- Poster presentation
- Open Access
Stat1 and phosphorylated Stat1 are increased in lymphocytes and monocytes of patients with systemic lupus erythematosus
© BioMed Central Ltd 2005
- Received: 11 January 2005
- Published: 17 February 2005
- Systemic Lupus Erythematosus
- Systemic Lupus Erythematosus Patient
- Interferon Gamma
- Stat1 Protein
- Rabbit Anti Mouse Antibody
Both interferon alpha (IFN-α) and interferon gamma (IFN-γ) are thought to be involved in systemic lupus erythematosus (SLE) immunopathogenesis. In their signal transduction, both cytokines lead to the tyrosine-phosphorylation and consequent nuclear translocation of the transcription factor Signal transducer and activator of transcription 1 (Stat1).
To evaluate Stat1 protein and Stat1 phosphorylation in SLE patients ex vivo and after stimulation with IFN-α as well as IFN-γ.
Peripheral blood mononuclear cells of 25 patients fulfilling ACR criteria for SLE and of 12 healthy individuals were prepared over Ficoll Paque gradients. Cells were either stained directly after preparation or after 15 min of incubation in medium with or without the addition of 100 U/ml IFN-α (Strathmann Biotech) or IFN-γ (R&D Systems). Intracellular staining was performed using either a monoclonal anti-Stat1 antibody and a FITC-labelled rabbit anti mouse antibody (Dako) with the Fix+Perm kit (An der Grub) or a directly PE-labelled monoclonal anti-phospho-Stat1 (pStat1) antibody (BD Biosciences Pharmingen) after fixation with 2% paraformaldehyde and permeabilization with 90% methanol. After staining, the cells were analyzed on a Becton Dickinson FACScan fluorocytometer. Gates were set for monocytes and for lymphocytes, and the logarithmic mean fluorescence intensity (mfi) was determined.
The amount of Stat1 protein, as measured by the mfi, was increased in lymphocytes of SLE patients as compared with healthy lymphocytes (21.4 ± 14.9 [mean ± standard deviation] versus 7.04 ± 1.54, P < 0.0001, t test), and in monocytes from SLE patients as compared with healthy monocytes (25.1 ± 13.2 versus 10.4 ± 2.54, P < 0.0001). Lymphocytic and monocytic Stat1 mfi correlated both for SLE patients (Pearson r = 0.57, P < 0.005) and for healthy individuals (r = 0.75, P < 0.005). The amount of phosphorylated Stat1, as measured by pStat1 mfi, was increased in SLE as compared with healthy lymphocytes (1.63 ± 0.40 versus 1.36 ± 0.22, P < 0.02), but not significantly increased in SLE monocytes as compared with healthy monocytes (4.03 ± 1.64 versus 3.24 ± 1.21, P = not significant). Nevertheless, the pStat1 mean fluorescence intensities of lymphocytes and monocytes were highly correlated, both for SLE patients and healthy individuals (r = 0.84, P < 0.0001, and r = 0.91, P < 0.0001, respectively).
As compared with incubation in medium alone, incubation with either IFN-α or IFN-γ increased the amount of pStat1 in SLE lymphocytes (from 1.57 ± 0.29 in medium alone to 1.82 ± 0.36 [P < 0.01, paired t test] with IFN-α and to 1.85 ± 0.40 [P < 0.002] with IFN-γ). In contrast, IFN-α, but not IFN-γ, increased the pStat1 mfi in healthy lymphocytes (from 1.41 ± 0.15 in medium alone to 1.83 ± 0.48 [P < 0.002] with IFN-α, but to 1.40 ± 0.18 [P = not significant] with IFN-γ). Likewise, SLE monocytes increased their pStat1 contents upon incubation with either IFN-α or IFN-γ (from 3.74 ± 1.18 to 4.66 ± 1.50 [P < 0.05] and 7.28 ± 4.14 [P < 0.001] for IFN-α and IFN-γ, respectively), while healthy monocytes responded to IFN-α (pStat1 mfi from 3.52 ± 1.11 to 5.23 ± 2.25, P < 0.01), but not IFN-γ (pStat1 mfi 3.96 ± 5.36, P < 0.001 versus medium).
Peripheral lymphocytes and monocytes of patients with SLE contain more Stat1 protein than those from healthy individuals and show increased Stat1 phosphorylation. While healthy peripheral bloob mononuclear cells phosphorylate Stat1 when stimulated by IFN-α only, SLE lymphocytes and monocytes are primed in a way that enables them to also react to IFN-γ.