Roles of p38 mitogen-activated protein kinase, NF-kappaB, and protein kinase C in proinflammatory cytokine mRNA expression by human peripheral blood leukocytes, monocytes, and neutrophils in response to Anaplasma phagocytophila

Abstract

Anaplasma phagocytophila, an obligately intracellular bacterium of granulocytes, causes human granulocytic ehrlichiosis. Within 2 h after addition of A. phagocytophila, interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and IL-6 mRNAs are induced in human peripheral blood leukocytes (PBLs) or monocytes in vitro. However, neutrophils generate only IL-1beta mRNA. In the present study, signaling pathways for induction of these three cytokines were examined. TNF-alpha and IL-6 mRNA expression by PBLs was inhibited with SB 203580 (a p38 mitogen-activated protein kinase [MAPK] inhibitor), MG-132 (a proteasome inhibitor), and SN-50 (an NF-kappaB inhibitor). Activation of p38 MAPK and NF-kappaB mRNAs in monocytes was detectable within 15 to 30 min after addition of A. phagocytophila. Expression of these two cytokine mRNAs in PBLs and monocytes was also dependent on protein kinase C (PKC), protein kinase A (PKA), and protein tyrosine kinase (PTK). IL-1beta mRNA expression by neutrophils was not dependent on p38 MAPK, and p38 MAPK was not activated in neutrophils incubated with A. phagocytophila. IL-1beta mRNA induction by PBLs, monocytes, and neutrophils was dependent on PKC and PKA. Neutrophil expression of IL-1beta mRNA was dependent on transglutaminase, phospholipase C, and PTK, all of which are also required for internalization of A. phagocytophila. However, monocyte expression of IL-1beta mRNA was less dependent on these enzymes. These results suggest that A. phagocytophila transduces different signals between its host neutrophils and monocytes for proinflammatory cytokine generation.

FIG. 1.

IL-1β, TNF-α, and IL-6 mRNA induction in human PBLs preincubated with several inhibitors and exposed to A. phagocytophila. PBLs (10⁷ cells/ml) were preincubated for 1 h with SN-50 (100 μg/ml), MG-132 (50 μM), SB 203580 (10 μM), PD 098059 (20 μM), and H-7 (25 μM) and exposed to A. phagocytophila (100 bacteria/cell) for 2 h. Total RNA was extracted and subjected to RT-PCR. The amounts of cDNAs were normalized against the amounts of G3PDH mRNA in corresponding samples. The PCR products (10 μl each) were resolved on agarose gels containing ethidium bromide. Molecular size markers (HaeIII fragments of φX174 replicative-form DNA) were included. The data (donor 3 data) are representative of the data from two independent experiments (donors 1 and 3) that gave similar results.

FIG. 2.

Effects of SB 203580 and H-7 on proinflammatory cytokine mRNA induction in human monocytes and neutrophils exposed to A. phagocytophila. Monocytes or neutrophils (10⁷ cells) were preincubated for 30 min with SB 203580 (10 μM) or H-7 (25 μM) and then incubated with host-cell-free A. phagocytophila (100 bacteria/cell) for 2 h. The total RNA was extracted and subjected to RT-PCR. The amounts of cDNAs were normalized against the amounts of G3PDH mRNA in corresponding samples. The PCR products were resolved on agarose gels containing ethidium bromide. Molecular size markers (HaeIII fragments of φX174 replicative-form DNA) were included. The data (donor 8 data) are representative of the data from three independent experiments (donors 6, 7, and 8) that gave similar results.

FIG. 3.

Western blot analysis of activation and phosphorylation of p38 MAPK in human monocytes (A) and neutrophils (B) in response to A. phagocytophila. A total of 2 × 10⁷ cells were incubated for different times (0, 15, 30, and 60 min) with A. phagocytophila (100 bacteria/cell) or HL-60 cell lysate (negative control), lysed, blotted on a nitrocellulose membrane, and probed with anti-phospho-p38 MAPK antibody (pp 38) or with anti-p38 MAPK antibody (p 38). As controls, nonphosphorylated (N) and phosphorylated (P) p38 MAPK extracts from C-6 glioma cells prepared without and with anisomycin (Cell Signaling Technology) treatment, respectively, were used. The positive bands were detected by immersing the blots in a DAB developing solution. The data (donor 12 data) are representative of the data from two independent experiments (donors 9 and 12).

FIG. 4.

Time course of NF-κB activation (A) and comparison of NF-κB activation and AP-1 activation (B) in human monocytes exposed to A. phagocytophila, examined by EMSA. Human monocytes (donor 5; 10⁷ cells/ml in each well) were incubated with A. phagocytophila (100 bacteria/cell) or E. coli LPS (1 μg/ml) (positive control) for 30 min. Nuclear extracts were prepared at different times (0, 0.5, 1, 2, and 4 h) and used for EMSA. The data (donor 5 data) are representative of the data from three independent experiments (donors 5, 6, and 7). N, uninfected monocytes.

FIG. 5.

Effects of several inhibitors on induction of proinflammatory cytokine mRNAs in PBLs (A), monocytes (B), and neutrophils (C) in response to A. phagocytophila. Cells (10⁷ cells/ml) were preincubated for 30 min with CHX (20 μg/ml), MDC (100 μM), H-89 (50 μM), verapamil (100 μM), W-7 (30 μM), neomycin (20 μM), and genistein (100 μM) and exposed to host-cell-free A. phagocytophila (100 bacteria/cell) for 2 h. For the oxytetracycline (OTC) (10 μg/ml) treatment, host-cell-free A. phagocytophila (100 bacteria/cell) was preincubated for 30 min. Total RNA was extracted and subjected to RT-PCR. The amounts of cDNAs were normalized against the amounts of G3PDH mRNA in corresponding samples. The PCR products were resolved on agarose gels containing ethidium bromide. Molecular size markers (HaeIII fragments of φX174 replicative-form DNA) were included. (A) PBL data (donor 3 data) representative of the data from three independent experiments (donors 2, 3, and 10) that gave similar results. (B) Monocyte data (donor 11 data) representative of the data from two independent experiments (donors 11 and 13). (C) Neutrophil data (donor 12 data) representative of the data from two independent experiments (donors 12 and 13).

FIG. 6.

Western blot analysis of phosphorylated p38 MAPK. Human monocytes preincubated for 30 min with kinase inhibitors were incubated with A. phagocytophila (100 bacteria/cell) for 30 min. The same amount of protein was electrophoresed, blotted on a nitrocellulose membrane, and probed with anti-phospho-p38 MAPK antibody (pp38) and with anti-p38 MAPK antibody (p38). Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G was added, and the positive bands were detected by using the enhanced chemiluminescence method. The data (donor 16 data) are representative of the data from five independent experiments (donors 15, 16, 20, 21, and 22) that gave similar results.

FIG. 7.

NF-κB activation in human monocytes preincubated with several inhibitors and exposed to A. phagocytophila. Human monocytes (10⁷ cells/ml in each well) were preincubated with H-7, H-89, genistein, and SB 203580 for 45 min and then exposed to A. phagocytophila (100 bacteria/cell) for 30 min. Nuclear extracts were prepared and used for EMSA. The data (donor 19 data) are representative of the data from five independent experiments (donors 15, 16, 17, 18, and 19) that gave similar results. The nonspecific competitor AP-2 and the specific competitor NF-κB were included in this assay to verify the effects of these competitors on binding of the NF-κB complex.