Abnormal expression of intracellular cytokines and chemokine receptors in peripheral blood T lymphocytes from patients with systemic sclerosis

Abstract

In patients with systemic sclerosis (SSc), there are conflicting findings regarding which is predominant between type 1 and type 2 immune responses. To determine the balance between type 1 and type 2 T lymphocytes in peripheral blood from SSc patients, we investigated the expression of intracellular cytokines, such as interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-13, and chemokine receptors such as CXCR3 and CCR4 by flow cytometry. The frequency of IFN-gamma-producing cells among CD8+ cells was significantly increased in patients with diffuse cutaneous SSc (n = 11, P < 0.0001) and limited cutaneous SSc (lSSc; n= 16, P < 0.0001) compared with normal controls (n = 17) while there was no significant difference in the frequency of IL-4- or IL-13-producing cells. In contrast, the frequency of IFN-gamma- or IL-4-producing cells among CD4+ cells was similar between the three groups. Similar results were obtained when absolute numbers were assessed. The frequency of IFN-gamma-producing cells among CD8+ cells inversely correlated with percentage DLco in SSc patients (r = - 0.650, P < 0.005). CXCR3+ CD8+ cells selectively produced IFN-gamma, and the frequency of CXCR3+ CD45RO+ cells among CD8+ cells was higher in lSSc patients (n = 14, P < 0.01) than in normal controls (n = 22). In contrast, there was no significant difference in the frequencies of CXCR3- or CCR4-expressing CD45RO+ cells among CD4+ cells. These results demonstrate the predominance of type 1 cytokine-producing cells (Tc1 cells) in peripheral blood CD8+ T cells from SSc patients, but no definite Th1/Th2 imbalance in CD4+ T cells. Tc1 cells may be associated with pulmonary vascular damage in SSc.

Fig. 1

Representative expression of IFN-γ and IL-4 in the cytoplasm of CD8⁺ and CD8⁻ (CD4⁺) T cells from a patient with diffuse cutaneous systemic sclerosis (dSSc), a patient with limited cutaneous SSc (lSSc), and a normal control (control). Whole blood samples were activated with phorbol myristate acetate and ionomycin in the presence of brefeldin A. Intracellular cytokine production was determined by flow cytometry with three-colour analysis. Plots represent live cells gated for expression of CD3. Quadrants were set according to the staining of control MoAbs.

Fig. 2

Frequencies of CD8⁺ T cells producing (a) IFN-γ, (b) IL-2, (c) IL-4 and (d) IL-13 in peripheral blood CD8⁺ T cells from dSSc patients, lSSc patients, and normal controls (control). Intracellular expression of cytokines was determined by flow cytometry, as described in Fig. 1. Bars indicate the medians.

Fig. 3

Frequencies of CD4⁺ T cells producing (a) IFN-γ, (b) IL-2, (c) IL-4 and (d) IL-13 in peripheral blood CD4⁺ T cells from dSSc patients, lSSc patients, and normal controls (control). Intracellular expression of cytokines was determined by flow cytometry, as described in Fig. 1. Bars indicate the medians.

Fig. 4

The inverse relation between the frequency of IFN-γ-producing cells among peripheral blood CD8⁺ T cells and percentage DLco in SSc patients. Intracellular expression of IFN-γ was determined by flow cytometry, as described in Fig. 1. r =−0·650; P < 0·005

Fig. 5

Representative expression of IFN-γ and IL-4 in the cytoplasm of CXCR3⁺ CD8⁺ T cells in peripheral blood from (a) a patient with SSc and (b) a normal control. Whole blood samples were activated with phorbol myristate acetate and ionomycin in the presence of brefeldin A. Intracellular cytokine production was determined by flow cytometry with three-colour analysis. Histograms represent live cells gated for expression of CD8 and CXCR3 (——). Staining of control MoAbs (------).

Fig. 6

Frequencies of CCR4⁺ or CXCR3⁺ cells in peripheral blood CD8⁺ or CD4⁺ memory T cells from dSSc patients, lSSc patients, and normal controls (control). CCR4 and CXCR3 expression was determined by flow cytometry with three-colour analysis. Bars indicate the medians.