Functional heterogeneity of murine macrophage precursor cells from spleen and bone marrow

We previously reported that highly purified bone marrow-derived macrophage precursors can exert strong spontaneous cytotoxicity against YAC-1 tumor cells, Candida albicans, and protozoa of the genus Leishmania. In the present paper, evidence is shown that macrophage precursors in normal untreated mice are not confined to the bone marrow compartment but can also be found in the spleen. These organ-associated cells, which have the same buoyant density as large granular lymphocytes, have been positively sorted by means of an indirect rosetting technique employing the macrophage-specific monoclonal antibodies F4/80 and M143. The rosetting fractions represented an extremely homogeneous population of macrophage precursors characterized by high candidacidal and natural killer activity and by a strong proliferative response to the macrophage-specific colony-stimulating factor CSF-1. Spleen- and bone marrow-derived macrophage precursors differed in their target selectivity. In addition, the mature macrophages derived in vitro from these two precursor populations displayed striking differences in their candidacidal activity. The implications of these findings in relationship to heterogeneity in the macrophage differentiation line are discussed.     

Organ-associated macrophage precursor activity: isolation of candidacidal and tumoricidal effectors from the spleens of cyclophosphamide-treated mice

We recently reported the modulating effects of a single injection of the anti-neoplastic drug cyclophosphamide (Cy; 150 mg/kg i.p.) on in vivo resistance against the experimental Candida albicans infection. Increased resistance to microbial challenge occurred 12 to 18 days after treatment. We now show that the increased resistance is paralleled by the appearance of potent nonadherent nonphagocytic effectors in the spleen (day 12) that are capable both of candidacidal activity and natural killer (NK) activity against YAC-1 cells. The cells mediating the two reactivities have a low buoyant density, a strong proliferating activity in response to the macrophage colony stimulating factor (CSF-1), and are unable to kill the NK-insensitive lines EL-4 and P815. A clear cut isolation of macrophage precursor cells from this Percoll low density fraction has been performed in an indirect rosette assay on the basis of their positivity for the surface markers recognized by the highly specific rat-anti-mouse macrophages, monoclonal antibodies M143 and F4/80. We obtained an extremely homogeneous population of cells in the early stage of macrophage differentiation that is responsible for all the candidacidal activity and for a major part of the NK activity observed in the spleen of Cy-treated mice, and which is extremely sensitive to CSF-1 induction.     

Human monocyte activation for cytotoxicity against intracellular Leishmania donovani amastigotes: induction of microbicidal activity by interferon-gamma

Macrophages are pivotal cells in interactions of man and leishmania. Leishmanial disease results from intracellular infection of macrophages: parasitized cells are seen in smears or biopsy specimens of lesions; macrophages cultured in vitro support replication of parasites. Paradoxically, parasite destruction is also mediated by macrophages, which become highly cytotoxic after exposure to immune lymphocytes or their lymphokine (LK) products. The precise molecular mechanisms by which lymphocytes or LK induce macrophage activation for leishmanicidal activity, however, are not yet known. We analyzed interactions of leishmania amastigotes with human monocytes cultured in vitro as a nonadherent cell pellet. Leishmania donovani and L. major replicated in freshly isolated monocytes. Monocytes treated with greater than 200 IU/ml of the LK, human Interferon-gamma (IFN-gamma), destroyed tumor cells and L. donovani, but not L. major. Phorbol myristate acetate, endotoxic bacterial lipopolysaccharide, and recombinant human IFN-alpha and IFN-beta did not induce cytotoxicity. The time course for induction of cytotoxicity contrasted sharply with that of previously described monocyte antileishmanial activity: IFN-gamma induced cytotoxicity even when added after infection with L. donovani; induction of cytotoxicity did not require that IFN-gamma be present throughout the period of culture after infection: a 30-min preinfection pulse of IFN-gamma was sufficient to induce 70% of maximal activity; and freshly isolated monocytes and cells cultured for up to 4 days in vitro prior to infection and IFN-gamma treatment were equally responsive to IFN-gamma. These studies provide convincing evidence for intracellular cytotoxicity for L. donovani by freshly isolated human monocytes. This system provides an important base for further analysis of induction and expression of cytotoxic mechanisms against leishmania and other intracellular organisms that cause human disease.     

Cell contact-mediated macrophage activation for antileishmanial defense. II. Identification of effector cell phenotype and genetic restriction

Host defense in cutaneous leishmaniasis, due to Leishmania tropica, is largely--if not exclusively--cell mediated. We observed in vitro that draining lymph node lymphocytes from L. tropica-infected C57BL/6 mice activate L. tropica-infected macrophages to kill the intracellular parasites (leishmanicidal effect). Because direct cell contact between lymphocytes and infected macrophages is required to achieve a maximum leishmanicidal effect, this effect cannot be attributed solely to lymphokines. Furthermore, because effector lymphocytes induced no detectable damage to infected macrophages, the effect also differs from conventional lymphocyte-mediated cytotoxicity. The present study identifies the phenotype of the effector lymphocyte and assesses the genetic restriction of the lymphocyte-macrophage interaction. Nylon wool column-enriched T lymphocytes from infected mice activate macrophages for antileishmanial effects; treatment of lymphocytes with anti-Thy-1.2 antibody plus complement abolishes this capacity. Furthermore, treatment with anti-Lyt-1 antibody plus complement (but not with anti-Lyt-2 plus complement) likewise abolishes the effector capacity of the lymphocytes. Parallel studies reveal that the percentage of Lyt-1+2- cells present in draining lymph nodes increases during the course of infection and reaches a peak with the onset of spontaneous resolution of the infection. Syngeneic, but not allogeneic, combinations of lymphocytes and infected macrophages result in macrophage activation. Furthermore, treatment of cells with appropriate anti-Ia monoclonal antibody abrogates the antileishmanial effects. These results indicate that Lyt-1+2- lymphocytes obtained from mice with spontaneously healing L. tropica infections can exert antileishmanial effects in vitro. This effect is genetically restricted--most likely to the I region of the MHC--and requires direct cell contact. The temporal relationship between the appearance of these effector lymphocytes in mice and the onset of disease resolution argues that they may also exert these antileishmanial effects in vivo.     

Macrophage precursors as natural killer cells against tumor cells and microorganisms

Macrophage precursors cells have been isolated from spleen and liver of mice and have characterized using F4/80 antibody, their proliferative response to CSF-1 and their maturation to macrophages. These nonadherent and nonphagocytic cells exert strong killing of Yac-1 tumor cells and of various microorganisms. Transplantation of these macrophage precursors into lethally irradiated allogenic hosts restores natural killer (NK) activity within 14 days. Macrophage precursors show enhanced NK activity when activated with interleukin 2. FACS analysis of F 4/80 presorted macrophage precursors reveals about 30% of the cells coexpressing NK 1.1. and F 4/80. These data support the assumption that at least a part of the NK cell compartment is derived from the myeloid lineage.     

Suppression of macrophage antimicrobial activity by a tumor cell product

Medium conditioned by tumor cells (TCM) and certain nonmalignant cells contains a trypsin-sensitive factor that suppresses macrophage oxidative metabolism. Because the killing of intracellular pathogens such as Toxoplasma gondii and Leishmania donovani by macrophages is largely oxygen-dependent, we tested the effect of TCM on the antiprotozoal activity of mouse peritoneal macrophages. After 24 hr of cultivation with TCM, in vivo and in vitro activated macrophages could no longer kill toxoplasmas or inhibit their replication. In vivo administration of TCM resulted in similar impairment. The leishmanicidal activity of resident and activated macrophages, when measured 6 hr after infection, was markedly suppressed by in vitro exposure to TCM. The addition of exogenous H2O2 in the form of glucose-glucose oxidase reconstituted the capacity of TCM-exposed macrophages to kill L. donovani promastigotes as quickly as control cells. Thus, TCM appears to deactivate macrophages by the functional criteria of suppressed antitoxoplasmal and antileishmanial activity, as well as by the biochemical criterion of suppressed oxidative metabolism.     

Isolation of protective T cells from BALB/cJ mice chronically infected with Leishmania donovani

BALB/cJ mice, which are homozygous for Lshs on chromosone 1, are genetically susceptible to Leishmania donovani (S3), but spontaneously reduce their parasite burdens late in the course of infection. Spleens from chronically infected mice (5 to 8 mo) were found to have T cells that responded to leishmanial Ag by proliferating and secreting immune IFN. An IFN-secreting T cell line derived from the spleen of a chronically infected mouse, after boosting with leishmanial Ag and treatment with Pentostam to kill residual parasites, was able to activate macrophages to kill amastigotes in vitro. Furthermore, after adoptive transfer of this cell line, naive BALB/cJ mice challenged with amastigotes demonstrated a 42-fold reduction in parasite burden compared to controls. Four of five clones derived from the protective T cell line secreted IFN and proliferated between days 7 and 14 after Ag stimulation. All clones were Ly1+2-, L3T4+. Another T cell line, which had Ly1+2-, L3T4+ phenotype, was derived from the lymph nodes of s.c. immunized, uninfected mice. It multipled but never produced IFN in response to leishmanial Ag and failed to protect macrophages against L. donovani infection in vitro or in vivo. This nonprotective T cell line and the fifth clone from the protective line, which also did not secrete IFN, proliferated between days 2 and 7 after Ag stimulation. In summary, leishmania-responsive helper/inducer T cells, which produced IFN but were slow to proliferate in response to Ag in vitro and which were able to activate macrophages to reduce amastigotes in vitro and in vivo, are present in chronically infected BALB/cJ mice and may mediate the decrease in parasite burden during chronic infection.     

Role of colony-stimulating factor-1 in macrophage activation in tumor-bearing mice.

We previously reported a dramatically increased number of macrophages in tumor-bearing mice. In this study, we investigated the involvement of CSF in that phenomenon. CSF-1 responding cells as macrophages precursors increased significantly in number in the spleens of tumor-bearing mice as compared with those in normal mice. Splenic cells and sera from the tumor-bearing mice respectively expressed CSF-1 in mRNA and serum protein levels, but failed to express the other CSF (granulocyte-macrophage-CSF or IL-3). Nonadherent splenic mononuclear cells (< 0.5% macrophages) from normal mice proliferated and differentiated into mature macrophages in culture within 7 days with recombinant mouse CSF-1 (rCSF-1). Both macrophages harvested from tumor-bearing mice and those activated in vitro with rCSF-1 expressed mostly Mac-1, -2 (and -3) Ag, showed yeast phagocytosis, produced IL-1 but not IL-2 or IL-3, and displayed potent cytotoxicity against NK cell resistant Meth-A tumor cells. These macrophages also expressed lipocortin I mRNA and secreted lipocortin I protein, and suppressed mitogenic responses of splenic lymphocytes. rCSF-1-activated macrophages derived from nonadherent splenic cells expressed both CSF-1 and CSF-1 receptor (c-fms) mRNA. Administration of rCSF-1 into normal mice induced hemopoietic and immunologic alternations similar to those observed in tumor-bearing mice. These results suggest that CSF-1 is involved in the dramatic increase of macrophages in tumor-bearing mice, possibly through an autocrine or paracrine loop.     

Prevention of relapse after chemotherapy in a chronic intracellular infection: mechanisms in experimental visceral leishmaniasis

In visceral leishmaniasis, chemotherapy probably seldom eradicates all parasites in tissue macrophages; nevertheless, most T cell-intact patients show long-lasting clinical cure after treatment despite residual intracellular infection. To characterize prevention of posttreatment relapse, amphotericin B was used to kill approximately 90-95% of Leishmania donovani in livers of mice deficient in mechanisms of acquired antileishmanial resistance. Recrudescence subsequently developed 1) in animals deficient in both CD4 and CD8 T cells as well as CD40L-mediated T cell costimulation, but not in a) CD4 or CD8 cells alone, b) NK cell lytic activity, or c) ICAM-1-recruited monocytes; and 2) in mice deficient in IFN-gamma, but not in the IFN-gamma-inducing cytokines, a) IL-12, b) IL-12 and IL-23, or c) IL-18. Posttreatment recrudescence also did not develop in animals deficient in macrophage phagocyte NADPH oxidase (phox) or inducible NO synthase (iNOS) alone or, surprisingly, in those deficient in both phox and iNOS. Therefore, regulation of the intracellular replication of residual Leishmania donovani that escape chemotherapy evolves to a host mechanism distinguishable from initial acquired resistance at the T cell, cytokine, and macrophage levels. Posttreatment, either CD8 or CD4 cells can direct the response, IL-12 is not required, and iNOS and phox, the activated macrophage's primary IFN-gamma-inducible leishmanicidal pathways, both become dispensable.     

Dendritic cell-based immunotherapy combined with antimony-based chemotherapy cures established murine visceral leishmaniasis

Dendritic cells (DCs) have been proposed to play a critical role as adjuvants in vaccination and immunotherapy. In this study we evaluated the combined effect of soluble Leishmania donovani Ag (SLDA)-pulsed syngeneic bone marrow-derived DC-based immunotherapy and antimony-based chemotherapy for the treatment of established murine visceral leishmaniasis. Three weekly injections of SLDA-pulsed DCs into L. donovani-infected mice reduced liver and splenic parasite burden significantly, but could not clear parasite load from these organs completely. Strikingly, the conventional antileishmanial chemotherapy (sodium antimony gluconate) along with injections of SLDA-pulsed DCs resulted in complete clearance of parasites from both these organs. Repetitive in vitro stimulation of splenocytes from uninfected or L. donovani-infected mice with SLDA-pulsed DCs led to the emergence of CD4(+) T cells with characteristics of Th1 cells. Our data indicate that DC-based immunotherapy enhances the in vivo antileishmanial potential of antimony or vice versa.     

Role of protein kinase C-alpha in the control of infection by intracellular pathogens in macrophages.

The protein kinase C (PKC) family regulates macrophage function involved in host defense against infection. In this study, we investigated the role of macrophage PKC-alpha in the uptake and subsequent fate of Leishmania donovani promastigotes and Legionella pneumophila infections. To this end, we used clones of the murine macrophage cell line RAW 264.7 overexpressing a dominant-negative (DN) mutant of PKC-alpha. While phagocytosis of L. donovani promastigotes was not affected by DN PKC-alpha overexpression, their intracellular survival was enhanced by 10- to 20-fold at 48 h postinfection. Intracellular survival of a L. donovani mutant defective in lipophosphoglycan repeating units synthesis, which normally is rapidly degraded in phagolysosomes, was enhanced by 100-fold at 48 h postinfection. However, IFN-gamma-induced leishmanicidal activity was not affected by DN PKC-alpha overexpression. Similar to macrophages from genetically resistant C57BL/6 mice, control RAW 264.7 cells were not permissive for the intracellular replication of Legionella pneumophila. In contrast, DN PKC-alpha-overexpressing RAW 264.7 clones were phenotypically similar to macrophages from genetically susceptible A/J mice, as they allowed intracellular replication of L. pneumophila. Permissiveness to L. pneumophila was not the consequence of a general defect in the microbicidal capacities because killing of a temperature-sensitive mutant of Pseudomonas aeruginosa was normal in DN PKC-alpha-overexpressing RAW 264.7 clones. Collectively, these results support a role for PKC-alpha in the regulation of innate macrophage functions involved in the control of infection by intracellular parasites.     

General suppression of macrophage gene expression during Leishmania donovani infection

Within the mammalian host, Leishmania donovani is an obligatory intracellular protozoan that resides and multiplies exclusively in the phagolysosomes of macrophages. The outcome of this infection is governed by the interaction between Leishmania and macrophage molecules that ultimately effect the expression of genes within both cells. To explore the effect of this intracellular infection on macrophage gene expression, a cDNA expression array analysis was performed to compare gene expression profiles in noninfected and L. donovani-infected macrophages. In this manner, it was possible to examine the effect of infection on the expression of several hundred well-characterized host cell genes in an unbiased manner. Interestingly, approximately 40% of the genes whose expression was detected in macrophages were down-regulated during infection with L. donovani. However, several genes were also induced during the infection process, some of which could play a role in recruitment of additional macrophages to the site of infection. Taken together, the general suppression of gene expression in addition to the selective induction of key genes is likely to play an important role in allowing the parasite to survive and proliferate within its host macrophage cell.     

The hamster as a model of human visceral leishmaniasis: progressive disease and impaired generation of nitric oxide in the face of a prominent Th1-like cytokine response

Active human visceral leishmaniasis (VL) is characterized by a progressive increase in visceral parasite burden, cachexia, massive splenomegaly, and hypergammaglobulinemia. In contrast, mice infected with Leishmania donovani, the most commonly studied model of VL, do not develop overt, progressive disease. Furthermore, mice control Leishmania infection through the generation of NO, an effector mechanism that does not have a clear role in human macrophage antimicrobial function. Remarkably, infection of the Syrian hamster (Mesocricetus auratus) with L. donovani reproduced the clinicopathological features of human VL, and investigation into the mechanisms of disease in the hamster revealed striking differences from the murine model. Uncontrolled parasite replication in the hamster liver, spleen, and bone marrow occurred despite a strong Th1-like cytokine (IL-2, IFN-gamma, and TNF/lymphotoxin) response in these organs, suggesting impairment of macrophage effector function. Indeed, throughout the course of infection, inducible NO synthase (iNOS, NOS2) mRNA or enzyme activity in liver or spleen tissue was not detected. In contrast, NOS2 mRNA and enzyme activity was readily detected in the spleens of infected mice. The impaired hamster NOS2 expression could not be explained by an absence of the NOS2 gene, overproduction of IL-4, defective TNF/lymphotoxin production (a potent second signal for NOS2 induction), or early dominant production of the deactivating cytokines IL-10 and TGF-beta. Thus, although a Th1-like cytokine response was prominent, the major antileishmanial effector mechanism that is responsible for control of infection in mice was absent throughout the course of progressive VL in the hamster.     

Macrophage activation for tumor cytotoxicity: induction of tumoricidal macrophages by supernatants of PPD-stimulated Bacillus Calmette-Guérin-immune spleen cell cultures.

Resident peritoneal macrophages from normal mice were activated for tumor cytotoxicity in vitro by co-cultivation with BCG1-immune spleen cells and PPD and by incubation with supernatants of PPD-stimulated BCG-immune spleen cell cultures (lymphokine supernatants). Lymphokine activation of macrophages occurred in unfractionated PC suspensions as well as in macrophage monolayers depleted of nonadherent PC. Tumor cytotoxicity by lymphokine-activated macrophages was evident by 3 to 4 hr of culture in active supernatants, reached maximal levels by 8 to 12 hr. and was absent by 20 hr. Continued incubation in lymphokines or even re-exposure after washing did not maintain macrophage cytotoxicity. The capacity of normal resident macrophages to be activated by lymphokines in vitro progressively decreased and was absent by 20 hr in culture. This decrease did not necessarily reflect cell death; macrophage viability as estimated by exclusion of trypan blue or by phagocytic responses did not change over the 20-hr culture period. The short lived nature of both macrophage tumoricidal capacity and capacity of precursor cells to be activated by lymphokines may function as negative feedback mechanisms in immune reactions.     

Leishmanicidal activity of stearylamine-bearing liposomes in vitro

Liposomes consisting of stearylamine (SA) and egg yolk phosphatidylcholine (PC) were studied for their cytotoxic activity against freshly transformed promastigotes and intracellular amastigotes of Leishmania donovani, the causative agent of visceral leishmaniasis. More than 99% of the parasites of strain AG83 were killed within 60 min by treatment with 22 mol% SA-PC liposomes (132 microg/ml total lipids). This was further confirmed by incubating the liposome-treated promastigotes at 22 C for 96 hr. The killing activity of the liposomes progressively decreased with lowering lipid concentration. However, weak cytotoxic activity was still detected at 6.6 microg/ml lipids. Leishmanicidal activity of the liposomes became stronger with increasing SA content but was reduced with the incorporation of cholesterol in the liposomes. A similar cytotoxic effect was observed on other Indian strains of L. donovani, for example PKDL and DD8, as well as on species such as Leishmania donovani S1, Leishmania donovani infantum, Leishmania tropica, Leishmania amazonensis, and Leishmania mexicana. However, freshly transformed promastigotes appeared to be more susceptible than the ones subcultured. The strong leishmanicidal activity of SA-PC liposomes was also demonstrated toward intracellular L. donovani amastigotes. The SA-bearing vesicles could effectively inhibit the growth and multiplication of the parasites within the macrophages. The cytolytic activity of these liposomes on leishmanial parasites and low toxicity on host macrophages may, thus, find application in the therapy of leishmaniasis.     

Inhibition of Leishmania Donovani Amastigote-To-Promastigote Transformation By Infected Hamster Spleen Lymphocyte Lysates*

SYNOPSIS Leishmania donovani amastigote-to-promastigote transformation is inhibited by homogenates of infected hamster liver and spleen. This inhibitory activity is localized in the 100,000 g pellet fraction. Tests with lysates of adherent (macrophyages) and nonadherent (lymphocytes) spleen cells indicated that the inhibitory activity resided in the lymphocytes, specifically in the 100,000 g pellet fraction.     

Colony-stimulating factors and regulation of macrophage tumoricidal and microbicidal activities

Conditioned medium from antigen- or mitogen-stimulated spleen cells, lymphokines, contained factors that induced formation of granulocyte and macrophage colonies in cultures of bone marrow cells (CSF). Lymphokines also contained factors that induced macrophage non-specific tumoricidal activity against fibrosarcoma 1023, antibody-dependent tumoricidal activity against lymphoma 18-8, and antimicrobial activities against amastigotes of the protozoan parasite, Leishmania tropica. The factors that regulated macrophage effector functions, however, were different from those that induced colony formation, and could be distinguished from CSF by Sephadex gel chromatography or heat sensitivity. To further analyze a role for CSF in induction of macrophage effector activities, conditioned medium from several nonlymphoid cell sources (L-929, WEHI-3, and endotoxin-treated lung cells) were assayed for CSF activities and capacity to induce tumoricidal and microbicidal activities. Conditioned medium that contained either macrophages CSF (CSF-1) or the factor that induced formation of both macrophage and granulocyte colonies failed to activate macrophages for effector activities against fibrosarcoma 1023, lymphoma 18-8, and L. tropica amastigotes (either resistance to infection or intracellular destruction). These data suggest that CSF has no direct role in activation of macrophages for tumoricidal and microbicidal activities against these targets.     

Suppressor T-cell hybridomas that exhibit various capacities to negatively regulate PFC responses in a T-cell dependent mouse model

Splenic leucocyte effector cells from 3-month-old C3H/HeJ mice which were antibody negative for influenza A virus were found to be spontaneously cytotoxic for mouse L929 fibroblasts infected with influenza virus, but not for uninfected cells. The cytotoxic activity was significantly enhanced if the effector cells were preincubated with splenic leucocytes from older 6- or 9-month-old antibody-negative mice before being added to the target cells. Enhancement appeared to be due to a cooperative effect between leucocytes from mice of different ages. Preincubation of effector cells with autologous sera markedly suppressed spontaneous cytotoxicity, but the suppression was significantly less with sera from 7- or 10-month-old mice than with sera from 3-month-old mice. Most cytotoxic activity was observed with a nylon wool-adherent subpopulation of leucocytes, although some cytotoxicity remained in the non-adherent fraction. The results suggested that spontaneous cytotoxicity was due to two subpopulations of cells, possibly adherent monocyte-macrophages and nonadherent natural killer cells.     

Antileishmanial activities of macrophages from C3H/HeN and C3H/HeJ mice treated with Mycobacterium bovis strain BCG

C3H/HeN and C3H/HeJ mice were infected ip with viable BCG, a macrophage-activating agent, and their peritoneal exudate macrophages exposed to Leishmania tropica amastigotes. Macrophages from BCG-infected C3H/HeN mice had both leishmanicidal activities described for lymphokine activation of C3H/HeN macrophages in vitro: increased resistance to L. tropica infection, followed by intracellular killing of the parasite. Macrophages from BCG-infected C3H/HeN mice were also activated to kill tumor cells in vitro. In contrast, macrophages from BCG-treated C3H/HeJ mice were not resistant to L. tropica infection, did not kill intracellular amastigotes over 72 hr in culture, and were not cytotoxic to tumor cells.     

Effect of immunization with lipid associated polysaccharide antigen and anti CD-2 antibodies on class II MHC expression and cellular immune response in BALB/C mice infected with Leishmania donovani

In a bid to characterize the antigens and immunization mechanisms which may be used to produce a protective response against L. donovani, role of lipid associated polysaccharide (LPS) antigen and whole antigen was evaluated. BALB/C mice were immunized with whole or LPS antigen in combination with one of three putative adjuvents (anti CD-2 antibody/FIA/0.85% Saline). LPS antigen emulsified in anti CD-2 antibody was found to induce significant antibodies in mice on day 28 against challenge with lethal dose of L. donovani. Immunoprophylactic properties of LPS and whole antigen was investigated on day 40 through cytokine elicitation (IL-2), MIF) in culture supernatants of spleen cells, but before that MHC-II expressed on macrophage was studied. The LPS antigen in combination with anti CD-2 antibody was found to be most immuno-reactive inducing higher MHC-II expression on macrophages which was associated with substantial rise in the level of MIF and IL-2. It coincided with decline in antibody titre in 100% mice immunized with LPS antigen while Leishmania injected as whole antigen failed to induce specific macrophage and T-cell response with all the above formulations. We surmise from our data that lipid associated polysaccharide antigen linked to anti CD-2 antibody has potential for eliciting protective immunity against Leishmania.