Infection of endothelial cells with Trypanosoma cruzi activates NF-kappaB and induces vascular adhesion molecule expression

Abstract

Transcriptional activation of vascular adhesion molecule expression, a major component of an inflammatory response, is regulated, in part, by the nuclear factor-kappaB/Rel (NF-kappaB) family of transcription factors. We therefore determined whether Trypanosoma cruzi infection of endothelial cells resulted in the activation of NF-kappaB and the induction or increased expression of adhesion molecules. Human umbilical vein endothelial cells (HUVEC) were infected with trypomastigotes of the Tulahuen strain of T. cruzi. Electrophoretic mobility shift assays with an NF-kappaB-specific oligonucleotide and nuclear extracts from T. cruzi-infected HUVEC (6 to 48 h postinfection) detected two major shifted complexes. Pretreatment with 50x cold NF-kappaB consensus sequence abolished both gel-shifted complexes while excess SP-1 consensus sequence had no effect. These data indicate that nuclear extracts from T. cruzi-infected HUVEC specifically bound to the NF-kappaB consensus DNA sequence. Supershift analysis revealed that the gel-shifted complexes were comprised of p65 (RelA) and p50 (NF-kappaB1). Northern blot analyses demonstrated both the induction of vascular cell adhesion molecule 1 and E-selectin and the upregulation of intercellular adhesion molecule 1 mRNA in HUVEC infected with T. cruzi. Immunocytochemical staining confirmed adhesion molecule expression in response to T. cruzi infection. These findings are consistent with the hypothesis that the activation of the NF-kappaB pathway in endothelial cells associated with T. cruzi infection may be an important factor in the inflammatory response and subsequent vascular injury and endothelial dysfunction that lead to chronic cardiomyopathy.

FIG. 1

T. cruzi infection induces or upregulates ICAM-1, VCAM-1, and E-selectin mRNA. Northern blot analyses of adhesion molecule expression by HUVEC that were uninfected, TNF-α treated, or infected for 24 h are shown (see Materials and Methods). ICAM-1 mRNA was constitutively expressed in untreated HUVEC and was upregulated in infected and TNF-α-treated cultures (top panel). VCAM-1 mRNA expression was undetectable in uninfected HUVEC and was induced after infection or TNF-α treatment (middle panel). There was a significant increase in E-selectin mRNA expression in both T. cruzi-infected and TNF-α-treated HUVEC, while uninfected cells expressed a low basal level of E-selectin message (bottom panel). An 18S rRNA probe was utilized to normalize the total RNA loading equivalency of each lane. Data shown are representative of three separate experiments. Lane C, uninfected cells as control; lane TNF-α, HUVEC treated with TNF-α; lane I, HUVEC infected with T. cruzi.

FIG. 2

Time course of ICAM-1, VCAM-1, and E-selectin mRNA expression with T. cruzi infection. Northern blot analyses of adhesion molecule expression by HUVEC that were uninfected or infected for 6, 24, 48, and 72 h. Data from three separate blots were quantified by densitometry and normalized to the 18S rRNA signal and expressed as a ratio. Student t test analysis indicated that the three adhesion molecules were significantly induced or upregulated from 6 to 72 h postinfection (P < 0.001) (see Materials and Methods). (A) ICAM-1 was upregulated from 6 to 72 h and was constitutively expressed in uninfected cells. (B) VCAM-1 was induced from 6 to 72 h postinfection. (C) E-selectin expression was increased at 6 h postinfection and remained upregulated until 72 h, the last time point analyzed.

FIG. 3

T. cruzi infection activates NF-κB in HUVEC. NF-κB was assayed in nuclear extracts of HUVEC by electrophoretic mobility shift assays with a ³²P-labeled, double-stranded consensus NF-κB oligonucleotide. Lanes 1, 3, and 5 represent nuclear extracts obtained from uninfected HUVEC after 6, 24, and 48 h in culture, respectively. Lanes 2, 4, and 6 represent nuclear extracts obtained from HUVEC infected for 6, 24, and 48 h, respectively. Two shifted complexes appeared in the nuclear protein-DNA interaction from infected HUVEC (S1, shifted complex 1; S2, shifted complex 2). Both shifted complexes were undetectable in uninfected HUVEC. The arrow at the bottom indicates free NF-κB probe.

FIG. 4

Identification of the shifted complexes as a result of T. cruzi infection. To confirm the specificity of nuclear protein binding to the NF-κB oligonucleotide, competition assays were performed. Nuclear extracts from HUVEC at 6 h postinfection formed two major protein-DNA complexes (S1 and S2) (lane 2) which were absent in uninfected HUVEC (lane 1). Complex formation was specifically inhibited by incubation with a 50-fold molar excess of unlabeled NF-κB probe (lane 3) but not by a 50-fold molar excess treatment with an unrelated oligonucleotide containing the SP-1 binding consensus sequence (lane 4), indicating that these nuclear proteins specifically bound to the NF-κB consensus sequence. To identify the subunit composition of NF-κB in the protein-DNA complexes induced after infection, supershift (SS) analyses were performed with polyclonal antibodies specific for p65 and p50. Anti-p65 caused a supershift of S1 (lane 5), while anti-p50 induced a supershift of S2 (lane 6). Nonimmunized rabbit serum did not cause any supershifted complexes (lane 7). TNF-α-treated HUVEC nuclear protein extract was used as a positive control and produced similarly shifted (lane 8) and supershifted (lane 9, anti-p65 supershift; lane 10, anti-p50 supershift) complexes. Data are representative of three separate experiments. The arrow at the bottom indicates free NF-κB probe.

FIG. 5

T. cruzi infection induces or upregulates E-selectin, VCAM-1, and ICAM-1 protein expression. Shown are photomicrographs of adhesion protein expression in infected HUVEC as demonstrated by immunocytochemistry. Uninfected HUVEC incubated with isotype-matched, negative-control, purified IgG1 mouse myeloma protein did not exhibit nonspecific staining (see Materials and Methods). Bar, 150 μm. (A) Uninfected HUVEC stained with anti-E-selectin antibody. There is minimal background staining and E-selectin expression. (B) HUVEC infected for 24 h and stained with anti-E-selectin antibody. E-selectin protein was upregulated. (C) Uninfected HUVEC stained with anti-ICAM-1 antibody. Cells constitutively expressed ICAM-1 protein. (D) HUVEC infected for 24 h and stained with anti-ICAM-1 antibody. ICAM-1 protein expression was upregulated. (E) Uninfected HUVEC stained with anti-VCAM-1 antibody. There is minimal VCAM-1 expression. (F) HUVEC infected for 24 h and stained with anti-VCAM antibody. VCAM-1 protein was upregulated.