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, 68 (12), 6917-23

Gene Expression and Production of Tumor Necrosis Factor Alpha, interleukin-1beta (IL-1beta), IL-8, Macrophage Inflammatory Protein 1alpha (MIP-1alpha), MIP-1beta, and Gamma Interferon-Inducible Protein 10 by Human Neutrophils Stimulated With Group B Meningococcal Outer Membrane Vesicles

Affiliations

Gene Expression and Production of Tumor Necrosis Factor Alpha, interleukin-1beta (IL-1beta), IL-8, Macrophage Inflammatory Protein 1alpha (MIP-1alpha), MIP-1beta, and Gamma Interferon-Inducible Protein 10 by Human Neutrophils Stimulated With Group B Meningococcal Outer Membrane Vesicles

J A Lapinet et al. Infect Immun.

Abstract

Accumulation of polymorphonuclear neutrophils (PMN) into the subarachnoidal space is one of the hallmarks of Neisseria meningitidis infection. In this study, we evaluated the ability of outer membrane vesicles (OMV) from N. meningitidis B to stimulate cytokine production by neutrophils. We found that PMN stimulated in vitro by OMV produce proinflammatory cytokines and chemokines including tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-8, macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-1beta. A considerable induction of gamma interferon (IFN-gamma)-inducible protein 10 (IP-10) mRNA transcripts, as well as extracellular IP-10 release, was also observed when neutrophils were stimulated by OMV in combination with IFN-gamma. Furthermore, PMN stimulated by OMV in the presence of IFN-gamma demonstrated an enhanced capacity to release TNF-alpha, IL-1beta, IL-8, and MIP-1beta compared to stimulation with OMV alone. In line with its downregulatory effects on neutrophil-derived proinflammatory cytokines, IL-10 potently inhibited TNF-alpha, IL-1beta, IL-8, and MIP-1beta production triggered by OMV. Finally, a neutralizing anti-TNF-alpha monoclonal antibody (MAb) did not influence the release of IL-8 and MIP-1beta induced by OMV, therefore excluding a role for endogenous TNF-alpha in mediating the induction of chemokine release by OMV. In contrast, the ability of lipopolysaccharide from N. meningitidis B to induce the production of IL-8 and MIP-1beta was significantly inhibited by anti-TNF-alpha MAb. Our results establish that, in response to OMV, neutrophils produce a proinflammatory profile of cytokines and chemokines which may not only play a role in the pathogenesis of meningitis but may also contribute to the development of protective immunity to serogroup B meningococci.

Figures

FIG. 1
FIG. 1
Effect of OMV on the induction of TNF-α production by human neutrophils. (A) Comparative ability of neutrophils and PBMC to express TNF-α mRNA in response to OMV. Samples of 7 × 107 PMN were cultured in the presence or absence of 5 μg of OMV per ml. After the times indicated, total RNA was extracted and Northern blot analysis for TNF-α and actin transcripts was performed. Then, 10 μg of total RNA was loaded per each gel lane. The figure shows the results of one representative experiment out of two performed with identical results. (B) Time course of TNF-α release by OMV-stimulated neutrophils. PMN (5 × 106/ml) were stimulated with OMV for the times indicated before determining the levels of TNF-α in the cell supernatants by ELISA. The figure shows the mean values ± the SEM for each time point, which were obtained from at least five experiments performed under the same conditions. The asterisks represent significant differences between OMV-treated and control PMN: ∗∗∗, P < 0.005; ∗∗∗∗, P < 0.001.
FIG. 2
FIG. 2
Effect of OMV on the induction of IL-1β production by human neutrophils. (A) Time course release of IL-1β by neutrophils stimulated with OMV. PMN were incubated with or without 5 μg of OMV per ml for up to 21 h at 37°C, and the resulting cell supernatants were processed for the determination of IL-1β levels by ELISA. The results are expressed as the mean ± the SEM for each time point of duplicated determinations from five independent experiments. The asterisks represent significant differences between OMV-treated and control PMN, i.e., “∗∗∗” indicates P < 0.005. (B) Effect of IFN-γ and IL-10 on the production of IL-1β in OMV-simulated PMN. PMN were preincubated with or without 100 U of IFN-γ or 10 ng of IL-10 per ml for 30 min and then cultured for 21 h after the addition of 5 μg of OMV per ml. The bars report the mean values ± the SEM of the percentage of enhancement or inhibition of IL-1β release by OMV-stimulated PMN, as exerted by IFNγ or IL-10, respectively, calculated from five experiments. The percent values were calculated from the difference in the amount of IL-1β produced in the absence or presence of IFN-γ or IL-10. ∗, P < 0.05; ∗∗∗, P < 0.005.
FIG. 3
FIG. 3
Chemokine mRNA expression in OMV-stimulated neutrophils. (A) Purified populations of PMN and PBMC isolated from the same donor were cultured with or without 5 μg of OMV per ml. Total RNA was then extracted, and chemokine mRNA levels were assessed by RPA. Then, 10 μg of total RNA was used for each condition. The autoradiography shown is representative of three different experiments. (B) Time course of chemokine mRNA expression. PMN were incubated with or without 5 μg of OMV per ml. At the time points indicated, the total RNA was extracted and analyzed for IL-8, MIP-1α, and MIP-1β mRNA expression by RPA. Hybridization signals were quantitatively analyzed in an InstantImager, as described in Materials and Methods. The experiment depicted in this figure is representative of two.
FIG. 4
FIG. 4
Kinetics of IL-8 and MIP-1β production by OMV-stimulated neutrophils. PMN were stimulated with 5 μg of OMV per ml for the times indicated. IL-8 (A) and MIP-1β (B) levels were then determined in the cell supernatants by ELISA. For each cytokine, the figure shows the mean value ± the SEM of duplicate assays for each time point, each obtained from five experiments performed under the same conditions. The asterisks represent significant differences between OMV-treated and control PMN: ∗, P < 0.05; ∗∗, P < 0.01; and ∗∗∗, P < 0.005.
FIG. 5
FIG. 5
Extracellular production of IP-10 by stimulated PMN. (A) PMN were stimulated with 5 μg of OMV per ml in the presence or absence of 100 U of IFN-γ per ml. Cell supernatants were collected at the indicated time points, and IP-10 protein levels were measured using a specific ELISA. Values are expressed as the means ± the SEM of averaged duplicate determinations for each time point, each obtained from five experiments. The asterisks represent significant differences between OMV plus IFN-γ-treated and control PMN (∗∗∗, P < 0.005). (B) PBMC (5 × 105/ml) from autologous donors were stimulated with 5 μg of OMV per ml in the presence or absence of 100 U of IFN-γ per ml. Cell supernatants were collected after 21 h, and antigenic IP-10 was then measured by ELISA. The figure shows the mean values ± the SEM of duplicate assays for each condition obtained from three independent experiments.
FIG. 6
FIG. 6
Effect of IFN-γ and IL-10 on the release of IL-8 and MIP-1β induced by OMV. (A) PMN were preincubated for 30 min with or without 100 U of IFN-γ per ml and then cultured for 21 h after the addition of OMV before determining the chemokine release. The figure shows the mean values ± the SEM of the percentage of enhancement of chemokine release determined by IFN-γ treatment. Percent values were calculated from the difference in the amount of cytokine produced in the absence or presence of IFN-γ calculated from eight experiments. The values of the constitutive cytokine secretion were not subtracted. The asterisk represents significant differences between IFN-γ-treated and control PMN (∗, P < 0.05; ∗∗, P < 0.01). (B) PMN were preincubated with or without 10 ng of IL-10 per ml for 30 min and then cultured for up to 21 h after the addition of OMV. The cell supernatants were then collected, and the levels of IL-8 and MIP-1β protein were determined. Values are the mean ± the SEM of the percentage of IL-10 inhibition, as calculated from eight independent experiments for IL-8 and MIP-1β, respectively. The asterisks represent significant differences between IL-10-treated and control PMN (∗, P < 0.05; ∗∗, P < 0.01).
FIG. 7
FIG. 7
Effect of neutralizing anti-TNF-α MAb on PMN-derived IL-8 and MIP-1β induced by OMV. PMN were stimulated with 5 μg of OMV or 200 ng of LPS per ml from N. meningitidis strain B for 21 h in the presence or absence of anti-TNF-α neutralizing antibodies. Cell supernatants were then analyzed for IL-8 and MIP-1β accumulation into the supernatants. The figure shows the mean values ± the SEM of the percentage of enhancement or suppression of chemokine release as determined by measuring the levels of anti-TNF-α MAb. The percent values were calculated from the difference in the amount of cytokine produced in the absence or presence of anti-TNF-α MAb, as determined from three experiments. The asterisks represent significant differences between anti-TNF-α MAb-treated and control PMN (∗, P < 0.05).

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