Pneumocystis carinii Does Not Induce Maturation of Human Dendritic Cells
Pneumocystis carinii is an opportunistic pulmonary pathogen that causes significant clinical complications in patients with immunodeficiency diseases. In particular, P. carinii pneumonia (PCP) is a severe complication in subjects with HIV infection. The importance of alveolar macrophages as a first line of host defense against P. carinii, the involvement of antigen-specific CD4+ and CD8+ T cells in the clearance of P. carinii, and inflammation-associated respiratory impairment during PCP have been demonstrated [5,6,10]. On the other hand, the ability to initiate primary immune responses is a unique property of dendritic cells (DCs), which are found in an immature state at the periphery, that function as sentinels of the immune system. Upon uptake of antigens, DCs undergo a maturation process that induces their migration to the draining lymph nodes where they present peptides and efficiently stimulate naïve T cells. DCs determine Thl versus Th2 polarization of CD4+ cells in coordination with activation of CD8+ cytotoxic T cells and therefore play a central role in the regulation of specific immune responses. On this basis, we decided to investigate the potential involvement of DCs in the host response against P. carinii infection.
MATERIALS AND METHODS
DCs were prepared as follows. Monocytes were isolated from human PBMC by Ficoll-Paque® density centrifugation. CD14+ cells were positively selected by magnetic sorting using anti-CD14-conjugated magnetic microbeads (Miltenyi, Bergisch Gladbach). The recovered cells were cultured at 0.5 × 106/ml in RPMI plus 10% fetal bovine serum (FBS) 50 ng/ml GM-CSF and 1000 U/ml IL-4 for 5 days. Maturation was induced by addition of 1.5 μg/ml of LPS (from Salmonella abortus equi, Sigma, Buchs) for 24h. Alternatively the cells were co-cultured with P. carinii trophozoites at a ratio of 1/5 and 2/1 (P. carinii/DC) for 24h.
P. carinii organisms were obtained from corticosteroids immunosuppressed Sprague Dawley female rats infected by transtracheal inoculation as previously reported. Trophozoites from lung homogenates were inoculated on HEL 299 cells sheeted on microcarrier beads in spinner flasks to obtain the microorganism almost free from host debris [7]. After harvesting, the organisms were washed twice by centrifugation, and the pellets were snap frozen until resuspension in PBS just before use for co-cultivation with immature DCs.
Immature DCs at day 5 of cultivation, DCs matured with LPS, and DCs co-cultured with P. carinii were analyzed by flow cytometry on a FACScan® instrument (Becton Dickinson, Basel). Cell suspensions were stained with mAb anti-CD40-FITC (Serotec, Oxford), anti-HLA-DR-FITC, anti-CD14-FITC, anti-CD80-PE (Becton Dickinson), anti-CD83-FITC (Immunotech, Marseille), and anti-CD86-FITC (PharMingen, Basel).
RESULTS AND DISCUSSION
After 5 days of cultivation in GM-CSF and IL-4, the quality of the DC preparation was tested by flow cytometric analysis of typical cell surface markers. As expected, the DCs were negative for surface expression of CD14. They expressed high levels of MHC class II (HLA-DR) and CD40 molecules and low levels of the costimulatory molecules CD80 (B7–1) and CD86 (B7–2). Importantly, the cells were negative for surface expression of the DC maturation marker CD83. After LPS treatment, DCs matured normally, as shown by the pronounced up-regulation of CD86 and CD83, and the clear up-regulation of CD80 and HLA-DR. In contrast, when DCs were co-cultured with P. carinii phenotypic maturation was not achieved. This was shown by the absence of up-regulation of CD83 and a minimal change in the surface expression of CD86 and HLA-DR.
Lack of phenotypic maturation of DCs after co-culture with P. carinii was in part unexpected because uptake of the organisms by macrophages is regulated by the mannose receptor [2]. Antigen capture in DCs has been shown to be mediated by the mannose receptor and by fluid uptake by macropinocytosis [9]. Therefore it seems likely that DCs are able to endocytose P. carinii. Actually, it is noteworthy that in our experiments DCs were sensitive to the presence of P. carinii, as evidenced by aggregation of cells in clumps after addition of the organisms in culture. That some kind of response was possibly induced, was suggested by the slight but appreciable and reproducible up-regulation of CD86 and HLA-DR. Interestingly, it has been recently reported that phenotypic maturation of DCs is achieved only when viable, but not killed, Toxoplasma gondii is provided, even though both of the forms are efficiently internalized [11]. In our study P. carinii organisms were snap-frozen before use. After resuspension of these organisms in PBS, it is most likely that they were not viable even if their cell integrity should have been preserved. So it may be that viable organisms were required in order to induce phenotypic maturation of DCs, comparable to that achieved after treatment with LPS.
Our results are also surprising if we consider that DCs have been recently shown to elicit specific and protective immune responses against Candida albicans, another HIV-related opportunistic fungus [3]. However, in that study maturation of DCs with stainings for surface markers were not documented. On the other hand, phenotypic maturation and functional activation of DCs may not always be correlated. It could be that P. carinii is able to activate DCs to properly stimulate T cells, despite poor up-regulation of maturation-related surface markers. Alternatively, additional factors which may be absent in our in vitro system, but present during P. carinii infection in vivo, could be required for optimal maturation of DCs. Another interesting question for future investigations will be whether P. carinii actively suppresses the maturation process of DCs in order to prevent their function in the induction of protective T-cell responses.
Given that alveolar macrophages have been shown to be insufficient for resistance against P. carinii [5], and that CD4+ T cells play a pivotal role in the protection against P. carinii infection [10], it is important to determine to what extent DCs are affected by P. carinii and the cytokine melieu critical for activating DCs and inducing their migration to the lymph nodes. Our results suggest that, as in the case of Leishmania mexicana [1], P. carinii by itself is unable to induce maturity of DCs. Therefore the activation of DCs during in vivo infection would rely on adjuvant activity of host-derived factors. This could be particularly relevant in patients with AIDS, in which sub-optimal cytokine conditions in the lung may not only fail to stimulate but also inhibit DC activation (as in the case of IL10). Alternatively, analogous to what are induced by measles virus and herpes simplex virus [4,8], active suppression of DC maturation may be involved in the ability of P. carinii to escape a proper immune response.
ACKNOWLEDGMENTS
This study was supported by grants from Istituto Superiore della Sanità (grant 50C.2), the Swiss National Foundation, Roche Research Foundation, Novartis Foundation and Retenanstalt Jubiläumsstiftung.
