S. pneumoniae did, while S. pneumoniae induced 50 times more IL-12 than H. influenzae. The ratio between the levels of IL-10/IL-12 was 0.1 for S. pneumoniae, and 108 for H. influenzae. The kinetics of cytokine secretion and cell surface activation marker expression on T cells, B cells and NK cells at different time points after stimulation with S. pneumoniae were investigated. The cell surface activation markers and cytokine secretion were detected at 4 h, 24 h, 48 h (cytokine secretion only), 72 h and one week. IL-1b, IL-10, IL-12 and TNF-a reached a maximum at 24 hours of stimulation, while IL-6, IL-8, IFN-g and TNF-b increased throughout the test period. The early activation marker CD69 could be detected on T cells, B cells and NK cells, and the expression reached maximal levels at 24 hours of stimulation. The CD25 and HLA-DR expression increased throughout the whole test period. Inbred Wistar rats were exposed to aerosol immunisation with two strains of whole killed S. pneumoniae for 2 weeks with a weekend in between, and four weeks apart. Five days after the last immunisation period, the animals were sacrificed, and serum and bronchoalveolar lavage (BAL) were collected. The antibody activity against two pneumococcal antigens, polysaccharide capsule 19F (PPS 19F), and pneumolysin toxoid (PL), was measured using an ELISA technique. Both a local (BAL) and a systemic (serum) antibody response against the two antigens could be detected after aerosol immunisation for IgA (BAL), IgG and IgM (both BAL and serum). The animals immunised with bacteria had significantly higher antibody responses in BAL and serum against PPS 19F than the unimmunised animals, while only the BAL IgG anti-PL antibody response was significantly higher for the immunised animals. When comparing the antibody responses from aerosol immunised rats to that of two intraperitoneally immunised rats, aerosol immunisation induced a higher local antibody response in BAL for IgA and IgG to the two antigens, while the BAL IgM levels were lower. Intraperitoneal immunisation, however, induced higher levels of antibody activity in serum as compared to aerosol immunisation.