Expression of chemokine genes in murine macrophages infected with Orientia tsutsugamushi - PubMed (original) (raw)

Expression of chemokine genes in murine macrophages infected with Orientia tsutsugamushi

N H Cho et al. Infect Immun. 2000 Feb.

Abstract

Scrub typhus, caused by Orientia tsutsugamushi infection, is characterized by local as well as systemic inflammatory manifestations. Inflammation is initiated by O. tsutsugamushi-infected macrophages and endothelial cells in the dermis. We investigated the regulation of chemokine induction in macrophage cell line J774A.1 in response to O. tsutsugamushi infection. The mRNAs for macrophage inflammatory proteins 1alpha/beta (MIP-1alpha/beta), MIP-2, and macrophage chemoattractant protein 1 were induced within 30 min, and their levels showed a transitory peak for 3 to 12 h. However, the lymphotactin, eotaxin, gamma interferon-inducible protein 10, and T-cell activation gene 3 mRNAs were not detected by RNase protection assays. Heat-killed O. tsutsugamushi induced a similar extent of chemokine responses. Induction of the chemokine genes was not blocked by the eukaryotic protein synthesis inhibitor cycloheximide, suggesting that de novo synthesis of host cell protein is not required for these transcriptional responses. The induction of chemokine mRNAs by O. tsutsugamushi was blocked by the inhibitors of NF-kappaB activation. Furthermore, O. tsutsugamushi induced the nuclear translocation and activation of NF-kappaB. These results demonstrate that heat-stable molecules of O. tsutsugamushi induce a subset of chemokine genes and that induction involves activation of the transcription factor NF-kappaB.

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Figures

FIG. 1

FIG. 1

Time course of _O. tsutsugamushi_-stimulated chemokine induction by the J774A.1 cell line. (A) Before and after incubation of J774A.1 cells with O. tsutsugamushi. The levels of chemokine mRNAs at each time point were assayed by the RNase protection assay. (B) Normalized expression level of each chemokine mRNA. (C) mRNA levels of chemokine genes induced by the infection of O. tsutsugamushi, analyzed by semiquantitative RT-PCR at each time point. M, φX174 DNA digested with _Hae_III; N, negative control (reactions performed without cDNA).

FIG. 2

FIG. 2

(A) Determination of chemokine mRNA induction in J774A.1 cells treated with polystyrene beads or CHX, by semiquantitative RT-PCR. (B) The band intensities were determined with TINA software, and the level of each chemokine mRNA expression was normalized with mRNA level of β-actin. J774A.1 cells stimulated for 6 h with medium alone (C), L-929 cell lysate (Lysate), O. tsutsugamushi (OT), polystyrene beads (PS), LPS derived from E. coli (LPS), cycloheximide (CHX), or cycloheximide and O. tsutsugamushi (CHX + OT).

FIG. 3

FIG. 3

(A) Semiquantitative RT-PCR to determine the effect of polymyxin B on the levels of _O. tsutsugamushi_-induced chemokine mRNAs in J774A.1 cells. (B) The band intensities were determined and normalized as for the experiment in Fig. 2. J774A.1 cells were stimulated for 6 h with medium (C), O. tsutsugamushi (OT), and LPS derived from E. coli (LPS) in the absence or presence of polymyxin B (PB).

FIG. 4

FIG. 4

Effect of PDTC and TPCK on the levels of _O. tsutsugamushi_-induced chemokine mRNAs in J774A.1 cells. (A) Levels of each chemokine mRNA were analyzed in total RNA samples prepared from uninfected cells (C), _O. tsutsugamushi_-infected cells (OT), and infected cells in the presence of PDTC (PDTC + OT) or TPCK (TPCK + OT) by RT-PCR analysis. (B) The intensities of bands were determined and normalized as specified for Fig. 2.

FIG. 5

FIG. 5

Activation of the transcription factor NF-κB by O. tsutsugamushi and effect of PDTC and TPCK on _O. tsutsugamushi_-induced activation of NF-κB. (A) NF-κB activation was analyzed by EMSA for nuclear extracts prepared from J774A.1 cells treated for 2 h with medium (C), L-929 cell lysate (Lysate), and O. tsutsugamushi (OT). The nuclear extracts from the cells pretreated with PDTC (PDTC + OT) or TPCK (TPCK + OT) for 1 h before infection with O. tsutsugamushi were also analyzed. A competitive inhibition assay was performed on nuclear extracts preincubated with the unlabeled NF-κB consensus oligonucleotide (50 × Competitor). (B) A supershift assay was also performed. Nuclear extract was preincubated with antibodies against the p65 subunit of NF-κB. N. S., nonspecific.

FIG. 6

FIG. 6

Chemokine responses to inactivated or active O. tsutsugamushi. (A) The levels of chemokine mRNAs were compared by semiquantitative RT-PCR after incubation of the J774A.1 cells for 6 h with medium (C), L-929 cell lysate (Lysate), or heat-inactivated (HOT) or live (LOT) O. tsutsugamushi. (B) The intensities of bands were determined and normalized as specified for Fig. 2.

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