Experimental cutaneous Leishmaniasis: a powerful model to study in vivo the mechanisms underlying genetic differences in Th subset differentiation

ARTICLE

Some aspects of the spectrum of pathological manifestations in humans infected with various Leishmania species have been successfully reproduced in inbred mice of different genetic backgrounds following infection with Leishmania major. This murine model of infection has thus been extensively used in attempts to correlate component(s) of the immune response associated with either spontaneous resolution of lesions or progressive disease.

Mice from most inbred strains are resistant to infection by L. major. In contrast, mice from BALB strains are unable to control infection and develop progressive disease [1]. Resistance and susceptibility have been correlated with the appearance of parasite-specific CD4⁺ Th1 or CD4⁺ Th2 cells, respectively. The dominant role of IFN-gamma produced by CD4⁺ Th1 cells in resistance to L. major has been demonstrated [2, 3]. In mice, the IFN-gamma produced by Th1 cells renders macrophages, the host cells for Leishmania, parasiticidal through the synthesis of the inducible nitric-oxide synthase leading to the production of toxic nitrogen radicals [4]. Susceptibility to L. major is also governed by Th2-derived cytokines with macrophage deactivating properties.

This murine model on infection with L. major is now considered as a powerful system to study the cellular and molecular mechanisms underlying genetically determined differences in the differentiation of CD4⁺ Th subsest in vivo. It has been demonstrated that Th1 and Th2 effector cells derive from a common CD4⁺ T cell precursor [5] and several stimuli have been reported to influence the pathway of maturation of CD4⁺ T cell precursors [6]. Among these, cytokines themselves critically regulate this process [6, 7]. Using CD4⁺ T cells transgenic for a unique TCR alpha/beta receptor, it was demonstrated that IL-12 and IL-4 are crucial for Th1 or Th2 cell maturation, respectively [6].

Development of polarized Th1 responses in genetically resistant mice

The importance of IL-12 for the establishment of Th1 cell responses in resistant mice following infection with L. major has been shown using either anti-IL-12 neutralizing antibodies or mice with disruption of the IL-12 gene [8, 9]. Conversely, treatment of BALB/c mice with exogenous recombinant IL-12 resulted in Th1 responses and resistance to L. major in these otherwise susceptible mice [10, 11].

Development of polarized Th2 responses in genetically susceptible BALB mice

Results obtained more than 10 years ago already supported a requisite role for IL-4 in mediating both Th2 cell differentiation and susceptibility to L. major in susceptible BALB/C mice [12]. In this context, we have demonstrated a burst of IL-4 mRNA expression in the draining lymph node CD4⁺ cells of BALB/c mice within 24 hours after infection with L. major [13]. It is noteworthy that this IL-4 burst occurred during the period when neutralizing IL-4 antibodies were capable of redirecting protective Th1 maturation in BALB/c mice [12, 14]. After this initial IL-4 mRNA burst, IL-4 mRNA expression returned to base line values before the occurrence of a second and permanent wave of IL-4 transcripts which reflects the establishment of a Th2 response. The cognate recognition of a single epitope of the Leishmania homolog of mammalian RACK1, designated LACK [15] was demonstrated to drive this early IL-4 response by a restricted population of MHC class II restricted CD4⁺ T cells that express the Vbeta4-Valpha8 TCR chains [16].

The importance of these Vbeta4-Valpha8 CD4⁺ T cells in Th2 cell maturation following infection with L. major was demonstrated using BALB/c mice deficient in Vbeta4⁺ cells following neonatal exposure to MMTV (SIM), a mouse mammary tumor virus encoding a superantigen leading to permanent deletion of Vbeta4⁺ cells. The early IL-4 mRNA burst after infection with L. major was absent in Vbeta4 CD4⁺ T cell-deficient BALB/c mice and Th2 cell development did not occur in these mice which did not suffer progressive disease [16]. Similarly to Vbeta4-deficient mice, BALB/c mice rendered tolerant to LACK as a result of the transgenic expression of this molecule under MHC class II promoters in the thymus were resistant to L. major and developed a Th1 response [17]. In addition, the induction of a specific unresponsive state in LACK-reactive Vbeta4-Valpha8 CD4⁺ T cells following treatment of BALB/c mice with altered LACK proteins that differ by a single amino acid from the natural I-A^d-restricted epitope antagonized early IL-4 response to the wild type LACK epitope, inhibited Th2 cell development, redirected Th1 cell maturation and resulted in long term protection [18]. Although it has been suggested that these LACK-reactive Vbeta4-Valpha8 CD4⁺ T cells were activated before exposure to LACK [19], our recent data revealing the functional plasticity of these cells in terms of cytokine production rather suggest that they are not differentiated memory cells [20].

Analysis of the accessory cells requirement for the expression of IL-4 transcripts in Vbeta4-Valpha8 CD4⁺ T cells following infection has further and unexpectedly demonstrated that B cells were essential (manuscript in preparation). The hypothesis that simultaneous recognition by T cells of their specific epitope on B and professional APC (i.e. dendritic cells) could signal B cells to produce cytokine(s) able to interfere with the Th1 inducing signals from DC in close vicinity is attractive and currently being investigated.

The use of the murine model of infection with L. major to reveal the opposite effects that IL-4 can exert on Th development

In contrast to its well known role in Th2 cell maturation, several results obtained with IL-4 transgenic and IL-4 deficient mice suggested that IL-4 may also promote Th1 cell development [21-25]. Studying the effects of exogenous IL-4 during the initiation of CD4⁺ T cell responses to OVA in vitro and to L. major in vivo we could show recently that when present only during the initial period of activation of dendritic cells (DCs), IL-4 instructed the differentiation of CD4⁺ T cells toward a Th1 phenotype and established resistance to L. major even in susceptible BALB/c mice. In both systems, this effect of IL-4 on Th1 cell development was totally dependent upon DC-derived IL-12 which, in turn, down-regulated early IL-4 production by LACK-specific Vbeta4Valpha8 CD4⁺ T cells in BALB/c mice [26]. In contrast, when also present during the period of T cell priming, IL-4 induced Th2 cell maturation and susceptibility to L. major, even in resistant Vbeta4⁺ cell-deficient BALB/c mice. Thus, since receptors for IL-4 and most other cytokines are expressed by a variety of cells, it is likely that, beyond cytokine concentration, the kinetics of cytokine action, including consecutive or preferential interactions with distinct cell subtypes, significantly contribute to their pleiotropic effects in vivo.

CONCLUSION

The murine model of infection with L. major has permitted us to demonstrate in vivo the existence and the importance of distinct CD4⁺ T cell subpopulation in vivo. This model is now considered as a powerful tool to study the cellular and molecular mechanisms operating in the selective development of peripheral CD4⁺ T cells in vivo. Knowing these mechanisms is essential for the development of novel interventional strategies for the prevention and treatment of serious infectious diseases.

Acknowledgements

Our groups are supported by grants from the Swiss National Science Foundation, the Deutsche Forschungsgemeinschaft and the French Ministry of Research.

Article accepted on 26/2/02

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