Dysregulation of macrophage signal transduction by Toxoplasma gondii: past progress and recent advances.

The opportunistic protozoan parasite Toxoplasma gondii is well known as a strong inducer of cell-mediated immunity, largely as a result of proinflammatory cytokine induction during in vivo infection. Yet, during intracellular infection the parasite suppresses signal transduction pathways leading to these proinflammatory responses. The opposing responses are likely to reflect the parasite's need to stimulate immunity allowing host survival and parasite persistence, and at the same time avoiding excessive responses that could result in parasite elimination and host immunopathology. This Review summarizes past and present investigations into the effects of Toxoplasma on host cell signal transduction. These studies reveal insight into the profound suppression of proinflammatory cytokine responses that occurs when the parasite infects macrophages and other cells of innate immunity.

Functional aspects of Toll-like receptor/MyD88 signalling during protozoan infection: focus on Toxoplasma gondii.

Toll-like receptor (TLR)/MyD88 signalling has emerged as a major pathway of pathogen recognition in the innate immune system. Here, we review recent data that begin to show how this pathway controls the immune response to protozoan infection, with particular emphasis on the opportunistic pathogen Toxoplasma gondii. The various ways that the parasite activates and suppresses TLR/MyD88 signalling defines several key principals that illuminate the complexities of the host-pathogen interaction. We also speculate how TLR/MyD88 signalling might be exploited to provide protection against Toxoplasma, as well as other protozoa and infection in general.

Toxoplasma gondii tachyzoites inhibit proinflammatory cytokine induction in infected macrophages by preventing nuclear translocation of the transcription factor NF-kappa B.

Control of microbial infection requires regulated induction of NF-kappaB-dependent proinflammatory cytokines such as IL-12 and TNF-alpha. Activation of this important transcription factor is driven by phosphorylation-dependent degradation of the inhibitory IkappaB molecule, an event which enables NF-kappaB translocation from the cytoplasm to the nucleus. In this study, we show that intracellular infection of macrophages with the protozoan parasite Toxoplasma gondii induces rapid IkappaB phosphorylation and degradation. Nevertheless, NF-kappaB failed to translocate to the nucleus, enabling the parasite to invade cells without triggering proinflammatory cytokine induction. Infected cells subsequently subjected to LPS triggering were severely crippled in IL-12 and TNF-alpha production, a result of tachyzoite-induced blockade of NF-kappaB nuclear translocation. Our results are the first to demonstrate the ability of an intracellular protozoan to actively interfere with the NF-kappaB activation pathway in macrophages, an activity that may enable parasite survival within the host.

Rapid recruitment of neutrophils containing prestored IL-12 during microbial infection.

Neutrophils are well known to rapidly migrate to foci of infection, where they exert microbicidal functions. We sought to determine whether neutrophils responding to in vivo infection with the protozoan pathogen Toxoplasma gondii were capable of IL-12 production as suggested by recent in vitro studies. Intraperitoneal infection induced a neutrophil influx by 4 h, accompanied by ex vivo IL-12 p40 and p70 release. Approximately 85% of the neutrophils displayed intracellular stores of IL-12, as determined by flow cytometry and confocal fluorescence microscopy. Neutrophils from IFN-gamma knockout mice also expressed IL-12, ruling out an IFN-gamma-priming requirement. Neither infected nor uninfected peritoneal macrophages displayed intracellular IL-12, but these cells were strongly IL-10(+). Infection per se was unnecessary for IL-12 production because peritoneal and peripheral blood neutrophils from uninfected animals contained IL-12(+) populations. Expression of the granulocyte maturation marker Gr-1 (Ly-6G) was correlated with IL-12 production. Mice depleted of their granulocytes by mAb administration at the time of infection had decreased serum levels of IL-12 p40. These results suggest a model in which neutrophils with prestored IL-12 are rapidly mobilized to an infection site where they are triggered by the parasite to release cytokine. Our findings place neutrophils prominently in the cascade of early events leading to IL-12-dependent immunity to T. gondii.

Larvae-induced plasma membrane wounds and glycoprotein deposition are insufficient for Trichinella spiralis invasion of epithelial cells.

Trichinella spiralis L1 larvae infect susceptible hosts by invading epithelial cells that line the small intestine. Invasion also occurs in vitro when larvae are inoculated into cultures of epithelial cells from several different animal species. To further investigate the mechanism of invasion, we studied the interaction of larvae with the rat epithelial cell line IEC-6. Larvae did not invade IEC-6 cells, but did cause the cells to take up parasite glycoproteins. Glycoprotein bearing cells remained viable and were detectable in monolayers for as long as 24 h, suggesting that the glycoproteins were not toxic for cells. Immunofluorescence revealed that parasite glycoproteins localized in the nuclei, mitochondria and cytoplasm and we found evidence for selection of certain molecules between nuclear and cytoplasmic compartments. Using fluorescent dextrans as fluid phase markers we found 17-38% of the cells in inoculated monolayers were engorged with dextran and that dextran was free in the cytoplasm. Increased dextran uptake was not lethal, required the presence of activated larvae, and was often associated with uptake of parasite glycoproteins. These observations suggest that larvae caused plasma membrane wounds. Our results showed that neither delivery of glycoproteins nor mechanical wounding is sufficient to allow entry of the parasite into resistant epithelial cells. Because both invasion-resistant and susceptible epithelial cells undergo non-lethal wounding, we propose that larvae-induced injury to epithelial cells may result in release of cell-specific mediators that signal larva to invade a particular cell line or, alternatively, to ignore it.

Perforin-mediated cytolysis plays a limited role in host resistance to Toxoplasma gondii.

Resistance of perforin knockout (PKO) mice to infection with Toxoplasma gondii was assessed in models of acute infection and during chronic disease. PKO mice vaccinated with the attenuated mutant, ts-4, displayed severely defective CTL responses against tachyzoite-infected targets. Lysis of the NK target, YAC-1, was also severely impaired in PKO mice following ts-4 vaccination. In contrast, wild-type mice developed high levels of CTL and NK lytic activity after ts-4 vaccination. Despite severely defective lytic activity, vaccinated PKO animals were completely resistant to challenge with the virulent strain RH, which normally causes a lethal acute infection. Resistance was attributable to production of IFN-gamma, which remained unimpaired in the PKO animals. In contrast, when PKO mice were infected with low virulence parasite strain ME49, which progresses to the cyst-forming stage after passage through an acute phase, accelerated mortality was observed beginning at 75 days postinfection. A three- to fourfold increase in brain cyst numbers was also found by day 30 in infected PKO animals. Nevertheless, the PKO strain produced normal levels of IFN-gamma after ME49 infection, ruling out impaired production of the latter cytokine as a cause of increased susceptibility. Together, these results show that perforin-dependent cytolytic function is not required for host resistance to lethal acute infection in preimmunized animals, but that the latter activity contributes to the control of infection during the chronic stage.

Deficiency in beta1,3-galactosyltransferase of a Leishmania major lipophosphoglycan mutant adversely influences the Leishmania-sand fly interaction.

To study the function of side chain oligosaccharides of the cell-surface lipophosphoglycan (LPG), mutagenized Leishmania major defective in side chain biosynthesis were negatively selected by agglutination with the monoclonal antibody WIC79.3, which recognizes the galactose-containing side chains of L. major LPG. One such mutant, called Spock, lacked the ability to bind significantly to midguts of the natural L. major vector, Phlebotomus papatasi, and to maintain infection in the sand fly after excretion of the digested bloodmeal. Biochemical characterization of Spock LPG revealed its structural similarity to the LPG of Leishmania donovani, a species whose inability to bind to and maintain infections in P. papatasi midguts has been strongly correlated with the expression of a surface LPG lacking galactose-terminated oligosaccharide side chains. An in vitro galactosyltransferase assay using wild-type or Spock membranes was used to determine that the defect in Spock LPG biosynthesis is a result of defective beta1,3-galactosyltransferase activity as opposed to a modification of LPG, which would prevent it from serving as a competent substrate for galactose addition. The results of these experiments show that Spock lacks the beta1, 3-galactosyltransferase for side chain addition and that the LPG side chains are required for L. major to bind to and to produce transmissible infection in P. papatasi.

Ligand-induced adhesion to activated endothelium and to vascular cell adhesion molecule-1 in lymphocytes transfected with the N-formyl peptide receptor.

Binding of FMLP to the neutrophil N-formyl peptide receptor (FPR) transmits signals through pertussis toxin-sensitive G proteins triggering Ca2+ flux, superoxide production, granule exocytosis, and neutrophil aggregation and adhesion involving the beta 2 (CD18) integrins. Expression of the FPR in mouse fibroblasts or human kidney cells has been shown to confer an N-formyl peptide-inducible Ca2+ flux in transfectants. Here we demonstrate that the transfected receptor can also support ligand-induced alterations in cellular adhesion. We established stable transfectants of mouse L1-2 pre-B cells with cDNA for human FPR (L1-2 FPR cells). The transfectants bind N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein with 1.4 x 10(5) sites per cell and a dissociation constant of 3.3 nM. Stimulation with FMLP induces a transient Ca2+ flux. FMLP also triggers adhesion of L1-2 FPR cells to TNF-alpha- or LPS-activated bEnd3 cells (mouse brain-derived endothelial cells) and to purified mouse VCAM-1. Binding is inhibited by Abs to VCAM-1 and to the alpha-chain of its lymphocyte receptor (the alpha 4 beta 1 integrin, VLA-4). Stimulation with FMLP does not induce a change in cell surface expression of alpha 4. Induced adhesion to VCAM-1 is rapid, detectable at the earliest times measurable (30 to 60 s after FMLP addition), and is inhibited by pertussis toxin. We conclude that FPR can mediate integrin activation not only in neutrophils but also in lymphocytes, and can trigger rapid adhesion via lymphocyte alpha 4 beta 1. The adhesion of lymphocytes is critical to their migration and targeting; our results suggest the possibility of manipulating adhesive responses through expression of chemoattractant receptors in lymphoid cells engineered for cellular therapy, allowing targeted adhesion and potentially migration in response to locally administered ligands.

Decreased renal excretion of beta-hexosaminidase in adults with insulin-dependent diabetes mellitus and normal renal function.

The activities of three lysosomal hydrolases and creatinine levels were measured in the plasma and urine of 11 adults (mean age, 28.1 years) with insulin-dependent diabetes mellitus and 14 non-diabetic controls (mean age, 27.9 years). All of the patients were free of diabetic complications and non exhibited microalbuminuria. Fractional enzyme excretion (FEE) values between the two groups of subjects were calculated and compared for the following enzymes: beta-hexosaminidase (N-acetyl-glucosaminidase), beta-glucuronidase and alpha-galactosidase. The FEE value was calculated as the ratio of enzyme clearance to creatinine clearance. Relative to the non-diabetic control group, the FEE value for beta-hexosaminidase was approximately 2-fold lower (P = 0.02) in the diabetic subjects (means, 0.424 vs. 0.242, respectively). The FEE values for beta-glucuronidase and alpha-galactosidase were not significantly different (P > 0.4) between the diabetic and control groups. These easily measured biochemical parameters in blood and urine and the resultant FEE value for beta-hexosaminidase may provide a means of assessing subtle deteriorative changes in renal function which occur in the early stage of diabetes before the onset of clinically evident complications.

Heparin enhances the interaction of infective Leishmania donovani promastigotes with mouse peritoneal macrophages. A fluorescence flow cytometric analysis.

Human visceral leishmaniasis results from the infection of macrophages by the protozoan parasite Leishmania donovani. Both forms of the parasite, the extracellular promastigote and the obligate intracellular amastigote, require cell surface molecules to ensure their recognition and uptake by the host cell, the macrophage. We have proposed previously that the heparin-binding protein on the surface of promastigotes is an adhesion molecule. The present report provides experimental evidence to support this hypothesis. Fluorescence flow cytometry using FITC-heparin was employed to study the heparin-binding protein of L. donovani promastigotes and amastigotes. We demonstrate the presence of the heparin-binding protein on the surface of amastigotes and document the heparin specificity of the binding protein for both forms of the parasite. Two-color fluorescence analysis was performed to compare R-PNA reactivity and FITC-heparin binding during the parasite's 7-day growth curve. Using this strategy we show that the expression of heparin binding activity coincides with the differentiation of the noninfective promastigote into the infective metacyclic from of the parasite. Macrophages that were challenged for 30 min with heparin-treated, FITC-labeled parasites became 2.82-fold more fluorescent than their counterparts which were exposed to non-heparin-treated FITC-labeled promastigotes. Finally, using Kolmogorov-Smirnov analysis we show that the adhesion of promastigotes to mouse peritoneal macrophages is significantly enhanced in the presence of 3.3 microM heparin. The experiments described in the present report provide evidence for the hypothesis that L. donovani's heparin-binding protein is a virulence factor that functions as an adhesion molecule in the parasite-macrophage interaction.

Leishmania donovani: cell-surface heparin receptors of promastigotes are recruited from an internal pool after trypsinization.

With the use of [3H]heparin, we recently demonstrated that Leishmania donovani promastigotes express a cell-surface receptor that is specific for the glycosaminoglycan heparin (Mukhopadhyay et al. 1989, The Biochemical Journal, 264, 517-525.). Treatment of the parasite with trypsin abolishes 75-90% of this [3H]heparin-binding activity. When trypsinized promastigotes were resuspended in fresh culture medium in the absence and presence of cycloheximide (10 micrograms/ml), approximately 25-30% of the original heparin-binding capacity was restored within 1 hr, indicating that recruitment of receptors from an internal pool occurred without de novo protein synthesis. Scatchard analysis of the regenerated receptor revealed that the number of regenerated binding sites per cell was 2.3 x 10(5); these sites have a binding affinity of 6.7 x 10(-7) M. Like the native heparin receptors on the surface of freshly isolated cells, the receptors recruited after trypsinization are also highly specific for heparin, as a 25-fold excess of four other glycosaminoglycans displaced less than 10% of bound [3H]heparin from the trypsinized cells. The structural requirements of the ligand heparin, namely the number of monosaccharide units and degree of sulfation, were compared for both the native and regenerated receptor: for both receptors, oversulfated polysaccharide heparin fragments of at least six to eight sugar residues were most efficient at displacing [3H]heparin. The concentrations of oligosaccharide fragments required to displace 50% of [3H]heparin were 0.32 and 0.035 microM for the hexa- and octasaccharides, respectively. Colloidal gold-labeled heparin was bound to promastigotes and visualized by electron microscopy. This analysis revealed that the heparin bound almost exclusively to the flagella of control cells (not subjected to trypsin) and those which had regenerated receptor after trypsinization. The physiological significance of this heparin-binding activity on the surface of promastigotes is discussed.

Use of 4-heptylumbelliferyl-beta-D-glucoside to identify Gaucher's disease heterozygotes.

Three fluorometric beta-glucosidase assays were compared for their ability to identify Gaucher's disease heterozygotes, using leukocytes as the source of enzyme: the pH 5.5-taurocholate assay of Peters et al.; the conduritol B epoxide dependent variation of that assay; and the newly developed method described herein. While the first two procedures utilize the standard substrate 4-methylumbelliferyl-beta-D-glucopyranoside to estimate beta-glucosidase activity, the new assay uses 4-heptylumbelliferyl-beta-D-glucoside as (C7UGlc) substrate. Use of this substrate enhances the specificity of the method for lysosomal glucocerebrosidase, thereby minimizing the contribution of the nonspecific cytosolic beta-glucosidase to estimates of substrate hydrolysis. Using Student's t test for the three assays examined, the C7UGlc assay procedure was determined to have the lowest p value (p less than 0.001) and highest t value (t = 4.95) for the discrimination between the mean glucocerebrosidase value of control and obligate Gaucher heterozygote samples. The high reliability and simplicity of the C7UGlc assay lends adequate reason to favor this assay for regular clinical diagnosis of Gaucher heterozygotes.