Changes in the cytokine profile of lupus-prone mice (NZB/NZW)F1 induced by Plasmodium chabaudi and their implications in the reversal of clinical symptoms

Abstract

We have previously observed that aged lupus-prone (NZB/NZW)Fl (BWF1) mice when infected with Plasmodium chabaudi show an improvement in their clinical lupus-like symptoms. In order to study the mechanisms involved in the long-lasting protective effect of the P. chabaudi infection in lupus-prone mice we analysed specific aspects of the cellular response, namely the profiles of cytokine mRNA expression and cytokine secretion levels in old BWF1 mice, in comparison with uninfected age-matched BWF1 mice and infected or uninfected BALB/c mice. Two months after infection, cells from BWF1 mice were stimulated with concanavalin A (Con A) and demonstrated a recovery of T cell responsiveness that reached the levels obtained with BALB/c cells. Old BWF1 mice showed high levels of interferon-gamma (IFN-gamma) and IL-5 production and correspondingly low levels of IL-2 and IL-4 secretion before infection with P. chabaudi. Infection did not modify the IFN-gamma levels of BWF1 T cells, whereas it considerably increased the secretion of the Th2-related cytokines IL-4, IL-5 and IL-10. In addition, only BWF1 T cells showed increased mRNA expression of tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta). This counter-regulatory cytokine network of infected BWF1 mice may be involved in the improvement of their lupus symptoms. The results of our investigations using the complex model of P. chabaudi infection can be extended and, by using more restricted approaches, it may be possible to explain the multiple regulatory defects of lupus-prone mice.

Fig. 1

Cytokine secretions by spleen cells from BWF₁ and BALB/c mice infected with Plasmodium chabaudi. Supernatants from concanavalin A-stimulated spleen cells were tested by ELISA for the presence of IFN-γ (a), IL-2 (b) IL-4 (c), IL-5 (d) and IL-10 (e). Results are expressed as the mean ± s.e.m. from six to eight supernatants per time point. (a) *P ≤ 0.015; †P ≤ 0.004; ‡P ≤ 0.002; §P ≤ 0.01, significantly different from infected BALB/c. (b) *P ≤ 0.002, significantly different from control BALB/c; ‡P ≤ 0.05, †P ≤ 0.0004, significantly different from, respectively, control BWF₁ and BALB/c. (c) *P ≤ 0.02, ‡P ≤ 0.008, significantly different from their respective uninfected controls; †P ≤ 0.0003, significantly different from control BALB/c. (d) *P ≤ 0.03, †P ≤ 0.016, ‡P ≤ 0.008, significantly different from control BWF₁. (e) *P ≤ 0.004, †P ≤ 0.006, significantly different from, respectively, control and infected BALB/c.

Fig. 2

T cell cytokine gene expression in BWF₁ and BALB/c mice infected with Plasmodium chabaudi. IFN-γ (a), IL-2 (b), IL-4 (c), IL-10 (d), tumour necrosis factor-alpha (TNF-α) (e) and transforming growth factor-beta (TGF-β) (f) gene expressions were assessed by semiquantitative polymerase chain reaction (PCR) analysis. RNA was extracted from 10⁶ freshly purified splenic T cells from infected or control BWF₁ and BALB/c mice. Different amounts of RNA were co-reverse transcribed in parallel with known concentrations of RNA obtained from the standard Th1 cell line (HDKI). All samples were amplified by PCR using specific primers for the housekeeping hipoxanthine phosphoribosyltransferase (HPRT) gene, followed by dot blot hybridization with an HPRT internal probe labelled with ³²P-γ-ATP. All samples, adjusted to equivalent amounts of 850 pg or 1450 pg of the HPRT transcript (see Materials and Methods) were amplified with IFN-γ-, IL-2-, IL-4-, IL-10-, TNF-α- and TGF-β-specific primers, in parallel with cDNA from the standard Th1 or Th2 cell line. Semiquantitative measurements were obtained by Phosphoimager scanning of the membranes from which the ct/min per dot were obtained. Results are expressed as RNA pg equivalents for each lymphokine calculated from the respective Th1 and Th2 standard curves.