Leishmania mexicana mexicana: attachment and uptake of promastigotes to and by macrophages in vitro

Promastigotes of Leishmania mexicana mexicana attach to mouse macrophages in vitro in the absence of serum by a wheat germ agglutinin-like ligand on the surface of the promastigote that binds to the N-acetyl glucosamine moiety of a receptor on the surface of the macrophage. The binding is temperature dependent, and the macrophage receptor is trypsin, cytochalasin B, and is assisted or inhibited as for attachment. Treatment of promastigotes with proteolytic enzymes uncovers a receptor for a serum component that binds strongly to a mouse macrophage receptor in vitro. The strain of mice donating the macrophages had little effect upon attachment and uptake except that A strain mouse macrophages attached fewer promastigotes in 10 min than those of outbred mice, but took up as many promastigotes over 90 min as those of outbred mice. Low responder Biozzi mouse macrophages took up more promastigotes than high responder Biozzi mouse macrophages. Normal unheated human, rabbit, and guinea pig sera lysed promastigotes and so inhibited their attachment to macrophages in vitro. Unheated immune serum showed an enhanced inhibition of attachment. Heated normal serum allowed attachment and uptake, while promastigotes treated with heated immune serum showed enhanced attachment to and uptake by macrophages. Treatment of macrophages in vitro with immune serum enhanced their ability to attach promastigotes and to engulf them. Repeated 90-min exposures of a population of promastigotes to uptake by mouse macrophages in vitro did not deplete the population of any sub-population more likely to be taken by macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)     

The involvement of the major surface glycoprotein (gp63) of Leishmania promastigotes in attachment to macrophages

The interaction between the macrophage and the parasite plays a central role in the continued success of Leishmania infection. The promastigote surface ligand, and its complementary macrophage membrane receptor, involved in attachment and phagocytosis are likely to exert considerable influence over the outcome of a new infection. In this study, we report experiments pertaining to one such parasite membrane protein. Initial examination of promastigote surface proteins by radiolabeling and two-dimensional-polyacrylamide gel electrophoresis revealed an abundant polypeptide with an apparent m.w. of 63,000. Lectin-binding studies indicated that it was a glycoprotein containing mannose, N-acetyl glucosamine, and N-acetyl galactosamine residues. Monospecific antiserum raised against this glycoprotein, gp63, decorated the entire promastigote plasmalemma. Univalent antibody fragments from this antiserum blocked the interaction between promastigotes and macrophages by inhibiting attachment. Anti-gp63-inhibition reduced parasite/macrophage binding to 30 to 35% of the control binding level. Additional evidence of the involvement of gp63 in attachment to macrophages was provided by studies that made use of gp63-containing proteoliposomes. These vesicles were avidly phagocytosed by macrophages. Uptake of the gp63-containing liposomes was suppressed by greater than 90% by both anti-gp63 F(ab) fragments and the oligosaccharide mannan, indicating that their phagocytosis was receptor dependent. These results demonstrate that the abundant glycoprotein gp63 plays an important role in attachment of promastigotes to macrophages, and attachment via this parasite ligand is sufficient to trigger phagocytosis.     

Expression of the major surface glycoprotein of Leishmania donovani chagasi in virulent and attenuated promastigotes

Using both hamster and mouse models of infection, we documented that the virulence of Leishmania donovani chagasi promastigotes decreases over time, when parasites are maintained in long term culture after isolation from an infected animal. Concomitant with this loss of virulence is a marked decrease in amount of the major promastigote surface glycoprotein, gp63, present in promastigotes. The latter was shown by a decrease in binding of polyclonal anti-gp63 serum to attenuated (cultivated long term) as compared to virulent (recently isolated) promastigotes, using immunofluorescence and Western blot assays. Binding of Con A to promastigote glycoproteins, separated by SDS-PAGE, documented a similar decrease. An alteration in the mechanism of promastigote attachment to macrophages was also noted: purified gp63 inhibited attachment of virulent promastigotes to human monocyte-derived macrophages, but it did not affect the attachment of attenuated promastigotes. Northern blot analysis showed that, despite marked differences in the amount of gp63 protein, the quantity of gp63 RNA was comparable in attenuated and virulent promastigotes. However, virulent promastigotes contained two major gp63 RNA species of 3.0 and 2.7 kb, whereas attenuated promastigotes had one predominant gp63 RNA of 2.7 kb and only minor amounts of 3.0 kb RNA. Thus, the decrease in gp63 expression in attenuated, contrasted to virulent, promastigotes is associated with qualitative, but not quantitative, differences in the gp63 messenger RNA.     

Effects of long-term in vitro cultivation on Leishmania donovani promastigotes

Promastigotes of Leishmania donovani that had been subcultured in modified Tobie's medium for 2 to 3 years showed decreased infectivity and lack of virulence for hamsters and mice compared to newly transformed promastigotes. Amastigotes derived from these long-term promastigote cultures decreased in number rapidly in hamsters, but only slightly in mice, over a 48-day period. In cultured mouse and hamster macrophages infected in vitro, amastigotes derived from long-term cultures rapidly decreased to low numbers, which persisted. The same pattern was seen in macrophages treated with catalase, an inhibitor of the oxygen-dependent killing mechanism of the macrophage. Promastigotes from long-term cultures also differed from virulent first-passage promastigotes in size, growth patterns in Tobie's medium, and in the quantities of some of their antigens.     

Evidence that Leishmania donovani utilizes a mannose receptor on human mononuclear phagocytes to establish intracellular parasitism

The pathogenic protozoan Leishmania donovani must gain entrance into mononuclear phagocytes to successfully parasitize man. The parasite's extra-cellular promastigote stage is ingested by human peripheral blood monocytes or monocyte-derived macrophages in the absence of serum, in a manner characteristic of receptor-mediated endocytosis. We have found remarkable similarities between the macrophage receptor(s) for promastigotes and a previously characterized eucaryotic receptor system, the mannose/fucose receptor (MFR), that mediates the binding of zymosan particles and mannose- or fucose-terminal glycoconjugates to macrophages. Ingestion of promastigotes by monocyte-derived macrophages was inhibited by several MFR ligands. Mannan (2.5 mg/ml) decreased ingestion by 63.7% (p less than 0.001), and the neoglycoproteins mannose-BSA and fucose-BSA (20 micrograms/ml) inhibited parasite ingestion by 46.5% and 39.6%, respectively (p less than 0.04). In contrast, promastigote ingestion by monocytes was unaffected by MFR ligands. These results are consistent with reports that MFR activity is present in monocyte-derived macrophages but not in monocytes. Furthermore, attachment of promastigotes to macrophages, assessed by using cytochalasin D to prevent phagocytosis, was reduced 49.8% by mannan. Reorientation of the MFR to the ventral surface of the cell was achieved by plating macrophages onto mannan-coated coverslips, reducing MFR activity on the exposed cell surface by 94% as assessed by binding of 125I-mannose-BSA. Under these conditions, ingestion of promastigotes was inhibited by 71.4% (p less than 0.006). Internalization of the MFR by exposure of macrophages to zymosan before infection with promastigotes resulted in a 62.3% decrease in parasite ingestion (p less than 0.006). Additionally NH4Cl, a weak lysosomotropic base that impairs MFR recycling, decreased macrophage ingestion of promastigotes by 38.2% (p less than 0.03, 30 mM NH4Cl). Subinhibitory concentrations of NH4Cl (10 mM) and of mannan (0.25 mg/ml) together inhibited parasite ingestion by 76.4% (p less than 0.002). These studies suggest that L. donovani promastigotes may utilize a receptor system on human monocyte-derived macrophages, the MFR, to efficiently parasitize the human host.     

Autoantibodies against bromelainized mouse erythrocyte: strain distribution of serum idiotype expression and relative peritoneal cell activity

Previously, we demonstrated that the naturally occurring mouse autoantibodies directed against bromelainized mouse red blood cells (BrMRBC) comprised a family of structurally related molecules bearing a common idiotypic determinant (CP) based on structural and idiotypic analysis of a series of anti-BrMRBC monoclonal autoantibodies derived from a fusion of peritoneal cells (PerC) with plasmacytomas. In the present studies, we have evaluated the quantitative expression of circulating CP idiotype related to autoantibodies against BrMRBC in relation to specific PerC anti-BrMRBC plaque-forming activity in an individual mouse of different strains. The data presented here show no direct relationship between serum CP idiotype expression and PerC anti-BrMRBC plaque-forming activity in an individual mouse of all strains tested. However, the circulating CP idiotype content is higher in strains, viz., CBA/J, NZB, C3H, BXSB, and Biozzi high responder (H) mice which exhibit a high perC autoantibody secretory activity against BrMRBC. The strains such as BALB/c, DBA2, SJL/J, CBA/N, and Biozzi low responder (L) express little or no circulating CP idiotype with a corresponding small or no PerC anti-BrMRBC activity. Furthermore, the PerC "auto"-immune phenomenon is markedly expressed in the normal CBA/J strain since these mice show a higher percentage ratio of CP idiotype over serum IgM (2.68%) as well as highest PerC anti-BrMRBC plaque-forming activity (11,319 +/- 18,029 plaques per million viable cells) compared to other normal and autoimmune strains tested. Nevertheless, the highest circulating serum CP idiotype (49.4 micrograms/ml) is observed in the autoimmune NZB mouse. The immunodeficient CBA/N mice fail to express detectable levels of CP idiotype in their serum. The experiments conducted in genetically selected outbred Biozzi (H and L) strain have revealed remarkable differences in serum CP idiotype expression as well as PerC anti-BrMRBC plaque-forming activity in these two lines. The expression of mouse PerC "auto"-immune phenomenon and quantitative circulation of CP idiotype in the serum seem to be related to regulatory mechanisms as for sheep erythrocytes and other natural antigens earlier demonstrated to be under polygenic regulation in Biozzi (H and L) mice.     

The mouse macrophage receptor for C3bi (CR3) is a major mechanism in the phagocytosis of Leishmania promastigotes

We examined the role of the macrophage receptor for C3bi, the CR3, in the phagocytosis of Leishmania major promastigotes and report that M1/70, a monoclonal antibody to the CR3, inhibited the binding of leishmania to macrophages both when the assays were performed in the presence of normal serum and in its absence. In serum, leishmania activate complement and fix C3. Fixation and subsequent cleavage to C3bi occurs rapidly, and by as early as 5 min both forms of the molecule can be identified on the parasites' surface. Complement fixation results in an enhanced phagocytosis of leishmania promastigotes by mouse macrophages. In the case of L. major, 63% of this serum-enhanced binding is inhibitable by M1/70. Binding assays were also performed in the absence of serum with the use of thoroughly washed promastigotes. The addition of M1/70 inhibited binding under these conditions by 54%. Two other rat monoclonal antibodies directed against different antigens on the macrophage plasma membrane did not inhibit binding. M1/70 did not inhibit the binding of promastigotes to rat bone marrow cells, nor did it inhibit IgG-SRBC binding to mouse peritoneal macrophages. These data indicate that the inhibition observed in the presence of M1/70 was specific for the CR3 and that the macrophage receptor for C3bi plays a major role in the phagocytosis of Leishmania major promastigotes, even in the absence of serum.     

Enhanced interleukin 1 (IL-1) production mediated by mouse serum amyloid P component

Purified serum amyloid P component (SAP), the major acute-phase reactant of mice, induces enhanced interleukin 1 (IL-1) production by elicited monocytes/macrophages in vitro. SAP also enhanced IL-1 elaboration by macrophages from lipopolysaccharide (LPS)-low responder mice and in the presence of polymyxin B, indicating that the small amounts of LPS present in the SAP preparation did not augment IL-1 production. Concentrations of SAP of 0.1 to 10.0 micrograms/ml enhanced IL-1 production by elicited and bacillus Calmette-Guerin (BCG)-activated peritoneal macrophages, but not by resident peritoneal macrophages. The inflammation-induced monocyte/macrophage population displayed selective binding of SAP. The mouse macrophage line P388D1, also could bind SAP and display enhanced IL-1 production in response to SAP. SAP did not bind to the macrophage cell line RAW264.7 nor did it enhance IL-1 secretion by this line. The results suggest that this acute-phase reactant has the potential to enhance inflammatory and immunological events mediated by IL-1.     

Leishmania mexicana pifanoi: in vivo and in vitro interactions between amastigotes and macrophages

Macrophages of the cell line J774 were used in a comparative study of virulence involving amastigote stages of Leishmania mexicana pifanoi isolated from macrophages (AMA-M) of the aforementioned cell line, amastigote forms grown in the UM-54-cell-free medium (AMA-C), and promastigote stages. The macrophage cultures were inoculated with AMA-M and AMA-C at the culture cell to parasite ratios of 1:3, 1:5, and 1:10. The macrophages were exposed to either kind of amastigotes for 24, 48, and 72 h. At the end of each of these periods, and for each dilution, the percentages of macrophages harboring the parasites within their cytoplasm and the mean numbers of intracellular parasite/macrophage were estimated on the basis of examination of 200 phagocytes. When either AMA-M or AMA-C were employed, after 24 h, the percentages of infected macrophages were, respectively, 84.5%, 89.0%, and 94.5% for the three aforementioned dilutions, the majority of the phagocytes containing 1-5 parasites. After 48- and 72-h exposures, the macrophages harbored 6-11 and 11-20 amastigotes/cell, respectively. Evidently intracellular multiplication of the amastigotes has taken place. In contrast to the results obtained with amastigote forms, after inoculations of the macrophages cultures with promastigotes at the dilutions previously used for amastigotes, only 48-78 phagocytes were found to contain intracellular stages within their cytoplasm. Many macrophages were parasite-free, especially when exposed to fewer promastigotes. Experiments in which 5 X10(6) promastigotes, AMA-M, or AMA-C were inoculated into the footpads of hamsters yielded the following results with regard to terminal footpad volumes: 1.57, 3.31, and 3.32 cm3, respectively. Evidently both kinds of amastigotes had equal virulence for hamsters; however, the promastigote stages were much les virulent for these experimental hosts.     

The macrophage-attachment glycoprotein gp63 is the predominant C3-acceptor site on Leishmania mexicana promastigotes

The interaction between Leishmania promastigotes and their vertebrate host's complement system results not only in parasite lysis but also, due to surface-bound complement components, in increased macrophage binding potential. In this study we demonstrate, with the use of isolated complement components, that activation is via the alternative complement pathway, initiated by direct deposition of C3 onto the parasite surface. The predominant C3 acceptor site on the promastigotes was initially identified as the glycoprotein gp63 by anti-C3 antibody immunoprecipitation of radioiodinated promastigotes following incubation in the alternative pathway initiators C3, and factors B and D. The C3-binding properties of gp63 were confirmed and quantified, in relation to other surface antigens, by incubating parasites in iodinated C3 and immunoprecipitating bound C3 with antibodies directed against different promastigote surface antigens. The other abundant surface antigen, the glycolipid 'excreted factor', did not show any C3-binding activity. Further demonstration was provided by incubating liposomes containing either gp63 or excreted factor in iodinated C3 and factors B and D. Only gp63-containing liposomes bound C3. Considering that both gp63 and the excreted factor have recently been implicated in attachment and uptake by macrophage, these findings may have considerable bearing in the determination of which of the macrophage surface receptors identify which parasite ligand.     

Effect of the Antiphagocytic Agent Cytochalasin B on Macrophage Invasion by Leishmania mexicana Promastigotes and Trypanosoma cruzi Epimastigotes*

Unstimulated mouse peritoneal exudate cells were cultured on coverslips in Medium 199 containing 10% (v/v) calf serum. Cytochalasin B dissolved in dimethyl sulphoxide (DMSO) and diluted in Medium 199 was added to cultures to give final concentrations of 1, 5 and 10 μg/ml. Equal numbers of Leishmania mexicana promastigotes, Trypanosoma cruzi epimastigotes and sheep red cells were added to 24 hr cultures incubated at 37 C. The macrophage monolayers were fixed and stained at various time intervals. L. mexicana promastigotes and sheep red blood cells were found to attach to macrophages in the presence of the drug but did not enter the cells. When the medium containing the Cytochalasin was replaced with normal medium phagocytosis of the adherent parasites and red cells followed rapidly. T. cruzi epimastigotes were found inside macrophages in both drug-treated and drug-free cultures although the number found to be intracellular in the latter was significantly greater. This study suggests that L. mexicana promastigotes enter macrophages by being phagocytosed, whereas T. cruzi epimastigotes can actively penetrate these cells.     

Parallel inheritance of tissue catalase activity and immunostimulatory action of amphotericin B in inbred mouse strains

Both amphotericin B (AmB) and its methyl ester derivative are potent immunoadjuvants that also stimulate murine B lymphocytes and macrophages in vitro. Most of the common inbred mouse strains show AmB-induced immunostimulation (AmB-high responders) but mice from the C57BL strains, regardless of H-2 genotype, are AmB-low responders. Lymphoid cells from AmB-high responder strains also exhibit greater resistance to H2O2 toxicity in vitro compared with cells from AmB-low responders. This result led to an evaluation of differences in the tissue catalase levels of AmB-high and -low responder strains. Results from several laboratories, including ours, indicate that C57BL mouse strains express low levels of tissue catalase activity in addition to low or absent immunostimulant effects of AmB. Several AmB-high responder strains have high spleen cell, macrophage, and liver catalase, and the mouse strain distribution of enzyme activity as well as the dominant inheritance of the low catalase phenotype is compatible with regulation by the Ce-1 locus in lymphoid organs as well as liver. Other evidence also suggests that H2O2 metabolism is important in lymphoid cell responses to AmB. For example, AmB stimulates a stronger respiratory burst in macrophages from AmB-high responder strains under the same conditions that inhibit burst activity in macrophages from low responders. Selective immune enhancement by AmB in high catalase mouse strains along with enhanced susceptibility to AmB toxicity in low responder C57BL mice with low tissue catalase activity suggests that cellular peroxidation is a major determinant of the genetic regulation of amphotericin-induced immunostimulation.     

Complement is an essential component of the immune response to adeno-associated virus vectors

Adeno-associated virus (AAV) vectors are associated with relatively mild host immune responses in vivo. Although AAV induces very weak innate immune responses, neutralizing antibodies against the vector capsid and transgene still occur. To understand further the basis of the antiviral immune response to AAV vectors, studies were performed to characterize AAV interactions with macrophages. Primary mouse macrophages and human THP-1 cells transduced in vitro using an AAV serotype 2 (AAV2) vector encoding green fluorescent protein did not result in measurable transgene expression. An assessment of internalized vector genomes showed that AAV2 vector uptake was enhanced in the presence of normal but not heat-inactivated or C3-depleted mouse/human serum. Enhanced uptake in the presence of serum coincided with increased macrophage activation as determined by the expression of NF-κB-dependent genes such as macrophage inflammatory protein 2 (MIP-2), interleukin-1β (IL-1β), IL-8, and MIP-1β. AAV vector serotypes 1 and 8 also activated human and mouse macrophages in a serum-dependent manner. Immunoprecipitation studies demonstrated the binding of iC3b complement protein to the AAV2 capsid in human serum. AAV2 did not activate the alternative pathway of the complement cascade and lacked cofactor activity for factor I-mediated degradation of C3b to iC3b. Instead, our results suggest that the AAV capsid also binds complement regulatory protein factor H. In vivo, complement receptor 1/2- and C3-deficient mice displayed impaired humoral immunity against AAV2 vectors, with a delay in antibody development and significantly lower neutralizing antibody titers. These results show that the complement system is an essential component of the host immune response to AAV.     

Stage-specific pathways of Leishmania infantum chagasi entry and phagosome maturation in macrophages

The life stages of Leishmania spp. include the infectious promastigote and the replicative intracellular amastigote. Each stage is phagocytosed by macrophages during the parasite life cycle. We previously showed that caveolae, a subset of cholesterol-rich membrane lipid rafts, facilitate uptake and intracellular survival of virulent promastigotes by macrophages, at least in part, by delaying parasitophorous vacuole (PV)-lysosome fusion. We hypothesized that amastigotes and promastigotes would differ in their route of macrophage entry and mechanism of PV maturation. Indeed, transient disruption of macrophage lipid rafts decreased the entry of promastigotes, but not amastigotes, into macrophages (P<0.001). Promastigote-containing PVs were positive for caveolin-1, and co-localized transiently with EEA-1 and Rab5 at 5 minutes. Amastigote-generated PVs lacked caveolin-1 but retained Rab5 and EEA-1 for at least 30 minutes or 2 hours, respectively. Coinciding with their conversion into amastigotes, the number of promastigote PVs positive for LAMP-1 increased from 20% at 1 hour, to 46% by 24 hours, (P<0.001, Chi square). In contrast, more than 80% of amastigote-initiated PVs were LAMP-1+ at both 1 and 24 hours. Furthermore, lipid raft disruption increased LAMP-1 recruitment to promastigote, but not to amastigote-containing compartments. Overall, our data showed that promastigotes enter macrophages through cholesterol-rich domains like caveolae to delay fusion with lysosomes. In contrast, amastigotes enter through a non-caveolae pathway, and their PVs rapidly fuse with late endosomes but prolong their association with early endosome markers. These results suggest a model in which promastigotes and amastigotes use different mechanisms to enter macrophages, modulate the kinetics of phagosome maturation, and facilitate their intracellular survival.     

Trypanosoma brucei: recognition in vitro of two developmental forms by murine macrophages

In vitro, murine macrophages attach to the midgut form of Trypanosoma brucei in the absence of exogenous proteins. Recognition appears to be specific and saturable. Attachment is mediated by a non-Fc, non-C3 receptor on the macrophage plasma membrane. The attachment mechanism is protease sensitive, temperature sensitive, requires the presence of divalent cations, and is functional in fetal calf serum-free medium. The midgut form is also lysed in normal rabbit serum by activating the alternative complement pathway. The form isolated from the blood is neither lysed in normal serum nor is it spontaneously recognized by the macrophage, in vitro. Partial trypsinization of the bloodstream form, however, results in both the triggering of alternative complement pathway lysis and spontaneous uptake into macrophages. Murine macrophages attach to the midgut form of T. brucei by a receptor on the macrophage plasma membrane which is capable of recognizing particulate activators of the alternative complement pathway.     

Interplay between parasite cysteine proteases and the host kinin system modulates microvascular leakage and macrophage infection by promastigotes of the Leishmania donovani complex

Kinins, the vasoactive peptides proteolytically liberated from kininogens, were recently recognized as signals alerting the innate immune system. Here we demonstrate that Leishmania donovani and Leishmania chagasi, two etiological agents of visceral leishmaniasis (VL), activate the kinin system. Intravital microscopy in the hamster cheek pouch showed that topically applied promastigotes induced macromolecular leakage (FITC-dextran) through postcapillary venules. Peaking at 15 min, the parasite-induced leakage was drastically enhanced by captopril (Cap), an inhibitor of angiotensin-converting enzyme (ACE), a kinin-degrading metallopeptidase. The enhanced microvascular responses were cancelled by HOE-140, an antagonist of the B2 bradykinin receptor (B2R), or by pre-treatment of promastigotes with the irreversible cysteine proteinase inhibitor N-methylpiperazine-urea-Phe-homoPhe-vinylsulfone-benzene (N-Pip-hF-VSPh). In agreement with the above-mentioned data, the promastigotes vigorously induced edema in the paw of Cap-treated J129 mice, but not Cap-B2R-/- mice. Analysis of parasite-induced breakdown of high molecular weight kininogens (HK), combined with active site-affinity-labeling with biotin-N-Pip-hF-VSPh, identified 35-40 kDa proteins as kinin-releasing cysteine peptidases. We then checked if macrophage infectivity was influenced by interplay between these kinin-releasing parasite proteases, kininogens, and kinin-degrading peptidases (i.e. ACE). Our studies revealed that full-fledged B2R engagement resulted in vigorous increase of L. chagasi uptake by resident macrophages. Evidence that inflammatory macrophages treated with HOE-140 became highly susceptible to amastigote outgrowth, assessed 72 h after initial macrophage interaction, further suggests that the kinin/B2R activation pathway may critically modulate inflammation and innate immunity in visceral leishmaniasis.     

Leishmania tropica: differences in the antigenicity of promastigotes and amastigotes

In vitro bioassays were used to compare T-cell responses induced by the intramacrophage amastigote stage and the sandfly-borne promastigote stage of Leishmania tropica. Lymph node cells from mice immunized with sonicated promastigotes or amastigotes incorporated in Freund's complete adjuvant were assayed for their ability to proliferate and to release interleukin 2 following in vitro challenge with promastigote or amastigote antigen. The levels of the proliferative and interleukin 2 synthetic responses of cells from promastigote and amastigote immunized mice were quite distinct. Cells from mice immunized with promastigotes demonstrated a vigorous in vitro response to the homologous antigen, but a reduced response to the potentially cross-reactive amastigotes. In contrast, cells from mice immunized with amastigotes mounted a weak response to the homologous antigen, but a consistently greater response to promastigote antigen. This unusual response was similar when 8 M urea extracts were used as immunogens and test antigens. In general, interleukin 2 production by immune mice paralleled the results from the T-cell proliferation assays. These results are discussed in relation to evasion of host immunologic detection by the intramacrophage amastigote stage.     

Leishmania mexicana: Energy metabolism of amastigotes and promastigotes

The utilisation of substrates by Leishmania mexicana amastigotes and promastigotes differed significantly. The rates of uptake and catabolism of nonesterified fatty acids were up to 10-fold higher with amastigotes. Almost all the available exogenous fatty acids were consumed during amastigote transformation and by stationary phase of promastigote growth. The results suggest that fatty acids are important energy substrates for amastigotes, whereas promastigote utilisation may reflect the requirement for these substrates in anabolism. Glucose was utilised by amastigotes and promastigotes but the rate of catabolism was up to 10-fold higher in promastigotes. Uptake of glucose occurred throughout amastigote transformation and growth in vitro of promastigotes. High-subpassage promastigotes exhibited markedly lower glucose but higher amino acid utilisation than low-subpassage promastigotes. Asparagine, glutamine, glutamate, leucine, lysine, methionine, and threonine were consumed in large quantities by amastigotes and promastigotes, whereas alanine and glycine were excreted. Proline was catabolised to CO₂ by amastigotes and promastigotes but only at a low rate, and it was excreted in large amounts throughout promastigote growth. The major end products of energy metabolism were found to be CO₂ and succinate with both forms of the parasite and there was a secretion of up to 12 and 16% of the total protein synthesised by transforming amastigotes and growing promastigotes, respectively. Catabolism in amastigotes and promastigotes was found to be sensitive to cyanide and amytal, whereas 2-mercaptoacetate and 4-pentenoate primarily affected β-oxidation in the amastigote.     

Lipophosphoglycan from Leishmania mexicana promastigotes binds to members of the CR3, p150,95 and LFA-1 family of leukocyte integrins

The abundant surface glycolipid, promastigote lipophosphoglycan (LPG), of Leishmania promastigotes was isolated and reconstituted onto the surface of hydrophobic silica beads. These beads bound to both macrophages and monocytes, suggesting that phagocytes possess a receptor(s) capable of recognizing LPG. LPG beads were unable to bind to macrophages isolated from individuals with a genetic deficiency in the CD18 complex of leukocyte integrins (CR3, p150,95, and LFA-1), suggesting that one or more of these receptors were required for binding of LPG. Individual members of the CD18 complex were depleted from macrophages by plating cells on surfaces coated with anti-receptor mAb. These experiments indicated that CR3 and p150,95 from the CD18 complex, were the predominant mediators of attachment of LPG. The phagocyte receptor CR3 expresses two distinct binding sites, one that binds peptide ligands, such as C3bi, and a second site, that recognizes bacterial LPS. Antibody inhibition experiments and competition binding studies with synthetic peptides and soluble LPG indicated that LPG is recognized by the nonpeptide, or "LPS" binding site on CR3.