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The present study was performed to characterize the immunologic potential of interstitial macrophages (INT) in comparison with alveolar macrophages (AL). The data showed that AL, compared with INT, have a more efficient phagocytic potential. In addition, they have a strong microbicidal activity and secrete large amounts of reactive oxygen radicals, nitric oxides, TNF, and IFN on appropriate stimulation. They also exert strong tumoricidal and parasiticidal activities. In contrast, INT are more efficient in releasing immunoregulatory cytokines such as IL-1 and IL-6. As determined by Ab staining, INT express more MHC class II molecules and are more effective in functioning as accessory cells for mitogen-stimulated lymphocyte proliferation compared with AL. Thus, AL appear to be particularly effective as nonspecific first line defense cells against infectious agents, whereas INT are equipped to cooperate with interstitial lymphocytes in inducing a specific immune reaction.

Alveolar macrophages (AL) are the first line of defense against inhaled pathogens and are exposed to virus during the course of a respiratory syncytial virus (RSV) infection. Interference of virus with alveolar macrophage functions may contribute to the risk of acquiring secondary bacterial infections during or after respiratory tract infections with RSV or other viral agents. We studied whether murine AL get infected with RSV and whether they support viral replication in vitro. In addition, the effects of RSV on microbicidal and on immunoregulatory functions were examined. Only a subpopulation of AL expressed viral F proteins after exposure of these cells to RSV. Infected AL released only small amounts of infectious virus into the supernatant. The extent of virus replication in AL seemed to be dependent in part on the amount of IFN induced by the virus, as has been demonstrated by infection of lung tissue macrophages and AL in vitro. In general, RSV infection of pulmonary macrophages appeared to be abortive. Nevertheless, release of reactive oxygen intermediates, phagocytosis, and killing of protozoa were reduced in RSV-infected AL in comparison to noninfected AL. In contrast, RSV stimulated secretion of TNF-alpha, IL-1, and IL-6 in an infectious-dose dependent manner. Along with the increased cytokine release, accessory functions of AL were increased after RSV exposure. Thus, exposure of AL to RSV appeared to stimulate their immunoregulatory functions, whereas the microbicidal activity of these cells seemed to be severely diminished.

Pulmonary macrophages play a crucial role in the defense of inhaled pathogens. We characterized functional properties of alveolar (AM) and interstitial (IM) macrophages from rats. AM exhibited a pronounced microbicidal capacity as shown by an elevated production of reactive oxygen intermediates (ROI), nitric oxide (NO), tumor necrosis factor (TNF)-, and tumor cytotoxicity when compared with IM. In contrast, IM were superior to AM regarding mechanisms mainly involved in the induction and maintenance of specific immune reactions (major histocompatibility complex [MHC] class II expression, interleukin [IL]-1 and IL-6). In this line, we were interested in whether the microbicidal potential of AM could be augmented by treating Lewis rats with rat recombinant interferon (IFN)- (5 × 102 to 1 × 105 U/animal) intratracheally, avoiding infection of interstitial lung macrophages or other organ-associated macrophages. The pulmonary cytokine application resulted in an activation of AM when macrophages from IFN-treated animals were compared with control macrophages from saline-treated rats 18 h after the treatment: (1) mediator release (ROI, NO, TNF-, IL-6), (2) tumoricidal activity; (3) dose-dependent increase of MHC class II expression. The local immunomodulation enhanced the resistance of normal and immunosuppressed rats against respiratory infections with Listeria monocytogenes. Taken together, local activation of lung macrophages is a feasible therapeutic strategy against pulmonary infections.

The efficiency of immunotherapy with murine recombinant interferon-Gamma (rIFN-Gamma) in mouse visceral leishmaniasis caused by Leishmania donovani was examined. To avoid the side effects encountered after the in vivo administration of high dosages of free IFN-Gamma, this lymphokine and muramyltripeptide (MTP-PE) were encapsulated into multilamellar liposomes. We established that a combination of 5 x 10high3U of IFN-Gamma and 6 Myg of MTP-PE, encapsulated in liposomes and given i.v. in C56BL/6 and BALB/c mice activates macrophages from spleen and liver in vivo to kill L. donovani in vitro. Neither empty liposomes nor the same concentration of free IFN-Gamma and/or MTP-PE injected i.v. resulted in a leishmanicidal activity of these macrophage populations. For verification of these results in an in vivo infection model, susceptible mice were infected with L. donovani and were treated with IFN-Gamma and MTP-PE encapsulated in multilamellar vesicles. Treatment consisted of multiple i.v. in jections beginning 4 and 2 days before infection (prophylactic), either simultaneously with the infection or at various times of the exacerbation and remission phases of visceral leishmaniasis. These mouse strains treated with IFN-Gamma and MTP-PE in liposomes had significantly fewer splenic parasites than untreated mice or control animals treated with free drugs or empty liposomes.

Systemic lupus erythematosus is an autoimmune disease, characterized by high titers of autoantibodies against many cell-membrane and intracellular antigens. Polyclonal B cell activation and alterations in the T cell compartment have been described. The present report deals with the organ-associated macrophage (M phi) system of two lupus-prone mouse strains and demonstrates that in both mouse strains the M phi compartment of liver and spleen is clearly expanded. In the liver the number of F 4/80plus M phi is strongly elevated. In addition, presence of early M phi precursors and of extramedullary organ-associated monocyte proliferation in response to colony-stimulating factor (CSF) is documented in liver and spleen of these mice. Further, in normal animals during the first two weeks of life extramedullar monocytopoiesis is present in liver and spleen, which is then down-regulated in the third week of life. In the two lupus-prone mouse strains down-regulation does not occur but extramedul lar monocyte proliferation is sustained at high level throughout life time. As possible correlates for the expansion of the M phi system elevated CSF-1 mRNA levels are demonstrated in kidney, spleen and liver of mice. The possible contribution of the expanded M phi system to B and T cell dysregulation is discussed.

A killing process is described where macrophages destroy 3H Thymidine prelabeled Leishmania parasites during the process of phagocytosis. This phagocytosis associated killing may represent a first line of defense and reduces drastically the amount of parasites homing in the macrophages. Treatment of the macrophages with anti-TNF antibody inhibited the phagocytosis associated killing. Soluble TNF had no effect on Leishmania parasites. Macrophages performed the killing process in the complete absence of any secreted TNF. Experiments using fixed macrophages and macrophage membranes revealed the existence of a 26 Kd membrane-TNF molecule. This membrane TNF was active against a TNF sensitive tumor cell line whereas the fixed cells or the isolated membranes had no cytotoxic effect on Leishmania parasites. This data indicates, that the Leishmanicidal effect was mediated through TNF but that apparently TNF itself was not the cytotoxic principle. Experiments using organ macrophages from Leishma nia infected susceptible C 57/B1 6 healer mice demonstrated that during the first 4 weeks of the infection the organ macrophages were totally unable to destroy Leishmania parasites independent of additional activation whereas the mechanism to destroy P815 tumor cells was unaffected. Similar data were obtained in therapy protocols using interferon gamma encapsulated in liposomes in order to target the substance to the macrophages. Also under these in vivo conditions the organ macrophages could not be activated and no beneficial effect was obtained by this activation treatment. In contrast, when therapy was started early (day 0 or 1) or late (5 weeks) after infection a highly significant reduction of the parasite burden was obtained.

Purified polysaccharides from cellcultures of the plant Echinacea purpurea were investigated for their ability to enhance phagocytes' activities regarding nonspecific immunity in vitro and in vivo. Macrophages (M phi) from different organ origin could be activated to produce IL-1, TNFalpha and IL-6, to produce elevated amounts of reactive oxygen intermediates and to inhibit growth of Candida albicans in vitro. Furthermore. in vivo the substances could induce increased proliferation of phagocytes in spleen and bone marrow and migration of granulocytes to the peripheral blood. These effects indeed resulted in excellent protection of mice against the consequences of lethal infections with one predominantly M phi dependent and one predominantly granulocyte dependent pathogen, Listeria monocytogenes and C. albicans, respectively. Specific immune responses to sheep red blood cells (antibody production) and to listeria (DTH) were not affected by the polysaccharides. The possibility of clinica l use is discussed.

Recent studies of our group presented evidence that the immunotherapy of murine visceral leishmaniasis with murine recombinant interferon gamma encapsulated in multilamellar vesicles reduced the parasite burden in spleen and liver. Mice treated with interferon gamma in liposomes had significantly fewer parasites than untreated mice, but the parasite burden could not be completely reduced. Therefore we performed experiments using a combination of interferon gamma and sodium stibogluconate (PentostamhighR) encapsulated in liposomes. The predilection of Leishmania species for macrophages renders this parasite and ideal target for liposome-encapsulated chemotherapeutic drugs like PentostamhighR or macrophage activators like interferon gamma. Liposomes, used here are preferentially taken up by spleen and liver macrophages when injected intravenously. The therapy with sodium stibogluconate and interferon gamma in liposomes resulted in a complete reduction of parasite burden in both organs, l iver and spleen. The chemotherapeutic in combination with the immuntherapeutic drug showed an increased effect compared to the influence of the single drugs. It is shown that this treatment of visceral leishmaniasis using combined liposomal drugs is much more effective than any single drug therapy.

Leishmaniasis is caused by an intracellular protozoan parasite. We examined the efficiency of immunotherapy with recombinant interferon gamma (IFN Gamma) in visceral leishmaniasis. In order to avoid the side effects encountered after the in vivo administration of high dosages of free IFN Gamma we encapsulated IFN Gamma and muramyltripeptide (MTP- PE) into multilamellar liposomes. The distribution of high123 J labelled IFN Gamma liposomes in C57BL/6 mice was investigated. We established that a combination of 5x10high3 U IFN Gamma and 6 Myg MTP- PE, encapsulated in liposomes, activates macrophages from spleen and liver in vivo to kill Leishmania donovani in vitro. For verification of these results in an in vivo infection model, susceptible mice were infected with Leishmania donovani and were treated with IFN Gamma and MTP-PE, encapsulated in multilamellar vesicles. Treatment consisted of multiple intravenous injections beginning prophylactic, simultaneously with the infec- tion and at va rious times of the exacerbation and remission phase. The targetting of the multilamellar vesicles to liver and spleen make them particularly suited for the delivery of macrophage activating substances used for treatment of Leishmania donovani infection. Mice treated with IFN Gamma and MTP-PE in liposomes had significantly fewer splenic parasites than the control groups.

Polysaccharides from plant cell cultures of Echinacea purpurea have been found to be very effective in activating macrophages both in vitro and in vivo. Macrophages stimulated with these polysaccharides produce tumor necrosis factor alpha, interleukin 1 and interferon beta. They are also activated to cytotoxicity against tumor cells and microorganisms. Applied intravenously these polysaccharides protect mice very efficiently from lethal infections with Candida albicans or Listeria monocytogenes.

We recently reported the modulating effects of a single injection of the anti-neoplastic drug cyclophosphamide 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 M 143 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.

The most important effectors of natural- as well as lymphokine-mediated cytotoxicity against microbicidal and fungal targets and protozoa such as Leishmania donovani are represented by cells of the monocyte-macrophage lineage. We recently described the bone marrow-derived macrophage precursor which is able to spontaneously and extracellularly kill protozoa of the genus Leishmania. These nonadherent, nonphagocytic macrophage precursor cells are present in the spleen of healthy mice only in a small quantity; however, high nmumbers have been isolated from the spleen of L. donovani infected mice. Macrophage precursors from spleen of diseased animals are able to kill spontaneously the promastigote as well as the amastigote form of L. donovani. the mechanism of the spontaneous leishmanicidal activity of macrophage precursor cells derived from spleens of L. donovani infected mice was investigated. This effector function could be defined in part as an antibody-dependent cellular cytotoxicity ( ADCC). In addition we assessed the role of CSFI containing L-cell donditioned supernatant at the leishmanicidal activity of these immature cells of the macrophage lineage. For that purpose, nonadherent cells from healthy mice were cocultivated with this CSFI containing medium for four days. These in vitro proliferated macrophage precursor cells from untreated mice showed an increased leishmanicidal activity. (-y-)

We recently described the bone marrow-derived macrophage precursor, which is able to spontaneously and extracellularly kill protozoa of the genus Leishmania. These nonadherent, nonphagocytic macrophage precursor cells are present in the spleen of healthy mice only in a small quantity. However, high numbers of proliferating macrophage precursors are isolated form the spleen of Leishmania donovani-infected mice. Macrophage precursors from spleens of diseased animals are able to kill spontaneously the promastigote as well as the amastigote form of L. donovani. The mechanism of the spontaneous leishmanicidal activity of macrophage precursor cells derived from spleens of L. This effector function could be defined in part as an antibody-dependent cellular cytotoxicity. In addition we assessed the role of CSF-1-containing L cell-conditioned supernatant at the leishmanicidal activity of these immature cells of the macrophage lineage. For that purpose, nonadherent spleen cells from healthy mice were cocultivated with this CSF-1-containing medium for 4 days. These in vitro proliferated macrophage precursor cells from untreated mice showed an increased leishmanicidal activity. Thereby we established a further activation mechanism for proliferating splenic macrophage precursor cells responsible for the observed killing of L. donovani pro- and amastigotes.

Recombinant mouse interferon gamma (IFN y) was encapsulated into multilamellar vesicles and the proportion of encapsulated IFN y determined by biological activity was 19%. The distribution of 125 J labelled IFN y liposomes in C57BL/6 mice was analysed. After an initial enrichment of liposomes in lung more than 60% of total 125 J IFN y was accumulated in spleen and liver. Furthermore it was observed if the encapsulation of IFN y in liposomes prevented the rapid decay of IFN y in serum of C57BL/6 mice after intravenous injection. We compared the serum decay curve of liposomal and free IFN y and it is shown that IFN y encapsulated in liposomes has an elongated availability in the serum. In addition we established that a combination of 10 high 2 U/ml IFN y and 1 ug/ml MTP-PE, encapsulated in liposomes, activates splenic and starch elicited peritoneal macrophages in vitro synergistically to kill Leishmania donovani promastigotes. After intravenous injection of liposomal IFN y (5 x 10 high 3 U) and MTP-PE (6 ug) in C57BL/6 mice splenic and liver macrophages were activated in vivo to kill Leishmania species in vitro. Neither an injection of the same amount of free substances nor injection of empty liposomes resulted in an increased leishmanicidal activity.

Homogenous populations of bone marrow derived murine macrophage precursors are able to respond to human rIL-2. A prolonged exposure to the lymphokine results in proliferation and in the final differentiation of macrophage precursors to mature macrophages, as shown by follow-up to the antigenic profile by means of cytofluorograph analysis. The frequency of the IL-2 responding cells was 1/48 as determined by limiting dilution analysis, and no cell death was evidenced in soft agar cultures. An analysis of the IL-2-binding sites conducted on bone marrow derived macrophage precursors revealed the presence of 4000 binding sites with intermediate affinity. Bone marrow-derived macrophage precursors did not react, even after prolonged culture in IL-2-conditioned media, with mAb recognizing the binding site or a binding site-associate molecule of a Mr 50000 to 60000 component of the IL-2R expressed by the IL-2-dependent T cell line CTLL-2. Crosslinking studies showed the presence of a single bin ding protein, with an apparent Mr of 70000. Macrophage precursors also proved able to internalize human rIL-2 within the first 20 minutes of incubation at 37 degree C. The implications of these findings are discussed.