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Human Polymorphonuclear Leukocytes Produce IL-12, TNF-α, and the Chemokines Macrophage-Inflammatory Protein-1α and -1β in Response to Toxoplasma gondii Antigens

Susan K. Bliss, Anthony J. Marshall, Yin Zhang and Eric Y. Denkers
J Immunol June 15, 1999, 162 (12) 7369-7375;
Susan K. Bliss
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Anthony J. Marshall
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Yin Zhang
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Eric Y. Denkers
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  • FIGURE 1.
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    FIGURE 1.

    Human PMN secrete both IL-12 (p70) and TNF-α during in vitro stimulation with T. gondii. Responder cells were obtained from healthy donors with no previous exposure to T. gondii. The PMN were cultured with STAg (ST) or media alone (Med) for 2 h, then supernatants were collected for cytokine measurement. Levels of IL-12 (p70) and TNF-α were measured by cytokine-specific ELISA (see Materials and Methods for details). The results represent mean ± SD values for triplicate wells. In this figure, donors 1–4 correspond to those listed in Table I.

  • FIGURE 2.
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    FIGURE 2.

    Kinetics of IL-12 and TNF-α production by PMN in response to T. gondii or LPS. Neutrophils were purified and stimulated with STAg (ST), LPS, or media (Med), and supernatants were harvested for cytokine measurement at the indicated time points after culture initiation. This figure shows the results from a single individual, but similar results were obtained from three additional donors.

  • FIGURE 3.
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    FIGURE 3.

    T. gondii up-regulates neutrophil MIP-1α and MIP-1β gene expression. PMN were purified and incubated with STAg (ST) or media (M). At the times indicated, cells were collected, RNA was isolated, and RT-PCR amplification was conducted as described in Materials and Methods. Amplified PCR products were separated on an agarose gel, stained with ethidium bromide, and photographed (A). A 100-bp ladder (mw, molecular weight) demonstrates that the PCR products possess the predicted size (MIP-1α, 195 bp; MIP-1β, 190 bp; β-actin, 661 bp). B, PCR products were amplified and subjected to Southern blot analysis employing internal chemokine-specific probes. The cell population used in this experiment was comprised of 94.7% neutrophils, 4.6% eosinophils, and 0.7% lymphocytes. Monocytes were not detected. This figure shows results using PMN from one representative donor but similar data were obtained over the course of three additional experiments using other donors.

  • FIGURE 4.
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    FIGURE 4.

    Effect of exogenous IFN-γ and TNF-α on MIP-1α and MIP-1β expression. Human neutrophils were isolated and stimulated with STAg (ST) or media (M) in the presence or absence of recombinant human IFN-γ (20 U/ml) or TNF-α (100 ng/ml). Cells were harvested after 3 h, and RT-PCR analysis was conducted as described in Materials and Methods. A, The amplification products separated by agarose gel electrophoresis followed by ethidium bromide staining are shown. B, Transcript levels relative to β-actin, determined by scanning densitometric analysis, are shown. In this experiment, the cell population consisted of 95.3% neutrophils, 2.6% monocytes, and 2.1% lymphocytes. This experiment was repeated twice with essentially identical results.

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    FIGURE 5.

    Neutralizing anti-TNF-α antiserum partially blocks T. gondii-induced neutrophil chemokine up-regulation. Human PMN were isolated and stimulated with media (lane 1), STAg (lane 2), STAg in the presence of anti-TNF-α antiserum (lane 3), and STAg with control serum (lane 4). Three hours after culture initiation, cells were harvested and transcript levels were determined by RT-PCR-assisted amplification followed by agarose gel separation and visualization with ethidium bromide (A). Transcript levels were estimated using scanning densitometry and expressed relative to β-actin levels (B). The cells employed in this study were comprised of 90% neutrophils, 9% eosinophils, and 1% lymphocytes. This experiment was repeated twice with similar results.

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    Table I.

    Donor cell populations after neutrophil enrichment of peripheral blood leukocytesa

    DonorCellular CompositionbViability at 12 h (%)c
    NeutrophilsEosinophilsMonocytesLymphocytesMediaSTAg
    1260.0 ± 18.4 (70%)88.0 ± 25.6 (24%)5.0 ± 0 (1%)17.0 ± 8.5 (5%)9694
    2231.0 ± 65.1 (92.4%)6.0 ± 2.8 (2.4%)4.5 ± 2.1 (1.8%)8.5 ± 0.7 (3.4%)9391
    3267.3 ± 32.5 (91.2%)16.7 ± 3.1 (5.3%)1.7 ± 1.5 (0.3%)12.6 ± 2.5 (3.3%)9796
    4288.1 ± 1.4 (96%)9.0 ± 1.4(3%)0 (0%)3.0 ± 1.5 (1%)9496
    • a PMN were isolated from each individual by centrifugation over a Percoll gradient as described in Materials and Methods.

    • b Cellular composition was determined visually after staining cytospin preparations with Diff-Quik.

    • c Viability was determined by trypan blue exclusion.

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The Journal of Immunology: 162 (12)
The Journal of Immunology
Vol. 162, Issue 12
15 Jun 1999
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Human Polymorphonuclear Leukocytes Produce IL-12, TNF-α, and the Chemokines Macrophage-Inflammatory Protein-1α and -1β in Response to Toxoplasma gondii Antigens
Susan K. Bliss, Anthony J. Marshall, Yin Zhang, Eric Y. Denkers
The Journal of Immunology June 15, 1999, 162 (12) 7369-7375;

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Human Polymorphonuclear Leukocytes Produce IL-12, TNF-α, and the Chemokines Macrophage-Inflammatory Protein-1α and -1β in Response to Toxoplasma gondii Antigens
Susan K. Bliss, Anthony J. Marshall, Yin Zhang, Eric Y. Denkers
The Journal of Immunology June 15, 1999, 162 (12) 7369-7375;
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