Role of gamma interferon in chemokine expression in the ileum of mice and in a murine intestinal epithelial cell line after Cryptosporidium parvum infection

Abstract

Cryptosporidium parvum is a protozoan parasite that infects intestinal epithelial cells and induces inflammation of the intestine. To better understand the inflammatory process occurring during cryptosporidiosis, we investigated in this study the kinetics of chemokine expression in the mucosa of mice by quantitative reverse transcription-PCR. Our results demonstrate that among the chemokine mRNAs studied, gamma interferon (IFN-gamma)-inducible protein 10 (IP-10), monokine induced by IFN-gamma (MIG), i-TAC, lymphotactin, macrophage inflammatory protein 1 beta (MIP-1 beta), and RANTES mRNAs were strongly up-regulated in infected neonate mice, which correlated with the immunofluorescence staining results showing T-cell and macrophage infiltration in the mucosa. Our in vitro data showed that intestinal epithelial cells infected by C. parvum or stimulated by the proinflammatory cytokines (IFN-gamma, interleukin-1 beta, and tumor necrosis factor alpha) produce a pattern of chemokine secretion similar to that observed in vivo, suggesting that these cells may take part in the initial production of chemokines. In order to identify the chemokines responsible for the recruitment of the inflammatory cells leading to a protective immune response, we compared the patterns of chemokine expression in a healing neonate mouse model and a nonhealing IFN-gamma knockout (GKO) mouse model of cryptosporidiosis. In the absence of IFN-gamma, the chemokine response was altered for IP-10, MIG, i-TAC, RANTES, and MIP-1 beta mRNAs, while the three ELR C-X-C chemokine mRNAs studied (lipopolysaccharide-induced C-X-C chemokine, MIP-2 alpha, and KC mRNAs) were strongly overexpressed. These results are consistent with the neutrophil recruitment observed in the lamina propria of GKO mice at day 9 postinfection but are not consistent with the hypothesis that these cells play an important role in the resolution of the infection. On the contrary, the altered response of chemokines responsible for the recruitment of macrophages and T cells in GKO mice suggests that these two populations may be critical in the development of a protective immune response.

FIG. 1.

Kinetics of C. parvum infection in the C57BL/6J neonate mouse model. The course of infection was determined by enumeration of oocysts in the intestine (⧫) (n = 5) and by quantitative RT-PCR (◊) performed with a pool of RNA extracted from the ilea of six mice, as described in Materials and Methods.

FIG. 2.

Kinetics of chemokine expression in the ilea of neonate mice infected by C. parvum. Three-day-old neonates were inoculated with 10⁶ oocysts of C. parvum. RNAs were extracted from infected (▪) and uninfected age-matched control (○) neonates as described in Materials and Methods. For each time point, pools of total RNA extracted from the ilea of five to seven neonates were combined. Levels of chemokine and β-actin mRNAs were determined by quantitative RT-PCR performed with internal standards. The values are the numbers of mRNA transcripts per microgram of total RNA. The lower limit of detection of the quantitative RT-PCR method used is 10³ mRNA molecules/μg of total RNA and is indicated by dashed lines on some of the graphs. The highest ratio of mRNA level in infected mice to mRNA level in control mice is indicated in every panel. The results are representative of the results of two experiments. Ltn, lymphotactin.

FIG. 3.

Chemokine mRNA expression after C. parvum infection of ICcl2 cells. Confluent monolayers of ICcl2 cells on six-well plates were infected with oocysts at a ratio of five oocysts per cell for 24 h (Cp). Control cells were cultured with medium alone or with heat-inactivated oocysts (ΔCp), as described in Materials and Methods. Chemokine mRNA levels were determined by qualitative RT-PCR. For most chemokines 35 amplification cycles were used; the exceptions were fractalkine (28 cycles), KC (21 cycles), and β-actin (21 cycles). Similar results were obtained in three repeated experiments.

FIG. 4.

Chemokine mRNA expression by ICcl2 cells treated with IL-1β, TNF-α, and IFN-γ. Confluent monolayers of ICcl2 cells grown in six-well plates were incubated with IL-1β (10 ng/ml), TNF-α (10 ng/ml), and IFN-γ (10 ng/ml) for 5 h. Control cells were cultured with medium alone. Chemokine mRNA expression was determined by qualitative RT-PCR. For most chemokines 35 amplification cycles were used; the exceptions were fractalkine (28 cycles), KC (21 cycles), and β-actin (21 cycles). The data are representative of the data obtained in three experiments.

FIG. 5.

Chemokine mRNA response during C. parvum infection in wild-type (WT) (solid bars) and GKO (open bars) neonates at day 4 p.i. Three-day-old neonates were inoculated with 10⁶ oocysts of C. parvum (Cp). Pools of total RNA extracted from the ilea of seven neonates were combined. Levels of chemokine and β-actin mRNAs were determined by quantitative RT-PCR performed with internal standards. The values are the numbers of mRNA transcripts per microgram of total RNA. The lower limit of detection of the quantitative RT-PCR method used is 10³ mRNA molecules/μg of total RNA and is indicated by dashed lines on some of the graphs. The ratios of mRNA levels for infected mice (+) to mRNA levels for control mice (−) are indicated in every panel. The results are representative of the results of two experiments. Ltn, lymphotactin.

FIG. 6.

Ileum histopathology of control and C. parvum-infected wild-type neonates and GKO mice. (A, E, and I) Control wild-type neonates; (C, G, and K) control GKO mice; (B, F, and J) wild-type neonates infected for 9 days; (D, H, and L) GKO mice infected for 9 days. (A to D) Immunofluorescence staining with anti-CD3; (E to H) immunofluorescence staining with anti-F4/80 (macrophage); (I to L) immunofluorescence staining with anti-neutrophils. All immunofluorescence-labeled samples were counterstained with Evans Blue. Magnification, ×200.