Chemokine secretion of human cells in response to Toxoplasma gondii infection

Abstract

The ubiquitous protozoan parasite Toxoplasma gondii is a major cause of morbidity and mortality in neonates and immunocompromised hosts. Both acute invasion and reactivation of latent infection result in an inflammatory reaction with lymphocytes, macrophages, and neutrophils. The mechanisms responsible for triggering the local host response to toxoplasmosis are not fully understood. Infection of monolayers of human HeLa epithelial cells and fibroblasts with T. gondii resulted in a marked increase in the expression of interleukin-8 (IL-8)-specific mRNA and secretion of the proinflammatory and chemoattractant cytokines interleukin-8 (IL-8), GROalpha, and MCP-1. Host cell invasion and lysis were required for this response, as tachyzoite lysates alone had no effect on IL-8 secretion. IL-8 release was dependent on the release of soluble host cell factors: IL-1alpha in HeLa cells and an additional mediator in fibroblasts. HT-29 epithelial cells, which lack IL-1alpha or another IL-8-inducing activity, did not release IL-8 after infection, although they were efficiently infected with T. gondii and increased IL-8 secretion in response to added IL-1alpha. These data suggest that proinflammatory chemokine secretion is an important host cell response to toxoplasmosis and that the release of IL-1alpha and other mediators from lysed host cells is critical for this chemokine response.

FIG. 1

Increased IL-8 secretion by human fibroblasts in response to infection with T. gondii. (A) IL-8 secretion. Fibroblast monolayers in 24-well plates were infected with 2.5 × 10⁵ tachyzoites/well (●) or left uninfected for controls (○). Supernatants were collected at the indicated times, and IL-8 concentrations were determined by ELISA. The increase in IL-8 secretion following T. gondii infection was significant at 48 and 72 h (P < 0.05, Student’s paired t test). (B) Viable cell counts. The number of viable cells in the fibroblast monolayers was determined by trypan blue dye exclusion following detachment of the cells with trypsin-EDTA. Values are reported as the percentage of uninfected monolayers at each time point (mean ± standard error of the mean; n = 4 to 6).

FIG. 2

Relationship of T. gondii inoculum size and cumulative IL-8 secretion. Fibroblast monolayers in 24-well plates were infected with 10³ to 10⁶ tachyzoite/well and incubated for 48 h. IL-8 concentrations are shown as means ± standard errors of the means (n = 4).

FIG. 3

Time dependence of maximal IL-8 secretion on the T. gondii inoculum. HeLa cell monolayers in 24-well plates were infected with 2.5 × 10⁵ (▾) or 1 × 10⁶ (●) tachyzoites/well, or left uninfected as controls (○), and incubated for 8 to 72 h. (A) IL-8 secretion. IL-8 concentrations were determined by ELISA. Data points represent means ± standard errors of the means (n = 4). IL-8 secretion at 48 and 72 h was significantly increased above controls (P < 0.02, Student’s paired t test). (B) Viable cell counts. The number of viable cells in the monolayers was determined by trypan blue dye exclusion following detachment of the cells with trypsin-EDTA. Values represent the means ± standard errors of the means of three or more determinations.

FIG. 4

Increased IL-8 mRNA expression after T. gondii infection of HeLa cells. Monolayers of HeLa cells in 25-cm² tissue culture flasks were infected with 5 × 10⁵ T. gondii tachyzoites and incubated for the indicated periods. Total RNA was extracted, and levels of IL-8 and β-actin transcripts were determined by RT-PCR. Twenty microliters of each PCR mixture was electrophoresed and stained with ethidium bromide. As a negative control, RNA was omitted from reverse transcription and PCR amplification (No RNA).