Increased levels of alternatively spliced interleukin 4 (IL-4delta2) transcripts in peripheral blood mononuclear cells from patients with systemic sclerosis

Abstract

Recent in vitro studies have shown that interleukin 4 (IL-4) induces and gamma interferon (IFN-gamma) inhibits collagen production. To define the TH1(IFN-gamma) and TH2(IL-4) cytokine profiles in systemic sclerosis (Sscl), a disease characterized by widespread fibrosis, we investigated IL-4 and IFN-gamma transcripts in peripheral blood mononuclear cells and plasma protein levels in 13 patients with Sscl. Two previously identified IL-4 transcripts, a full-length transcript and an alternatively spliced (truncated) transcript (designated IL-4delta2), were identified in patients and normal controls. Significantly increased levels of total IL-4 transcripts (full-length plus IL-4delta2 transcripts) were found in patients with Sscl in comparison to those found in healthy controls (P = 0.003), and this increase was primarily due to an increase in the level of the alternatively spliced IL-4delta2 form. The IL-4delta2/full-length-IL-4 transcript ratio was significantly increased in Sscl patients (P < 0.0001, versus healthy controls). Sequencing analysis revealed that the frequency of IL-4 clones carrying the IL-4delta2 transcript was also substantially increased in patients with Sscl. Plasma IL-4 protein levels were increased in Sscl patients compared to those in healthy controls (P = 0.001) and correlated with total IL-4 transcript levels. The up-regulation of the fibrogenic IL-4 (a TH2 cytokine) in Sscl suggests a pathogenic role for IL-4 in this disease.

FIG. 1

Plasma IL-4 and IFN-γ protein levels in patients with Sscl and healthy controls. Both cytokines were measured by sandwich ELISA. Horizontal lines indicate median values. IL-4 levels were higher in patients with Sscl than in healthy controls (Mann-Whitney test; two-tailed P = 0.001). P value for IFN-γ is 0.5.

FIG. 2

RT-PCR results for IL-4 and IFN-γ in PBMC from patients with Sscl and healthy controls. Representative transcripts are shown. C, corresponding MIMIC; M, 100-bp ladder.

FIG. 3

PBMC IL-4 and IFN-γ transcript levels in patients with Sscl and healthy controls. Total IL-4 transcripts were semiquantitated by densitometry, as described in Materials and Methods. Full-length-IL-4 and IFN-γ transcripts were quantitated by MIMIC PCR. Horizontal lines indicate median values. Results were normalized for β-actin as a measure of PBMC equivalents. Total IL-4 transcript levels were higher in patients with Sscl than in healthy controls (Student’s t test; two-tailed P = 0.003). P values for full-length IL-4 and IFN-γ were 0.07 and 0.0004, respectively.

FIG. 4

IL-4δ2/full-length-IL-4 transcript ratio in PBMC from patients with Sscl and healthy controls. The ratio was calculated by densitometry, as described in Materials and Methods, and it was significantly higher in patients with Sscl than in healthy controls (Mann-Whitney test; P < 0.0001). Error bars represent SDs.

FIG. 5

IL-4 clones with full-length IL-4 and spliced IL-4δ2 transcripts. Representative PCR products were visualized after gel electrophoresis. M, 100-bp ladder.