Kinetics of interferon-gamma secretion and its regulatory factors in the early phase of acute graft-versus-host disease

Abstract

Increased serum levels of interferon-gamma (IFN-gamma) have been observed in acute graft-versus-host disease (GVHD). Recent in vitro studies have demonstrated that interleukin-12 (IL-12) and interleukin-18 (IL-18) synergistically up-regulate IFN-gamma secretion. In this communication, we investigated the factors relevant to IFN-gamma secretion in acute GVHD. A murine model of acute GVHD was established by injecting donor spleen cells into severe combined immunodeficiency (SCID) mice. A series of specimens, including sera, livers and spleens derived from the GVHD mice, were investigated with histological examination, enzyme-linked immunosorbent assay (ELISA), flow cytometry, and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). IFN-gamma secretion increased in serum 3 days after spleen cell transfer, peaked on day 7, and then gradually decreased close to the baseline level by day 35. A synchronized increase of activated T cells and mRNA expression of IL-12, IL-18 and their respective receptors was observed after spleen cell transfer. However, only the kinetic expression pattern of IL-12 receptor (IL-12R) beta2 chains was closely correlated with that of IFN-gamma, while IL-12 dropped to the baseline level earlier than IFN-gamma. Therefore, IFN-gamma expression in the early phase of acute GVHD is a mono-peak and self-restricted pattern. Its secretion is closely related with T-cell activation, the presence of IL-12, IL-18 and their respective receptors. However, the limiting factors for IFN-gamma secretion seem to be IL-12 and IL-12R beta2 chains.

Figure 3

Representative experiments on the expression of IL‐12p40, IL‐18 and the receptors at the mRNA level in spleens of control and GVHD mice. RNA was isolated and transcribed into cDNA for PCR amplification. PCR product was hybridized with an internal oligonucleotide probe, and the signals were measured with densitometry. (a) PCR products of β‐actin after 28 cycles amplification were separated on an agarose gel stained with ethidium bromide, showing a nearly equal intensity for every sample. M: DNA marker. Lanes 1, 2, 3, 4, 5 and 6 represent specimens obtained, respectively, from control mouse, and experimental mice 3, 7, 14, 28, and 35 days after injection of donor spleen cells. (b) hybridized PCR products of IL‐12p40, IL‐18, β1 and β2 chains of IL‐12R, and IL‐18R. Lanes 1, 2, 3, 4, 5 and 6, see explanation in (a). (c) the optical density of each hybridized PCR signal was determined by densitometry. The OD values were divided by that of the corresponding β‐actin signal, and the adjusted OD values of IL‐12p40, IL‐18 and their respective receptors show a variation with sample and time.

Figure 1

Sustained T‐cell activation in the early phase of acute GVHD. Spleen cells from donor and GVHD suffering mice were prepared as single cell suspension, and stained with anti‐CD3 FITC or anti‐CD3 FITC in combination with anti‐CD25 PE. Cells were gated with anti‐CD3 FITC, and therefore, only T cells were included for analysis. (a) CD3⁺ CD25⁺ cells increased proportionally in all mice after donor spleen cell transfer, and this increase was maintained in the whole observation period. (b) Representative FACS analysis results showing the increase of CD3⁺ CD25⁺ cells in mice after donor spleen cell transfer.

Figure 2

Mono‐peak pattern increase of IFN‐γ expression in the blood in the early phase of acute GVHD. Serum samples were collected at various time points after donor spleen cell transfer, and IFN‐γ was measured with ELISA. The minimal detectable dose of IFN‐γ by this kit is 3·0 pg/ml.

Figure 4

Correlation between the secretion of IFN‐γ and the expression of IL‐12R β2 chains. A Pearson linear correlation test was performed between the mean concentration of IFN‐γ at each time point and the corresponding adjusted OD value of IL‐12R β2 chain. A positive correlation was observed (R = 0·97, P = 0·002 < 0·01).