Effects of prednisolone treatment on cytokine expression in patients with leprosy type 1 reactions

Abstract

Leprosy type 1 reactions (T1R) are due to increased cell-mediated immunity and result in localized tissue damage. The anti-inflammatory drug prednisolone is used for treatment, but there is little good in vivo data on the molecular actions of prednisolone. We investigated the effect of prednisolone treatment on tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-10, and transforming growth factor beta1 (TGF-beta1) mRNA and protein expression in blood and skin biopsies from 30 patients with T1R in India. After 1 month of prednisolone treatment the sizes of the skin granulomas were reduced, as were the grades of cells positive for TNF-alpha and IL-10 in skin lesions. Increased production of TGF-beta1 was seen in skin lesions after 6 months of prednisolone treatment. Expression of mRNA for TNF-alpha, IL-1beta, and TGF-beta1 was reduced, whereas no change in IL-10 mRNA expression was detected during treatment. The circulating cytokine profiles were similar in patients with and without T1R, and prednisolone treatment had no detectable effects on cytokine expression in the blood. The data emphasize the compartmentalization of pathology in T1R and the importance of the immune response in the skin. Clinical improvement and cytokine expression were compared. Surprisingly, patients with improved skin and nerve function and patients with nonimproved skin and nerve function had similar cytokine profiles, suggesting that clinical improvement is not directly mediated by the cytokines studied here. This in vivo well-controlled study of the immunosuppressive effects of prednisolone showed that the drug does not switch off cytokine responses effectively.

FIG. 1.

Levels of TNF-α, IL-10, and total TGF-β1 responses to MLSA in PBMC from patients with T1R and nonreactional patients before and during treatment. PBMC were cultured for 20 h in the presence of MLSA. Cytokine levels in culture supernatants were detected with the ELISA. Each line represents a patient. Statistical differences compared with zero time are indicated as follows: two asterisks, P < 0.01; and three asterisks, P < 0.001.

FIG. 2.

Representative microphotographs of tissue sections stained for TNF-α: cryosections of biopsies taken from a patient with T1R before prednisolone treatment was started (A) and after 1 month of prednisolone treatment (B). Bar = 10 μm. Harris hematoxylin counterstaining was used. Original magnification, ×400.

FIG. 3.

Fluorescent double staining for cytokines and CD68 in skin biopsies from patients with T1R before treatment. Three color fluorescence confocal images were obtained for TNF-α and IL-10 (blue) (first panel of each row), CD68 (green) (second panel of each row), and cell nuclei (propidium iodide [PI]) (red) (third panel of each row). The three images were superimposed (fourth panel of each row). The arrows indicate a double-positive cell. Bar = 10 μm. Original magnification, ×630.

FIG. 4.

Cytokine mRNA levels in sequential skin biopsies from patients with T1R and nonreactional patients before and during treatment. Levels of cytokine mRNA for TNF-α, IL-1β, TGF-β1, IL-10, and HPRT1 in skin biopsies were quantified using real-time PCR assays. The results are expressed as the ratio of cytokine mRNA to HPRT1 mRNA. Each dot represents a patient. Significant differences compared with zero time are indicated as follows: one asterisk, P < 0.05; and two asterisks, P < 0.01.