RESUMO
Healing of leishmaniases is dependent on activation of parasitized macrophages (Mphi) by IFN-gamma, which is secreted by Leishmania-specific Th1 cells. IL-12 enhances IFN-gamma production by Th1 cells and is crucial for cure. The host cells of Leishmania sp., Mphi, are a main source of IL-12 in vivo. We report that infection of quiescent murine Mphi with L. mexicana or L. major amastigotes does not induce IL-12 production. Moreover, infection suppresses IL-12 secretion by Mphi activated by LPS, by CD40 cross-linking or cognate interaction with Th1 cells. IL-12 secretion is also suppressed in Mphi activated after phagocytosis of latex beads. Suppression is independent of engagement of CR3 or FcgammaR during phagocytosis, is not mediated by IL-10 and does not alter steady state IL-12p40 mRNA levels. In addition, suppression of IL-12 secretion does not depend on Mphi activation concurrent to infection. In contrast, NO production was not inhibited. Thus, Mphi effector functions are differentially affected and this may be a general effect of phagocytosis of non-activating particles. The possible implications of this effect on the infection are discussed.
Assuntos
Interleucina-12/biossíntese , Leishmania mexicana/imunologia , Macrófagos/imunologia , Fagocitose/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Sequência de Bases , Antígenos CD40/metabolismo , Primers do DNA/genética , Feminino , Técnicas In Vitro , Interferon gama/farmacologia , Interleucina-12/genética , Leishmaniose Cutânea/genética , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Óxido Nítrico/biossíntese , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas RecombinantesRESUMO
Leishmania are protozoan parasites which invade mammalian macrophages and multiply as amastigotes in phagolysosomes (parasitophorous vacuoles). Using L. mexicana and bone marrow-derived macrophages (BMM), the question is addressed whether infected BMM induced to express major histocompatibility complex class II molecules can present defined antigens to specific T helper type 1 cells. As a model antigen, a membrane-bound acid phosphatase (MAP), a minor protein associated with intracellular vesicles in amastigotes, was either overexpressed at the surface of the parasites or overexpressed in a soluble form leading to antigen secretion into the parasitophorous vacuole. Presentation of MAP epitopes by these three types of amastigotes was then compared for macrophages containing live parasites or amastigotes inactivated by drug treatment. It is shown that surface-exposed and secreted MAP can be efficiently presented to T cells by macrophages harboring live amastigotes. Therefore, the parasitophorous vacuole communicates by vesicular membrane traffic with the plasmalemma of the host cell. The intracellular MAP of wild-type cells or the abundant lysosomal cysteine proteinases are not or only inefficiently presented, respectively. After killing of the parasites, abundant proteins such as overexpressed MAP and the cysteine proteinases efficiently stimulate T cells, while wild-type MAP levels are not effective. We conclude that intracellular proteins of intact amastigotes are not available for presentation, while after parasite inactivation, presentation depends on antigen abundance and possibly stability. The cell biological and possible immunological consequences of these results are discussed.
Assuntos
Apresentação de Antígeno , Antígenos de Protozoários/imunologia , Leishmania mexicana/imunologia , Macrófagos/imunologia , Proteínas de Membrana/biossíntese , Linfócitos T/imunologia , Vacúolos/parasitologia , Animais , Antígenos de Protozoários/biossíntese , Feminino , Leishmania mexicana/crescimento & desenvolvimento , Ativação Linfocitária , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Linfócitos T/parasitologia , Vacúolos/imunologiaRESUMO
In a previous publication, we described the purification of a membrane-bound acid phosphatase of Leishmania mexicana as a heterogeneously N-glycosylated protein of an apparent molecular mass of 70000-72000 expressed in both the promastigote and the amastigote stage of the parasite [19]. Screening of a genomic DNA library of L. mexicana with degenerate oligonucleotides designed according to the NH2-terminus of the protein led to the cloning of the lmmbap gene, which is present in one copy per haploid genome. The open reading frame predicts a protein of 516 amino acids composed of a signal sequence, a large hydrophilic region, a trans-membrane alpha-helix and a short cytoplasmic tail. The sequence of the hydrophilic region is homologous to acid phosphatases from other organisms. While in wild-type promastigotes, the acid phosphatase is located in the endosomal/lysosomal compartment between the flagellar pocket and the nucleus, overexpression leads to its abundant exposure on the cell surface. In cells transfected with a construct lacking the region corresponding to the trans-membrane and the cytoplasmic parts, the resulting altered acid phosphatase is efficiently secreted into the culture medium. The potential of this system for studies on membrane trafficking in kinetoplastid organisms is discussed.
Assuntos
Fosfatase Ácida/genética , Genes de Protozoários , Leishmania mexicana/genética , Glicoproteínas de Membrana/genética , Organelas/enzimologia , Fosfatase Ácida/biossíntese , Fosfatase Ácida/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Sequência Conservada , Endossomos/enzimologia , Imunofluorescência , Leishmania mexicana/enzimologia , Leishmania mexicana/ultraestrutura , Lisossomos/enzimologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/isolamento & purificação , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Testes de Precipitina , Proteínas Recombinantes/biossíntese , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Leishmania mexicana amastigotes proliferate in the phagolysosomes of mammalian macrophages. The parasites abundantly synthesize lysosomal cysteine proteinases, which are encoded by the lmcpb gene family. One of these genes was overexpressed in Escherichia coli, and the purified recombinant protein was used as an antigen to induce and establish a T helper 1 (Th1) cell line. The T cells recognize epitopes shared by the native cysteine proteinases and the recombinant protein. Infected bone marrow-derived macrophages induced to express major histocompatibility complex class II molecules by interferon (IFN)-gamma do not affect parasite viability. These macrophages fail to stimulate the proliferation of the T cell line. In contrast, strong T cell stimulation is observed after the parasites are killed by treatment with L-leucine methylester, or after activation of macrophages by IFN-gamma and tumor necrosis factor-alpha. It is concluded that infected macrophages efficiently present this lysosomal Leishmania antigen once the parasites are inactivated and degraded. This observation may be of considerable relevance for the outcome of Leishmania infections provided that it can be extended to other parasite antigens.
Assuntos
Antígenos de Protozoários/imunologia , Cisteína Endopeptidases/imunologia , Leishmania mexicana/imunologia , Leishmaniose Cutânea/imunologia , Macrófagos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Apresentação de Antígeno , Sequência de Bases , Células Cultivadas , Cisteína Endopeptidases/química , DNA Complementar , Ativação Linfocitária , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Linfócitos T Auxiliares-Indutores/citologiaRESUMO
It is well established that Leishmania mexicana amastigotes contain large amounts of cysteine proteinases in their extended lysosomes. In this study it is shown that the cell-free supernatant of homogenized lesion tissue from infected mice contains large amounts of acid proteinases. The majority of this enzymatic activity also corresponds to cysteine proteinases from L. mexicana amastigotes. Immunoelectron microscopy of mouse lesion sections suggests, that frequently amastigotes lyse and release lysosomal cysteine proteinases into the parasitophorous vacuole of infected macrophages. The cysteine proteinases are also found extracellularly in the tissue presumably as a result of macrophage rupture and appear to persist in the lesion tissue, where they may damage host cells and the extracellular matrix.