Leishmaniasis in beige mice.

The courses of two protozoal diseases, cutaneous and visceral leishmaniasis, were examined in three groups of C57BL/6J mice. One group of mice was homozygous recessive for the beige gene (bg/bg). Beige mice are the genetic homologue of the human Chédiak-Higashi syndrome and, among other defects, are profoundly deficient in natural killer cell activity. Wild-type (+/+) mice, which respond to experimental cutaneous or visceral leishmaniasis by eventually eliminating their parasites, and heterozygous beige (bg/+) mice served as controls; both are phenotypically normal in natural killer cell activity, which is particularly high in the spleen. In bg/bg mice, the course of Leishmania tropica, a causative agent of cutaneous leishmaniasis, was similar to that in control mice after both primary and challenge inoculations. All groups of mice expressed similar humoral and cellular immune responses to L. tropica antigen. However, bg/bg mice failed to eliminate amastigotes of Leishmania donovani, a causative agent of visceral leishmaniasis, from their spleens over an observation period of 56 days, in contrast to bg/+ and +/+ controls. Similar levels of anti-leishmanial antibody were produced by all groups of mice, and all mice responded comparably to footpad injections of L. donovani antigen. The results of this study suggest a possible role for natural killer cells in recovery from L. donovani but not from L. tropica infection.     

in vitro : The Adsorption of Antibody and Antigen by Spleen Cells • Some Further Experiments

Spleen cells treated in vitro with rabbit antiserum against human serum albumin (HSA) are capable of specifically adsorbing ¹³¹I-HSA. The antibody responsible for this effect has been termed `cytophilic antibody' (Boyden and Sorkin, 1960).

Absorption of anti-HSA serum under appropriate conditions with normal spleen cells results in a greater loss of cytophilic antibody activity than of precipitable antibody.

Preparations made from freeze-dried cells are also capable of adsorbing cytophilic antibody. However, treatment of cells (either fresh or freeze-dried) at 56° results in loss of capacity to take up cytophilic antibody.

Treatment at 56° of cells which have adsorbed cytophilic antibody results in elution of the cytophilic antibody. Eluates prepared in this way have a much higher proportion of cytophilic antibody activity to precipitable antibody than has the original antiserum.

These results are discussed particularly in relation to some apparently conflicting reports of other workers.

     

Cytophilic antibodies in mice contact-sensitized with oxazolone: Immunochemical characterization and preferential binding to a trypsin-sensitive macrophage receptor

Oxazolone-specific cytophilic antibodies in the sera of non-immune CBA mice and mice contact-sensitized with oxazolone, were studied with a rosette test employing peritoneal exudate macrophages and oxazolone-coupled sheep erythrocytes. Macrophage rosettes, produced by direct or indirect cytophilic antibodies, were found to depend on optimally hapten-coupled erythrocytes. Sera obtained 1 week after contact sensitization with oxazolone contained principally 7S IgG2a cytophilic antibodies. Monomeric 7S IgM antibodies cytophilic for macrophages may have been present as well. Primary contact sensitization and boosting was found uniquely to lead to high titres of hyperimmune oxazolone cytophilic antibodies predominantly binding to trypsin-sensitive macrophage receptors.

It has been previously shown that specifically sensitized macrophages mediate a component of delayed skin reactions in mice contact sensitized with oxazolone. Since these reactions can be transferred by immune serum or by normal macrophages coated with immune serum, and since acquisition of this passive sensitization can be abolished by prior trypsinization of these cells, it is suggested that 7S IgG2a and/or 7S IgM cytophilic oxazolone antibodies attaching to trypsin-sensitive macrophage receptors, mediate the specific macrophage component of contact sensitivity in mice.

     

In Vitro: The Adsorption of Antigen by Spleen Cells previously treated with Antiserum*

Rabbit spleen cells, which have been treated in vitro with certain rabbit or guinea-pig antisera (e.g. against human serum albumin) and washed, are capable of specifically adsorbing radio-isotope labelled antigen. The antibody responsible for this effect appears to be distinct from the main precipitating antibody in the serum and the term `cytophilic antibody' has been suggested. The activity of the cytophilic antibody is not destroyed by heating at 56° C. for half an hour.

The cytophilic antibody is not adsorbed by red cells. Spleen cells which have been treated with methanol before treatment with antiserum take up less antigen than non-methanol-treated cells tested in the same way. However, the addition of fresh normal spleen cells to the methanol-antibody-treated cells before the addition of antigen increased the uptake of the latter.

     

Macrophage behaviour during the complaisant phase of murine pertussis

During the complaisant phase of murine pertussis, Bordetella pertussis survived within lung macrophages. Bactericidal activity in the complaisant mouse, which was shown to be activated by viable superinfection, could also be elicited by alcohol killed B. pertussis. Isolated complaisant cells in vitro, although not superior to normal cells in phagocytosis, showed an enhanced antibacterial activity against large numbers of viable B. pertussis. The relationship of cytophilic antibody to this enhanced bactericidal activity is unknown, but circulating protective antibody was relatively unimportant at this stage of infection. Although a higher titre of opsonins was found in complaisant mice, opsonization with optimal quantities of normal or complaisant phase serum neither increased phagocytosis nor affected the outcome of intracellular digestion.     

Leishmania amazonensis Amastigotes Trigger Neutrophil Activation but Resist Neutrophil Microbicidal Mechanisms

Neutrophils are the first cells to infiltrate to the site of Leishmania promastigote infection, and these cells help to reduce parasite burden shortly after infection is initiated. Several clinical reports indicate that neutrophil recruitment is sustained over the course of leishmaniasis, and amastigote-laden neutrophils have been isolated from chronically infected patients and experimentally infected animals. The goal of this study was to compare how thioglycolate-elicited murine neutrophils respond to L. amazonensis metacyclic promastigotes and amastigotes derived from axenic cultures or from the lesions of infected mice. Neutrophils efficiently internalized both amastigote and promastigote forms of the parasite, and phagocytosis was enhanced in lipopolysaccharide (LPS)-activated neutrophils or when parasites were opsonized in serum from infected mice. Parasite uptake resulted in neutrophil activation, oxidative burst, and accelerated neutrophil death. While promastigotes triggered the release of tumor necrosis factor alpha (TNF-α), uptake of amastigotes preferentially resulted in the secretion of interleukin-10 (IL-10) from neutrophils. Finally, the majority of promastigotes were killed by neutrophils, while axenic culture- and lesion-derived amastigotes were highly resistant to neutrophil microbicidal mechanisms. This study indicates that neutrophils exhibit distinct responses to promastigote and amastigote infection. Our findings have important implications for determining the impact of sustained neutrophil recruitment and amastigote-neutrophil interactions during the late phase of cutaneous leishmaniasis.     

Demonstration of the role of cytophilic antibody in resistance to malaria parasites (Plasmodium berghei) in rats.

This paper reports the results of a study of the nature of the immune response against Plasmodium berghei parasites by inbred rats. A macrophage-cytophilic antibody specific for malarial antigens was identified and characterized. Detection of the antibody on the macrophage surface was accomplished by the parasite adherence tests and by the indirect fluorescent antibody technique. Isolation and purification of the macrophage-cytophilic and opsonic antibodies from hyperimmune rat serum was accomplished by QAE-Sephadez A-50 elution chromatography, and of the macrophage-cytophilic antibody by adsorption with and elution from syngeneic macrophages as well. Characterization of the cytophilic antibody as immunoglobulin G1 was done by immunoelectrophoresis and by Ouchterlony-type double diffusion in gel. Passive protection tests in weanling inbred rats have demonstrated that the opsonizing antibody conferred some protection against P. berghei. The macrophage-cytophilic antibody, on the other hand, was not protective alone but acted synergistically with the opsonizing antibody.     

Role of macrophages in tumour immunity: I. Co-operation between macrophages and lymphoid cells in syngeneic tumour immunity

Macrophages from DBA/2 mice hyperimmunized with irradiated syngeneic L5178Y or SL2 lymphoma cells inhibited the growth of these cells in vitro and killed them within 48 hours of culture. The reaction was immunologically specific. Non-immune macrophages could be rendered capable of immunologically-specific growth inhibition of target cells in vitro (arming) (a) by direct contact of hyperimmune lymphoid cells and macrophages, and (b) by incubating macrophages with a specific macrophage-arming factor (SMAF) derived by incubating spleen cells from singly immunized mice with irradiated lymphoma cells. Singly immunized spleen cells did not arm macrophages by direct cell-to-cell contact. A temporal relationship was seen between the presence of immune macrophages in hyperimmunized mice and the ability of the spleen cells to arm normal macrophages. Furthermore, macrophages could be armed in vivo by a single i.p. injection of hyper immune spleen cells. The presence of arming factors cytophilic for macrophages, but not for cells of non-macrophage origin, is briefly discussed.     

Elution of macrophage-bound immunoglobulins by temperature changes in vitro

Some mouse anti-sheep-erythrocyte macrophage cytophilic antibodies are more readily adsorbed to cells at 4° than at 37°. By treating cells with serum at 4°, washing them well and eluting bound material at 30°, it was found possible to isolate cytophilic immunoglobulins. These were found to have fast γ-mobility, but were heterogeneous upon preparative ultracentrifugation and gel-filtration. The macrophage eluate contained relatively high titres of anti-sheep-erythrocyte cytophilic antibodies.     

Susceptibility of 3H-thymidine-prelabelled Leishmania donovani amastigotes and promastigotes to the cytotoxic activity of peritoneal, splenic and liver macrophages

C57BL/6 macrophage populations from spleen and liver, the main organs for the manifestation of visceral leishmaniasis, were investigated for their ability to perform spontaneous phagocytosis-associated killing of 3H-thymidine (3H-TdR)-prelabelled L. donovani amastigotes and promastigotes. The results showed that organ macrophages from spleen and liver killed L. donovani amastigotes and promastigotes spontaneously with high efficiency. This consistent finding was first detectable at 2-3 h, and the reaction was completed at 12 h. This type of killing was strongly enhanced when spleen and liver macrophages were activated. This phagocytosis-associated killing mechanism may contribute, to a large extent, in maintaining the infection under control in vivo, by drastically reducing the amount of parasites that is required to establish intracellular parasitism. To be able to assay phagocytosis-associated destruction of both promastigotes and amastigotes, a reproducible system for the production in vitro of Leishmania donovani amastigotes by the macrophage cell-line J774 was developed. The DNA of the Leishmania amastigotes was labelled with 3H-TdR with high efficiency. The spontaneous label release of prelabelled L. donovani amastigotes was comparable to that of prelabelled promastigotes over an assay period of 24 h.     

Entry and survival of Leishmania amazonensis amastigotes within phagolysosome-like vacuoles that shelter Coxiella burnetii in Chinese hamster ovary cells.

Coxiella burnetii, a rickettsia, and Leishmania amazonensis, a protozoan flagellate, lodge in their host cells within large phagolysosome-like vacuoles. In the present study, C. burnetii-infected Vero or CHO cells were superinfected with L. amazonensis amastigotes to determine if these parasites can home to and survive within heterologous vacuoles. Six hours after superinfection, Leishmania amastigotes were located almost exclusively within large Coxiella-containing vacuoles. Thereafter, the numbers of parasites in the vacuoles increased at the same rate as those in cells infected with L. amazonensis alone. Furthermore, in cultures shifted to 25 degrees C, some of the amastigotes transformed into promastigote-like forms that moved their flagella within the adoptive vacuoles. Thus, L. amazonensis amastigotes not only entered Coxiella vacuoles, most likely by fusion of donor and recipient vacuoles, but temporarily survived, differentiated, and replicated therein. This appears to be the first account of the temporary cohabitation of two living pathogens within the same vacuole in a mammalian cell.     

Leishmania donovani Ornithine Decarboxylase Is Indispensable for Parasite Survival in the Mammalian Host

Mutations within the polyamine biosynthetic pathway of Leishmania donovani, the etiological agent of visceral leishmaniasis, confer polyamine auxotrophy to the insect vector or promastigote form of the parasite. However, whether the infectious or amastigote form of the parasite requires an intact polyamine pathway has remained an open question. To address this issue, conditionally lethal Δodc mutants lacking ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, were created by double targeted gene replacement within a virulent strain of L. donovani. ODC-deficient promastigotes and axenic amastigotes were auxotrophic for polyamines and capable of robust growth only when exogenous putrescine was supplied in the culture medium, confirming that polyamine biosynthesis is an essential nutritional pathway for L. donovani promastigotes. To assess whether the Δodc lesion also affected the ability of amastigotes to sustain a robust infection, macrophage and mouse infectivity experiments were performed. Parasite loads in murine macrophages infected with each of two independent Δodc knockout lines were decreased ~80% compared to their wild-type counterpart. Furthermore, α-difluoromethylornithine, a suicide inhibitor of ODC, inhibited growth of wild-type L. donovani amastigotes and effectively cured macrophages of parasites, thereby preventing host cell destruction. Strikingly, however, parasitemias of both Δodc null mutants were reduced by 6 and 3 orders of magnitude, respectively, in livers and spleens of BALB/c mice. The compromised infectivity phenotypes of the Δodc knockouts in both macrophages and mice were rescued by episomal complementation of the genetic lesion. These genetic and pharmacological studies strongly implicate ODC as an essential cellular determinant that is necessary for the viability and growth of both L. donovani promastigotes and amastigotes and intimate that pharmacological inhibition of ODC is a promising therapeutic paradigm for the treatment of visceral and perhaps other forms of leishmaniasis.     

Roles of free GPIs in amastigotes of Leishmania

Glycosylated phosphatidylinositols (GPIs) are abundant cell surface molecules of the Leishmania. Amastigote-specific GPIs AmGPI-Y and AmGPI-Z, both ethanolamine (EtN)-containing glycolipids, were identified in Leishmania amazonensis. A paucity of GPI-anchored proteins in amastigotes of L. amazonensis made the kinetoplastid suitable for evaluating the importance of free (i.e. unconjugated to protein or polysaccharide) GPIs. A strain deficient in both AmGPI-Y and AmGPI-Z was produced by stable transfection of wild-type Leishmania with a GPI-phospholipase C gene. Phosphatidylinositol deficiency was not detected in the transfectants. GPI-deficient promastigotes infected murine macrophages in vitro and differentiated into amastigotes whose growth was arrested within the host cells. Cytostasis of amastigotes was also observed during axenic culture of GPI-deficient parasites. In a hamster model of leishmaniasis, GPI-deficient promastigotes produced smaller lesions with 20-fold fewer amastigotes than infections with control parasites. Together, these observations indicate that EtN-GPIs may be essential for amastigote viability, replication, and/or virulence. Implicit in these observations is the notion that drugs targeted against the GPI biosynthetic pathway might be of value in the management of human leishmaniasis.     

Elimination of Leishmania donovani amastigotes by activated macrophages.

Tissue macrophages are the obligatory host cells for Leishmania donovani, the causative agent of visceral leishmaniasis. In this study we sought to determine whether activated macrophages, as defined by the functional criterion of tumor cell cytotoxicity, were also able to exert a microbicidal effect on ingested L. donovani amastigotes. We found that mouse peritoneal macrophages activated by a variety of means exerted a cytotoxic effect on tumor cell targets but were not able to kill L. donovani amastigotes unless the infected macrophages were exposed continually to an activating stimulus. Corynebacterium parvum, Mycobacterium tuberculosis H37Ra, and lymphokine-activated peritoneal macrophages from C57BL/6J mice were cytotoxic for EMT6 tumor cell targets. However, L. donovani Sudan strain 1S amastigotes were not killed by these macrophages unless the activated state was maintained by daily addition of lymphokine to the infected monolayers for several days postinfection. The killing of amastigotes was dependent on the time of exposure to lymphokine, as well as on the concentration of lymphokine added to the culture.     

Protective effect of glucan against visceral leishmaniasis in hamsters.

The effect of pre- or posttreatment with glucan, a reticuloendothelial stimulant, on the course of Leishmania donovani infection was assessed in highly susceptible hamsters. Intravenous administration of glucan before or after L. donovani infection significantly suppressed proliferation of amastigote-stage parasites in liver and spleen. Glucan-activated peritoneal macrophages in vitro also significantly reduced multiplication of the intracellular parasite. Ultrastructural studies revealed a well-defined hepatic granulomatous response to glucan, with hypertrophic Kupffer cells and reduced numbers of intracellular parasites compared to the control group. In additional studies, groups of hamsters were immunized by intravenous injections of glucan with Formalin-killed promastigote-stage L. donovani cells and challenged 60 days after the last immunizing injection. This treatment regimen significantly prolonged the mean survival time of those hamsters which died after infection, relative to untreated control groups. Hamsters stimulated with the glucan-killed promastigote preparation also exhibited significant reductions in splenic amastigotes on days 10 and 21 postinfection compared with all other control groups, but on day 35, splenic amastigotes did not differ significantly from those of control animals. Our composite observations provide evidence for glucan-enhanced nonspecific resistance of hamsters to visceral leishmaniasis.     

Identification of antibody classes and Fc receptors responsible for phagocytosis of Trypanosoma musculi by mouse macrophages

The phagocytosis of Trypanosoma musculi by macrophages in the presence of specific antibodies was investigated. In 14-day-infected mice, opsonic antibodies were detected in serum, and phagocytosis of parasites by peritoneal macrophages was observed. The mechanism of T. musculi phagocytosis was analyzed. The binding of trypanosomes to peritoneal macrophages and J774 cells was observed in the presence of serum from hyperimmune mice and from mice infected 14 or 28 days earlier, but not in the presence of control mouse serum or sera from 7-day-infected mice. Binding was partially inhibited by mouse monoclonal immunoglobulins G1 (IgG1) or IgG2a and almost completely inhibited by a mixture of both. Binding was also partially inhibited by the anti-Fc gamma 1/gamma 2b receptor monoclonal antibody 2.4G2. Binding of T. musculi was also induced by fractions of serum from 28-day-infected mice obtained by protein A-Sepharose chromatography. Only the IgG1-rich fraction eluted at pH 6.0 and the IgG2a-rich fraction eluted at pH 4.5 promoted binding which could be almost completely inhibited by monoclonal IgG1 and IgG2a. These data indicate that IgG1 and IgG2a anti-T. musculi antibodies are responsible for the phagocytosis of T. musculi by mouse macrophages and both Fc gamma 2a and Fc gamma 1/gamma 2b receptors are involved. Such a mechanism is likely to account for the elimination of parasites in T. musculi-infected mice.     

Analysis of macrophage interactions with cryopreserved amastigotes of Leishmania tropica.

Amastigotes of Leishmania tropica were harvested from infected footpads of BALB/cJ mice and cryopreserved, using a controlled-rate freezer. This technique produced inocula with reproducible viability and infectivity for peritoneal macrophages from C3H/HeN mice. Cryopreserved amastigotes, stored for up to 17 weeks in a liquid nitrogen freezer, were similar to freshly harvested parasites in susceptibility to lymphokine-induced intracellular killing. Interactions of cryopreserved parasites with macrophages were best evaluated at 72 h, since macrophages preferentially ingested viable amastigotes and rapidly cleared ingested nonviable parasites.     

Role of macrophages in tumour immunity: II. Involvement of a macrophage cytophilic factor during syngeneic tumour growth inhibition*

Growth inhibition by immune or armed macrophages of target L5178Y or SL2 (DBA/2) lymphoma cells leading to their death required an immunologically specific cell-to-cell contact, and was not mediated via a soluble product of the macrophages. The lymphoma cells were not irreversibly damaged if left in contact for up to 24 hours with the immune macrophages and grew normally when removed, but while in contact there was little or no growth of the cells. Immune macrophages progressively lost their cytotoxic capacity after prolonged exposure to the target cells. The antigenic recognition of specific lymphoma cells by immune macrophages could be blocked by the presence of allo-immune serum directed against the lymphoma cells, but this was not affected by treatment of the macrophage monolayers with rabbit anti-mouse γ-globulin, and only slightly affected by high concentrations of trypsin. The involvement of cytophilic factors during the cytotoxic reaction is discussed in relation to the presence of γ-globulin or non-immunoglobulin factors on immune macrophage membranes and after the arming of non-immune macrophages by contact with hyperimmune spleen cells or with SMAF (the specific macrophage-arming factor) found in supernatants of mixed cell cultures of sensitized lymphoid cells and specific lymphoma cells.     

Characteristics of cytophilic antibody in the mouse

Immunoglobulin M hemagglutinin and antibody cytophilic for macrophages were produced simultaneously in mice after primary antigenic stimulation with sheep erythrocytes in saline. Antigen emulsified in Freund's complete adjuvant induced formation of cytophilic antibody (CA) in high titer. Purified cytophilic globulin was isolated by permitting it to attach to macrophages in vitro and eluting it at 56 C. The chromatographic elution pattern, molecular weight, and resistance to mercaptoethanol suggested that CA is a member of the immunoglobulin G class of immunoglobulins.     

Comparative study of American cutaneous leishmaniasis and diffuse cutaneous leishmaniasis in two strains of inbred mice.

Two Leishmania strains, AZV (isolated from a typical case of American cutaneous leishmaniasis) and AMP (from a case of diffuse cutaneous leishmaniasis), were studied in C57BL/6 and BALB/c mice. After infection with 10(4) amastigotes of either strain, C57BL/6 mice developed self-resolving lesions lasting 20 to 23 weeks and showed both delayed hypersensitivity response to leishmanial antigen and specific agglutinating antibodies. On the other hand, BALB/c mice infected with 10(4) AZV or AMP amastigotes developed chronic, large, ulcerated lesions and showed impaired cellular and humoral responses to the parasite. When BALB/c and C57BL/6 mice received 10(2) AMP amastigotes, patterns of infection were similar to those observed after inoculation of 10(4) amastigotes. In vitro studies revealed that spleen cells from AZV- or AMP-infected C57BL/6 mice showed an increased DNA-synthetic response to leishmanial antigen, concanavalin A, and phytohemagglutinin. Spleen cells from AZV- or AMP-infected BALB/c mice showed an increased response to concanavalin A and diminished responses to leishmanial antigen, phytohemagglutinin, and lipopolysaccharide.