Donovani-infected Macrophages Research Articles

Uptake of Biodegradable Gel-Assisted LBL Nanomatrix by Leishmania donovani-Infected Macrophages

The aim of this study was to develop novel gel-assisted layer-by-layer (LBL) nanomatrix with high payload of doxorubicin (DOX) and to assess its efficacy against Leishmania donovani. The biodegradable LBL nanomatrix was fabricated using LBL technique using polyions (protamine and sodium alginate) on decomposable core. The developed system was characterized in vitro in terms of layer-by-layer growth and payload efficiency. The efficacy of optimized formulations was evaluated against L. donovani strain in terms of inhibitory concentration (IC50). Uptake studies by infected macrophages were investigated both qualitatively and quantitatively using fluorescence microscopy and flow cytometry. The autogelling property subsequent to core removal inside the nanomatrix resulted in high payload efficiency of DOX (i.e., >70%). The reversal in charge followed the same trend with additional layers, and the magnitude of the charge remained constant up to five complete bilayers of polyions. The DOX can be effectively encapsulated, delivered, and subsequently taken up by L. donovani-infected macrophage cells. The matrix is completely internalized into macrophages showing improved efficacy (IC50 of formulation is almost <or=1.9-fold as compared to plain drug, P<0.05) against intracellular amastigotes. Having ample of opportunity to manipulate surface architecture, this system demonstrates unique platform as a low cost ideal substitute for visceral leishmaniasis to expensive lipid-based formulations.

Miltefosine Promotes IFN-γ-Dominated Anti-Leishmanial Immune Response

Leishmania donovani, a protozoan parasite, resides and replicates as amastigotes within macrophages. The parasite inflicts the disease visceral leishmaniasis by suppressing host cell function. Neither a therapeutic vaccine nor an effective anti-leishmanial drug to reverse the immunosuppression is available. Although miltefosine (hexadecylphosphocholine or HPC) is a promising orally bioavailable anti-leishmanial drug, its efficacy is seriously compromised by contra-indications in pregnant women. Further rational redesigning of the drug requires studies on its mechanism of action, which is unknown at present. Because miltefosine is proposed to have immunomodulatory functions, we examined whether miltefosine exerts its anti-leishmanial functions by activating macrophages. We observed that miltefosine's anti-leishmanial function was significantly compromised in IFN-gamma-deficient macrophages suggesting the importance of endogenous IFN-gamma in miltefosine-induced anti-leishmanial functions of macrophages. Miltefosine induced IFN-gamma, neutralization of which reduced the anti-leishmanial functions of macrophages. IFN-gamma responsiveness is reduced in L. donovani-infected macrophages but is significantly restored by miltefosine, as it enhances IFN-gamma receptors and IFN-gamma induced STAT-1 phosphorylation but reduced activation of SHP-1, the phosphatase implicated in the down-regulation of STAT-1 phosphorylation. Miltefosine induced protein kinase C-dependent and PI3K-dependent p38MAP kinase phosphorylation and anti-leishmanial function. Miltefosine promotes p38MAP kinase-dependent anti-leishmanial functions and IL-12-dependent Th1 response. Leishmania donovani-infected macrophages induced Th2 response but miltefosine treatment reversed the response to Th1-type. Thus, our data define for the first time the mechanistic basis of host cell-dependent anti-leishmanial function of miltefosine.

Quassin alters the immunological patterns of murine macrophages through generation of nitric oxide to exert antileishmanial activity

The aim of this study was to characterize the in vitro antileishmanial activity of quassin, a traditional Chinese herbal medicine. The cytotoxic effect of quassin was studied in murine peritoneal macrophages at various concentrations using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide method. The role of quassin as an antileishmanial agent was evaluated by microscopic counting of intracellular amastigotes in macrophages stained with Giemsa. To understand the effector mechanism of quassin-treated macrophages against leishmanial parasites, western blot and real-time PCR analysis of inducible nitric oxide (NO) synthase 2 (iNOS2) were done followed by measurement of NO generation by Griess reaction. The effect of quassin on the production of Th1 cytokines such as interleukin (IL)-12 and tumour necrosis factor (TNF)-alpha and Th2 cytokines such as IL-10 and transforming growth factor-beta was measured by ELISA, and the mRNA expression of these cytokines was analysed by real-time PCR. Quassin at a dose of 25 microg/mL (64.36 microM) showed less cytotoxicity to the host murine peritoneal macrophages but at the same dose was effective enough to control the intracellular parasitic load compared with higher doses of quassin. Leishmania donovani is known to exert its pathogenic effects mainly by the suppression of NO generation and subversion of the cellular inflammatory responses in the macrophages. Quassin was found to induce a potent host-protective immune response by enhancing NO generation and iNOS2 expression both at a protein and mRNA level and by up-regulating pro-inflammatory cytokines such as TNF-alpha and IL-12 in L. donovani-infected macrophages with concurrent inhibition of anti-inflammatory responses. These findings strongly support the effectiveness of quassin as a potent immunomodulatory tool for controlling the establishment of leishmanial parasite within the host macrophages.

Comparison of the Effects ofLeishmania majororLeishmania donovaniInfection on Macrophage Gene Expression

The intracellular parasite Leishmania causes a wide spectrum of human disease, ranging from self-resolving cutaneous lesions to fatal visceral disease, depending on the species of Leishmania involved. The mechanisms by which different Leishmania species cause different pathologies are largely unknown. We have addressed this question by comparing the gene expression profiles of bone marrow-derived macrophages infected with either Leishmania donovani or L. major promastigotes. We found that the two species had very similar effects on macrophage gene expression. Both species caused a small (<2.5-fold) but statistically significant repression of several hundred genes. In addition, both species strongly induced and repressed about 60 genes. Comparing the effects of the two species showed that only 26 genes were regulated differently by L. major as opposed to L. donovani, including those for metallothioneins 1 and 2, HSP70 and -72, CCL4, Gadd45beta, Dsp1, matrix metalloprotease 13, T-cell death-associated gene 51 (Tdag51), RhoB, spermine/spermidine N1-acyl transferase 1 (SSAT), and Cox2. L. donovani-infected macrophages were also found to express higher levels of Cox2 protein and prostaglandin E synthase mRNA than L. major-infected macrophages. While both species have previously been shown to trigger prostaglandin E synthesis by bystander cells, this study suggests that infected macrophages themselves express prostaglandin E-synthesizing genes only in response to L. donovani.

Morphine-induced Neuroimmunomodulation in Murine Visceral Leishmaniasis: The Role(s) of Cytokines and Nitric Oxide

Opioid modulation of host resistance to infectious diseases is well documented; however, not much is known during visceral leishmaniasis (VL). Low doses of morphine, administered subcutaneously in Leishmania donovani-infected BALB/c mice, on days 0 and +15, significantly (p < 0.05) suppressed (1 mg/kg/day) or even sterile-cleared (2 mg/kg/day) the infection; paradoxically, high doses (10 and 30 mg/kg/day) exacerbated the infection. In vitro, low concentration (1 x 10(-9) and 1 x 10(-11) M) morphine treatment of L. donovani-infected mouse peritoneal macrophages (PM), endowed them with significant (p < 0.05) leishmanicidal activity, whereas a high-concentration (1 x 10(-5) M) treatment augmented intramacrophage parasite growth. Naloxone pre-treatment of infected-mice (4 mg/kg x 2) and of infected-PM (1 x 10(-5) M), blocked only the morphine low dose/concentration-induced protective effect. The splenocytes from protected mice and morphine low concentration-treated infected-PM, elaborated significantly (p < 0.05) enhanced levels of interleukin-12, interferon-gamma, tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor and nitrite in the culture medium; a high dose/concentration suppressed their elaboration. Curiously, only morphine high dose/concentration-treated infected mice splenocytes and infected PM, produced significantly (p < 0.05) increased quantity of transforming growth factor-beta1. Aminoguanidine, significantly (p < 0.05) blocked the morphine low dose/concentration-induced protective effect, in vivo and in vitro. This first study demonstrates dose-dependent biphasic modulatory effects of morphine in L. donovani-infected mice and PM, in vitro, apparently via nitric oxide-dependent mechanisms. These results thus demonstrate the implications of opiate abuse on the efficacy assessment of antileishmanial drugs and vaccines, and on the reactivation of latent VL in areas where both drug abuse and VL are rampant.

Leishmania donovani-Induced Ceramide as the Key Mediator of Akt Dephosphorylation in Murine Macrophages: Role of Protein Kinase Cζ and Phosphatase

Leishmania donovani is an intracellular protozoan parasite that impairs the host macrophage immune response to render it suitable for its survival and establishment. L. donovani-induced immunosuppression and alteration of host cell signaling is mediated by ceramide, a pleiotropic second messenger playing an important role in regulation of several kinases, including mitogen-activated protein kinase and phosphatases. We observed that the endogenous ceramide generated during leishmanial infection led to the dephosphorylation of protein kinase B (PKB) (Akt) in infected cells. The study of ceramide-mediated Akt phosphorylation revealed that Akt was dephosphorylated at both Thr308 and Ser473 sites in infected cells. Further investigation demonstrated that ceramide was also responsible for the induction of PKCzeta, an atypical Ca-independent stress kinase, as well as the ceramide-activated protein phosphatases (e.g., protein phosphatase 2A [PP2A]). We found that Akt dephosphorylation was mediated by ceramide-induced PKCzeta-Akt association and PP2A activation. In addition, treatment of L. donovani-infected macrophages with PKCzeta-specific inhibitor peptide could restore the translocation of phosphorylated Akt to the cell membrane. This study also revealed that ceramide is involved in the inhibition of proinflammatory cytokine tumor necrosis factor alpha release by infected macrophages. These observations strongly suggest the importance of ceramide in the alteration of normal cellular functions, impairment of the kinase/phosphatase balance, and thereby establishment of leishmaniasis in the hostile macrophage environment.

Regulation of Impaired Protein Kinase C Signaling by Chemokines in Murine Macrophages during Visceral Leishmaniasis

The protein kinase C (PKC) family regulates macrophage function involved in host defense against infection. In the case of Leishmania donovani infection, the impairment of PKC-mediated signaling is one of the crucial events for the establishment of parasite into the macrophages. Earlier reports established that C-C chemokines mediated protection against leishmaniasis via the generation of nitric oxide after 48 h. In this study, we investigated the role of MIP-1alpha and MCP-1 in the regulation of impaired PKC activity in the early hours (6 h) of infection. These chemokines restored Ca2+-dependent PKC activity and inhibited Ca2+-independent atypical PKC activity in L. donovani-infected macrophages under both in vivo and in vitro conditions. Pretreatment of macrophages with chemokines induced superoxide anion generation by activating NADPH oxidase components in infected cells. Chemokine administration in vitro induced the migration of infected macrophages and triggered the production of reactive oxygen species. In vivo treatment with chemokines significantly restricted the parasitic burden in livers as well as in spleens. Collectively, these results indicate a novel regulatory role of C-C chemokines in controlling the intracellular growth and multiplication of L. donovani, thereby demonstrating the antileishmanial properties of C-C chemokines in the disease process.

Leishmania donovani Affects Antigen Presentation of Macrophage by Disrupting Lipid Rafts

Leishmania donovani-infected splenic macrophages and P388D1 (P388D1(I)) failed to activate T cells in response to low dose of exogenous peptide. The membrane fluidity of P388D1(I) was greater than that of the normal counterpart P388D1(N), but could be reduced either by exposing the cell below phase transition point or by loading cholesterol into membrane (L-P388D1(I)), and this was associated with enhanced Ag-presenting ability of P388D1(I). Presentation of endogenous leishmanial Ag, kinetoplastid membrane protein-11, was also defective, but could be corrected by loading cholesterol into membrane. Because membrane rafts are important for Ag presentation at a low peptide dose, raft architecture of P388D1(I) was studied using raft (CD48 and cholera toxin-B) and non-raft (CD71) markers in terms of their colocalization with I-A(d). Binding of anti-CD48 mAb and cholera toxin B subunit decreased significantly in P388D1(I), and consequently, colocalization with I-A(d) was not seen, but this could be restored in L-P388D1(I). Conversely, colocalization between I-A(d) and CD71 remained unaffected regardless of the presence or the absence of intracellular parasites. P388D1(N) and L-P388D1(I), but not P388D1(I), formed peptide-dependent synapse with T cells quite efficiently and this was found to be corroborated with both intracellular Ca2+ mobilization in T cells and IL-2 production. This indicated that intracellular parasites disrupt the membrane rafts, possibly by increasing the membrane fluidity, which could be corrected by making the membrane rigid. This may be a strategy that intracellular L. donovani adopts to evade host immune system.

Proteasome-mediated Degradation of STAT1α following Infection of Macrophages with Leishmania donovani

Activation of the Janus-activated kinase 2 (JAK2)/STAT1alpha signaling pathway is repressed in Leishmania-infected macrophages. This represents an important mechanism by which this parasite subverts the microbicidal functions of the cell to promote its own survival and propagation. We recently provided evidence that the protein tyrosine phosphatase (PTP) SHP-1 was responsible for JAK2 inactivation. However, STAT1 translocation to the nucleus was not restored in the absence of SHP-1. In the present study, we have used B10R macrophages to study the mechanism by which this Leishmania-induced STAT1 inactivation occurs. STAT1alpha nuclear localization was shown to be rapidly reduced by the infection. Western blot analysis revealed that cellular STAT1alpha, but not STAT3, was degraded. Using PTP inhibitors and an immortalized bone marrow-derived macrophage cell line from SHP-1-deficient mice, we showed that STAT1 inactivation was independent of PTP activity. However, inhibition of macrophage proteasome activity significantly rescued Leishmania-induced STAT1alpha degradation. We further demonstrated that degradation was receptor-mediated and involved protein kinase C alpha. All Leishmania species tested (L. major, L. donovani, L. mexicana, L. braziliensis), but not the related parasite Trypanosoma cruzi, caused STAT1alpha degradation. Collectively, results from this study revealed a new mechanism for STAT1 regulation by a microbial pathogen, which favors its establishment and propagation within the host.

Leishmania donovani-induced expression of suppressor of cytokine signaling 3 in human macrophages: a novel mechanism for intracellular parasite suppression of activation.

Leishmania donovani protozoan parasites, the causative agent of visceral leishmaniasis, establish an infection partly by interfering with cytokine signaling in the host macrophages. Therefore, we investigated the expression of the suppressor of cytokine signaling (SOCS) genes in human macrophages infected with L. donovani. The expression of SOCS3 mRNA was induced transiently after exposure to live or heat-killed parasites, but not purified lipophosphoglycan, while that of other SOCS genes remained unchanged. SOCS3 gene expression was not dependent on phagocytosis or on cytokines released by L. donovani-infected macrophages, such as interleukin-1beta or tumor necrosis factor alpha. In addition, Leishmania used a different signaling pathway(s) than bacterial lipopolysaccharide to induce SOCS3 mRNA, as indicated by the kinetics of induction and sensitivity to polymyxin B inhibition. Finally, phosphorylation of the STAT1 transcription factor was significantly reduced in L. donovani-infected macrophages and required de novo transcription. The induction of SOCS3 provides a potent inhibitory mechanism by which intracellular microorganisms may suppress macrophage activation and interfere with the host immune response.

Chemokine-induced leishmanicidal activity in murine macrophages via the generation of nitric oxide.

This study explored the role of the proinflammatory chemokines macrophage inflammatory protein (MIP)-1alpha and macrophage chemoattractant protein (MCP)-1 for development of antileishmanial activity. There was substantial inhibition in nitrite generation in Leishmania donovani-infected macrophages. A marked elevation of nitrite generation and induction of inducible nitric oxide (NO) synthase (iNOS) mRNA was found in chemokine-primed parasite-infected macrophages. Tumor necrosis factor-alpha, which is the priming signal for NO production, was also up-regulated under similar experimental conditions. The priming with chemokine inhibited the multiplication of L. donovani amastigotes within the intramacrophageal milieu. The antileishmanial effect of chemokines was almost completely abrogated when the macrophages were preincubated with l-N-monomethyl arginine, the specific inhibitor of iNOS. The results of this investigation suggest that the CC chemokines MIP-1alpha and MCP-1 orchestrate an antileishmanial armamentarium via the induction of an NO-mediated regulatory mechanism to control the intracellular growth and multiplication of the Leishmania protozoan.

The kinetoplastid membrane protein 11 of Leishmania donovani and African trypanosomes is a potent stimulator of T-lymphocyte proliferation.

Kinetoplastid membrane protein 11 (KMP-11) from Leishmania donovani is an abundant 11-kDa surface membrane glycoprotein. Lymph node cells from mice of six different H-2 haplotypes immunized with KMP-11 or with L. donovani promastigotes were stimulated to proliferate in vitro KMP-11. Primed purified T cells required antigen presentation since they were not stimulated unless KMP-11-pulsed or L. donovani-infected macrophages were added. Promastigotes of a wide variety of Leishmania species and procyclic forms of African trypanosomes stimulated proliferation of KMP-11-primed or L. donovani promastigote-primed lymph node cells. All of the Leishmania promastigotes and African trypanosomes tested contained an 11-kDa protein, as detected by immunoblotting with KMP-11-specific monoclonal antibodies. The widespread distribution of the 11-kDa (KMP-11) molecules and their ability to stimulate strong T-lymphocyte proliferation in a non-H-restricted fashion suggest that they may be important molecules for induction of cell-mediated immune responses.

Neoglycosylated liposomes as efficient ligands for the evaluation of specific sugar receptors on macrophages in health and in experimental leishmaniasis.

Receptors interacting with terminal sugars as ligands are involved in the binding of Leishmania donovani promastigotes to the macrophage surface and their subsequent internalization. Mannose and glucose are specifically involved in the binding process. Decreased binding occurs to macrophages already infected with L. donovani either in vivo or in vitro. When mannose- or glucose-bearing liposomes are used as ligands the binding shows similar trends and the percentage inhibition of binding with mannose-bearing liposomes increases when compared to that for the glucose-bearing ones. The decreased binding of the ligand seems to be due to a decrease in the number of receptors after infection. The affinity of the ligands for the binding sites either on the normal macrophages or on the infected macrophages apparently remains the same. The results based on the incorporation of [3H]phenyl alanine and supported by the binding of glycosylated liposomes to both infected and non-infected macrophages suggest that protein synthesis, in general, is suppressed in L. donovani-infected macrophages thus affecting also mannose/glucose receptor protein synthesis, resulting in fewer receptors on the macrophage surface.

Leishmania donovani-infected macrophages: characterization of the parasitophorous vacuole and potential role of this organelle in antigen presentation.

Leishmania donovani amastigotes, the etiological agents of visceral leishmaniasis, are obligate intracellular parasites residing in membrane-bound compartments of macrophages called parasitophorous vacuoles (PV). The study of these organelles is of paramount importance to understanding how these parasites resist the microbicidal mechanisms of macrophages and how they escape the immune response of their hosts. Confocal microscopy of mouse bone marrow-derived macrophages infected with L. donovani amastigotes and stained for various prelysosomal/lysosomal markers and for major histocompatibility complex (MHC) molecules was used to define PV with respect to the endocytic compartments of the host cells and to address the issue of their potential role in antigen processing and presentation. Forty-eight hours after infection, many PV contained cathepsins B, D, H and L and they were all surrounded by a membrane enriched for the lysosomal glycoprotein lgp120/lamp 1 but apparently devoid of the cation-independent mannose 6-phosphate receptor, a membrane protein generally absent from the lysosomes. These data suggested that PV acquire within 48 hours the characteristics of a lysosomal compartment. However, both macrosialin and the GTP-binding protein rab7p (specific markers of the prelysosomal compartment) were found to be highly expressed in/on PV membrane. Thus, at this stage, PV appear to exhibit both lysosomal and prelysosomal features. Infected macrophages activated with IFN-gamma before or after infection showed PV strongly stained for MHC class II molecules but not for MHC class I molecules. This suggests that, if infected macrophages can act as antigen-presenting cells for class I-restricted CD8+ T lymphocytes, Leishmania antigens must exit the PV. MHC class II molecules reached the PV progressively, indicating that they were not plasma membrane-bound molecules trapped during internalization of the parasites. The redistribution of class II observed in infected cells did not alter their quantitative expression on the plasma membrane at least during the first 48 hours following the phagocytosis of the parasites. The invariant chains, which are transiently associated with class II molecules during their intracellular transport and which mask their peptide-binding sites, did not reach PV or were rapidly degraded in these sites, suggesting that PV-associated class II are able to bind peptides. This last assumption is strengthened by the fact that class II located in PV could bind conformational antibodies that preferentially recognize class II with tightly associated peptides.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular infection by Leishmania donovani inhibits macrophage apoptosis.

The phagocytic macrophage plays a critical role in host immune responses to microbial infection, and represents a major source of inflammatory and growth cytokines. Intramacrophage infection by the protozoan parasite Leishmania donovani results in increased viability of the host cell in the absence of exogenous growth factor. We demonstrate that infection of bone marrow-derived macrophages (BMMs) by L. donovani promastigotes or treatment of BMMs with lipophosphoglycan LPG, the major surface molecule of the promastigote, inhibits apoptosis in the macrophage induced by the removal of macrophage (M)-CSF. This effect was also achieved by supernatants collected from L. donovani-infected macrophages, implicating the elaboration of a soluble factor by infected cells as the mediator of this inhibition. To identify candidate factors, reverse transcription PCR was employed to characterize the mRNA cytokine profile of infected macrophages. L. donovani infection of BMMs was found to induce gene expression for granulocyte-macrophage CSF, TNF-alpha, TGF-beta, and IL-6, but not M-CSF or IL-1 beta. Of the cytokines induced by L. donovani, rTNF-alpha and recombinant granulocyte-macrophage CSF were shown to inhibit apoptosis of BMMs induced by the removal of M-CSF. The amount of these cytokines in L. donovani-infected cell supernatants was quantified by ELISA. The mechanism by which L. donovani may inhibit apoptosis is discussed.

Leishmania donovani infection enhances macrophage viability in the absence of exogenous growth factor

Bone marrow-derived macrophages rapidly die in the absence of macrophage growth factor (M-CSF). However, as demonstrated here, bone marrow-derived macrophages infected with Leishmania donovani exhibit increased viability in the absence of exogenous growth factor. Forty-eight hours after inoculation with promastigotes or amastigotes, infected cell cultures contained 180 and 95% more cells, respectively, than control cultures. This effect was specific to Leishmania infection, as uptake of latex beads or avirulent promastigotes by macrophages did not enhance cell viability. L. donovani-infected macrophages also displayed increased phagocytic capacity, as compared with control macrophages and macrophages grown continuously in M-CSF-containing medium. Supernatants collected from infected cells elaborated a factor(s) that enhanced macrophage viability but did not stimulate macrophage DNA synthesis. This activity of L. donovani-infected cell-conditioned medium could be abrogated by preincubation of macrophages with cycloheximide before inoculation with the parasite, implying that macrophage protein synthesis is required for the elaboration of this factor(s).

Antileishmanial drug targeting through glycosylated polymers specifically internalized by macrophage membrane lectins.

Antileishmanial chemotherapy is hampered by the location of the parasite within the phagolysosome of the macrophage, which restricts the bioavailability of many potentially useful antileishmanial drugs. In this study, the possibility of using antileishmanial drugs targeted to the infected macrophages by means of a chemical linkage to a neutral mannose-substituted poly-L-lysine carrier molecule was explored. The study was performed in an in vitro model with Leishmania donovani-infected murine macrophages. The antileishmanial activities of various synthetic constructs were compared with those of the free drugs and the pentavalent antimonial Pentostam, which was used as the positive control. The 50% effective dose of allopurinol riboside linked to the mannosylated poly-L-lysine was below 7.5 x 10(-6) M, while it was up to 3 x 10(-4) M for the free drug, indicating that the drug bound to the polymer was 50 times more active than the free drug. Control experiments with other constructs (e.g., allopurinol riboside linked to the mannose-free polymer) confirmed that the enhancement of activity was indeed achieved by means of the mannose homing device.

C-fos and tumor necrosis factor gene expression in Leishmania donovani-infected macrophages.

Leishmania donovani is an obligate intracellular protozoan which resides in macrophages and impairs a number of macrophage functions. We have undertaken to study this host cell-parasite interaction by examining the ability of L. donovani to impair the transmission of information from the cell surface to the nucleus and thus influence normal gene expression. We demonstrate that, in response to lipopolysaccharide, expression of both the c-fos and tumor necrosis factor genes was impaired in L. donovani-infected macrophages. Indomethacin reversed the parasite-mediated downregulation of the tumor necrosis factor gene but not the c-fos gene, suggesting that the impaired expression of these two genes occurred through different mechanisms. Direct stimulation of protein kinase C with oleoyl-2-acetoyl-3-glycerol did not abrogate inhibition of c-fos gene expression by L. donovani; however, L929 cell-conditioned medium induced a similar level of c-fos gene expression in both infected and noninfected macrophages. Impairment of c-fos gene expression by L. donovani thus appeared to be selective, depending on the external stimuli used to induce its expression. These data argue that L. donovani was capable of impairing macrophage gene expression in a selective rather than a general manner.

C-fos and Tumor Necrosis Factor Gene Expression in Leishmania donovani-Infected Macrophages

Leishmania donovani is an obligate intracellular protozoan which resides in macrophages and impairs a number of macrophage functions. We have undertaken to study this host cell-parasite interaction by examining the ability of L. donovani to impair the transmission of information from the cell surface to the nucleus and thus influence normal gene expression. We demonstrate that, in response to lipopolysaccharide, expression of both the c-fos and tumor necrosis factor genes was impaired in L. donovani-infected macrophages. Indomethacin reversed the parasite-mediated downregulation of the tumor necrosis factor gene but not the c-fos gene, suggesting that the impaired expression of these two genes occurred through different mechanisms. Direct stimulation of protein kinase C with oleoyl-2-acetoyl-3-glycerol did not abrogate inhibition of c-fos gene expression by L. donovani; however, L929 cell-conditioned medium induced a similar level of c-fos gene expression in both infected and noninfected macrophages. Impairment of c-fos gene expression by L. donovani thus appeared to be selective, depending on the external stimuli used to induce its expression. These data argue that L. donovani was capable of impairing macrophage gene expression in a selective rather than a general manner.

Eicosanoid metabolism by Leishmania donovani-infected macrophages: mouse strain responses in prostanoid synthesis.

Arachidonic acid (20:4) conversion to prostanoids was examined in murine peritoneal macrophages infected in vitro with Leishmania donovani. Four strains of mice differing in resistance to in vivo L. donovani infection were studied. Normal macrophages from all strains converted 20:4 to prostanoids and this was augmented by L. donovani infection. Although cells from each strain synthesized elevated levels of prostaglandin-E2 (PGE2), there were differences with respect to relative increases of this product, with infected macrophages from the C3H/HeJ strain showing the smallest increase above basal levels. These results indicate that macrophages from mouse strains with distinct levels of in vivo resistance to L. donovani all respond to infection with augmented prostanoid synthesis. Although some heterogeneity in strain-specific PGE2 responses to in vitro infection were observed (with respect to increases in PGE2 synthesis above basal levels), it is unlikely that differential resistance of these strains to in vivo infection is strictly related to these relative differences. It seems likely that other genetically controlled factors may have a major impact on disease expression.