Nonadherent Spleen Cells Research Articles

Cytotoxic Activity and Lymphocyte Subtypes in Mice Selected for Maximal and Minimal Inflammatory Response after Transplantation of B16F10 and S91 Melanoma Cells.

AIRmax and AIRmin mice strains were selected according to the intensity of their acute inflammatory responsiveness. Previous studies have shown that AIR mice differ in their resistance to chemically induced skin tumors and in the development of melanoma metastases, in addition to differences in neutrophil and NK cells activity. In the present work, we aimed to evaluate whether the difference of susceptibility to murine melanoma is associated with NK cytotoxic activity against Yac.1 cells and lymphocyte subsets. Mice were subcutaneously inoculated with B16F10 or S91 melanoma cells. After 7, 14, or 30 days, the animals were euthanized to analyze the number of lymphocyte subsets, cytotoxic activity, and number of cytokine-producing spleen cells. AIRmax mice presented a higher number of CD4+/CD25+ cells than that of AIRmin mice following inoculation of B16F10 cells, whereas inoculation of S91 cells reduced CD4+/CD25+ and increased TCD8+ cell subsets in the AIRmax mice. AIRmax mice had a higher number of interleukin (IL)-10- and IL-12-producing cells and a lower number of interferon-γ–producing cells than those of AIRmin mice at 30 days. The cytotoxic activity of nonadherent spleen cells was similar in both the AIR strains. These results suggest that melanoma cells can induce different responses in AIR mice, possibly owing to alterations in regulatory mechanisms, such as the action of CD4+/CD25+ regulatory T cells and IL-10, in AIRmax mice.

Natural killer activity of the spleen cells of Ehrlich tumor-bearing mice treated with Copaifera multijuga extract

AIMS: Copaifera multijuga Hayne oleoresin is commonly used in traditional medicine owing to its anti-inflammatory, antiseptic, antitumor, and antibacterial properties. However, little is known about the effect of the compounds from the bark of this plant. In this study, the immunomodulatory effect of the ethanolic extract of C. multijuga bark via natural killer activity of non-adherent spleen cells of Ehrlich tumor-bearing mice was evaluated.METHODS: Male Swiss mice were inoculated subcutaneously with 1×106 Ehrlich tumor cells (Ehrlich and Ehrlich/C. multijuga group) or phosphate buffered saline solution (control group and C. multijuga group) and treated orally daily with C. multijuga extract (200 mg kg-1, 0.1 mL per mouse, for the Ehrlich/C. multijuga and C. multijuga groups) or phosphate buffered saline solution (control group and Ehrlich group). The four experimental groups consisted in eight mice each and were organized in two sets, one treated for seven days and another treated for 14 days, totalizing 64 mice throughout the experiment. Twenty-four hours after the last oral administration, the mice were euthanized and the spleen tissue was isolated to prepare a non-adherent spleen cell suspension and to evaluate natural killer activity. Data are presented as the cell lysis percentage of Yac.1 target cells by non-adherent spleen cells.RESULTS: Treatment for seven days increased natural killer activity in the Ehrlich/C. multijuga group (21.20±8.89, p<0.05) compared to the control group (3.14±2.71, p<0.05); however, this effect was not maintained in the groups treated for 14 days (Control: 6.02±6.98, Ehrlich: 4.82±7.72, C. multijuga: 2.07±2.10, Ehrlich/C. multijuga: 2.01±1.63, p>0.05).CONCLUSIONS: Treatment for seven days with an ethanolic extract of C. multijuga bark enhanced the natural killer activity of non-adherent spleen cells from Ehrlich tumor-bearing mice.

Downregulation of Cellular Energy Sensor during Disease condition results in Alteration of Lipid Homeostasis in Immune Cells

AMP‐activated protein kinase (AMPK), an energy sensing metabolic switch of mammalian cells, was observed to be down regulated during T cells mediated autoimmune disease. A catalytic subunit of AMPK, alpha1, was predominantly expressed in T‐ and antigen presenting cells. During EAE disease, activity of AMPK was significantly downregulated in thymocytes, spleen cells and brain. Decreased AMPK activity results increased lipid biosynthesis and their content in total T cells, adherent and non‐adherent spleen cells at disease state. Increased lipids biosynthesis leads to the higher proliferative state of these cells was evident from active form of Akt, p42/44 and reduced levels of p21 and p27. AMPK activators restored lipid alteration in total spleen cells by reversing the AMPK activity and inhibited production of pro‐inflammatory cytokines under in vitro stimulation. All together, our results demonstrated that downregulation of AMPK‐mediated altered lipid metabolism in immune cells, resulted an inflammatory environment may be one of the critical aspects of pathogenesis during EAE or Multiple Sclerosis and identified AMPK as a novel therapeutic target for autoimmune diseases.Grants support: Mainly by RG 3810‐A‐1 and PP1283 from the MS Society (SG) and in part by (NS‐22576, NS‐34741, NS‐37766 and NS‐40810) from NIH (IS).

Gpnmb Is Induced in Macrophages by IFN-γ and Lipopolysaccharide and Acts as a Feedback Regulator of Proinflammatory Responses

The process of inflammation requires the selective expression of a suite of genes in cells of the macrophage lineage. To identify candidate regulators of inflammation, we used cDNA microarrays to compare the transcriptome of inflammatory macrophages (thioglycolate-elicited peritoneal macrophages), bone marrow-derived macrophages, nonadherent spleen cells, and fibroblasts. We identified genes that were macrophage restricted and further elevated in inflammatory macrophages, and characterized the function of one such gene, gpnmb. Gpnmb mRNA expression was enriched in myelomonocytic cell lines and macrophage-related tissues and strongly up-regulated during macrophage differentiation. Epitope-tagged GPNMB expressed in RAW264.7 cells exhibited a perinuclear distribution and colocalized with the Golgi marker coat protein beta. Upon activation of macrophages with IFN-gamma and LPS, GPNMB translocated from the Golgi apparatus to vesicular compartments scattered toward the periphery. Gpnmb overexpression in RAW264.7 cells caused a 2-fold reduction in the production of the cytokines IL-6 and IL-12p40 and the inflammatory mediator NO in response to LPS. DBA mice, which have an inactivating point mutation in the gpnmb gene, exhibited reduced numbers of myeloid cells, elevated numbers of thioglycolate-elicited peritoneal macrophages, and higher levels of proinflammatory cytokines in response to LPS. Thus, GPNMB acts as a negative regulator of macrophage inflammatory responses.

Role of Tumor Necrosis Factor Alpha (TNF-α) and Interleukin-10 in the Pathogenesis of Severe Murine Monocytotropic Ehrlichiosis: Increased Resistance of TNF Receptor p55- and p75-Deficient Mice to Fatal Ehrlichial Infection

Intraperitoneal (i.p.) infection with a high dose of a highly virulent Ehrlichia strain (IOE) results in a toxic shock-like syndrome characterized by severe liver injury and systemic overproduction of tumor necrosis factor alpha (TNF-alpha) by CD8+ T cells. We examined the role of TNF-alpha and TNF receptors in high-dose-IOE-induced shock/liver injury. TNF receptor (TNFR) I/II-/- mice lacking both the p55 and p75 receptors for this cytokine were more resistant to IOE-induced liver injury than their wild-type background controls. TNFR I/II-/- mice survived longer, dying between 15 and 18 days, with evidence of mild liver necrosis/apoptosis. In contrast, wild-type mice were not rescued from the lethal effect of IOE by TNF-alpha neutralization. TNF-alpha-depleted mice developed severe liver injury and succumbed to disease between days 9 and 11 postinfection, similar to sham-treated, infected wild-type mice. Although IFN-gamma production in the spleens of IOE-infected TNFR I/II-/- and TNF-alpha-depleted mice was higher than that detected in wild-type controls, these mice had higher bacterial burdens than infected controls. Following high-dose IOE challenge, TNFR I/II-/- and TNF-alpha-depleted mice have an early increase in IL-10 levels in sera and spleens, which was produced mainly by adherent spleen cells. In contrast, a late burst of interleukin-10 (IL-10) was observed in control mice. Nonadherent spleen cells were the major source of IL-10 in IOE-infected wild-type mice. We conclude that TNFR I/II and TNF-alpha participate in Ehrlichia-induced shock and host defense by regulating liver injury and controlling ehrlichial burden. Our data suggest that fatal ehrlichiosis could be a multistep process, where TNF-alpha is not solely responsible for mortality.

Protective Effect of Curcumin in an F1-Hybrid Model of Acute Graft-vs.-Host Disease.

Curcumin (diferuloylmethane), a pigment from the rhizomes of Curcuma Longa, has anti-oxidant, anti-tumor and anti-inflammatory properties. It has been shown that curcumin can inhibit the development of experimental allergic encephalomyelitis, a Th1-mediated, autoimmune, demyelinating disease that affects the central nervous system in mice. Because acute GVHD involves the development of a Th1-mediated immune response, and is characterized by the development of a potent, systemic, inflammatory response, we wished to determine whether curcumin might also protect mice from developing acute GVHD in the C57BL/6→(C57BL/6 x DBA/2)F1-hybrid model. We found that ip injections of curcumin (100 micrograms dissolved in 25 microliters of DMSO), given once a day, every 2 days, protected recipients from developing acute, lethal GVHD. Two thirds of the treated group survived well past day 100 post-transplantation. Control GVH mice treated with DMSO alone became moribund within three weeks of transplantation, the time at which mice with acute GVHD typically succumb. To ensure that the protective effects of curcumin were not due to abortion of the graft, we analyzed T cell engraftment in some of the long-term survivors. In the surviving mice tested on days 135 and 275 post-transplantation, we were unable to detect any cells of recipient origin in the spleen. Furthermore, the percentage of donor-derived CD4+ cells ranged from 32–47% and the percentage of CD8+ T cells ranged from 25–42% in the non-adherent spleen cell fraction. Data from these experiments suggest that curcumin can protect mice from developing acute, lethal GVHD.

Efficient Gene Transfer into Spleen Cells of Newborn Mice by a Replication-Competent Retroviral Vector

A murine leukemia virus-derived replication-competent retroviral vector with a translational cassette for the enhanced green fluorescence protein (EGFP) was previously found to function efficiently in cell culture (Jespersen et al., 1999, Gene 239, 227–235). We here report that infection of newborn NIH Swiss mice gives rise to EGFP expression in a majority of spleen cells within the first days after infection. Among the nonadherent spleen cells, B, T, and NK lymphocytes were found to be efficiently marked by EGFP by flow cytometry analysis, whereas the adherent spleen cells were negative in most animals. Analysis at time points up to 60 days after infection reveals a decline in EGFP-positive spleen cells over time. Viremia analysis and PCR analysis of spleen DNA indicate that viruses that have lost the translation cassette predominate at later stages. The results provide a model for efficient gene delivery to spleen cells in a time window of 1 to 2 weeks after infection of newborns. Although this type of vector will not in itself be applicable in the clinic, we envision that efficient in vivo gene delivery will be valuable by analysis of differentiation and proliferation of hematopoietic cells in animal models and by development and evaluation of effectors interfering with these processes.

ABSENCE OF SEASONAL EFFECT ON THE IMMUNOMODULATORY ACTION OF BRAZILIAN PROPOLIS ON NATURAL KILLER ACTIVITY

Propolis, a beehive product widely used in folk medicine as an antiinflammatory agent, has been attracting researchers' attention to scientifically elucidate its biological properties and therapeutic activities. The aim of this paper was to study the possible effect of propolis on natural killer activity, since propolis immunomodulatory action has been suggested, especially on non-specific immunity. Propolis was produced by africanized honeybees (Apis mellifera L.), collected throughout a whole year, and pooled by season. Hydroalcoholic solutions of propolis were prepared with each pool and administered to rats by gavage over three days. Natural killer activity of non-adherent spleen cells was evaluated by the 51Cr-release cytotoxicity assay against Yac-1 target cells. Our results indicated that the natural killer activity was increased in spleen cells from propolis-treated animals. There were no significant differences related to the seasonal effect on the immunomodulatory action of propolis.

Bacterially induced activation of interleukin-18 in porcine intestinal mucosa

Interleukin (IL)-18 is a cytokine with structural and functional properties similar to IL-1β and IL-12, respectively. It is activated by caspase-1 cleavage, like IL-1β, and induces interferon (IFN)-γ, like IL-12. In order to study the role of IL-18 in the immune response to infectious diseases of mucosal surfaces we cloned and expressed porcine IL-18 and developed antibodies to the protein. Porcine IL-18 retains the caspase-1 cleavage site present in other mammalian IL-18 proteins, but has two potential N-linked glycosylation sites not found in those proteins. Porcine interleukin-18 mRNA and protein are expressed in immune tissues including lymph nodes and gut associated lymphoid tissues. Specific cell types containing IL-18 include lung and splenic macrophages, nonadherent spleen cells and intestinal epithelial cells. Although IL-18 transcription is moderately induced by lipopolysaccharide, the magnitude and total expression level are small compared to those of interleukin-1β. In vivo and ex vivo infection of intestinal mucosa with Salmonella choleraesuis resulted in a decrease in size of IL-18, consistent with cleavage of the preprotein by caspase-1. Thus, IL-18 is present in mucosal tissues where it could play a role in the immune response to invading pathogens.

Augmentation of natural killer cell activity in vitro against tumor cells by wild plants from Jordan

Thirteen aqueous extracts prepared from Jordanian plants, that are currently used in traditional medicine to treat various types of cancer, were tested in mice for their ability to augment natural killer (NK) cell function in vitro in generating cytotoxicity against YAC tumor targets. Lymphoid cells at a concentration of 5×10 6/ml were incubated in medium alone or in medium containing different dilutions of either plant extract or purified interferon α for 20 h and tested for NK activity. Maximum NK activity (62.3%) was obtained at 1:50 dilution of Nigella sativum fresh aqueous extract, 48.5% at 1:100 dilution for Allium sativum (and 38.3% at 1:50 dilution for Onopordum acanthium. Fresh aqueous plant extracts appeared to be more potent than old dried aqueous extract or ethanolic extracts. NK augmentation by plant extracts using nylon wool non-adherent spleen cells was slightly higher than the whole spleen cells.

Nitric-Oxide-Dependent Systemic Immunosuppression in Animals with Progressively Growing Malignant Gliomas

The role of nitric oxide (NO) and adherent spleen cells in systemic immunosuppression developing in animals carrying malignant glioma isografts was analyzed. Rats harboring a subcutaneous glioma isograft for 3 weeks were immunized with glioma cells genetically engineered to express IFN-γ. One week later spleen cells were tested for immune responsiveness in vitro. A decreased cytotoxic activity of NK-cells and T-cells compared to tumor-free animals immunized in parallel was shown. Spleen cell proliferative responses to tumor cells, SEA, and anti-CD3 were all significantly suppressed, as was the production of IFN-γ and IL-10. Plastic adherent spleen cells from tumor-bearing rats suppressed the SEA-induced proliferative response and the production of IFN-γ and IL-10 by nonadherent spleen cells from tumor-free rats. A major part of this suppression appears to be dependent on the production of NO because suppression was efficiently counteracted in vitro by the NO-synthase inhibitor N-nitro-l-arginine methyl ester. Moreover, a significantly increased level of nitrite in culture supernatants correlated with the observed suppression. We conclude that the systemic immunosuppression associated with growing gliomas is in part mediated by mechanisms dependent on NO overproduction in adherent spleen cells.

The role of the thymus in modulating gammadelta T cell suppressor activity during experimental Trypanosoma cruzi infection.

We have previously shown that splenic gammadelta T cells from young but not aged BALB/c mice possess suppressor activity in vivo and in vitro during the acute phase of Trypanosoma cruzi infection. The present work was undertaken to investigate the suppressor activity of gammadelta T cells from T. cruzi-infected euthymic or athymic mice and the role of the thymus in modulating non-adherent spleen cell suppressor activity during the acute phase of infection. Splenic gammadelta T cells from aged or athymic BALB/c mice reconstituted with total spleen cells or non-reconstituted did not exhibit suppressor activity when added to full allogeneic, mixed lymphocyte cultures. In contrast, splenic gammadelta T cells from young euthymic BALB/c mice showed suppressor activity in vitro. Thymectomy reduced the splenic gammadelta T cell suppressor activity of young BALB/c mice in a time-dependent manner, following a T. cruzi challenge. The continuous provision of thymocytes to aged mice, young thymectomized mice or total spleen cell-reconstituted athymic mice could re-establish the gammadelta T cell suppressor activity. Of particular significance was the observation that the depletion of gammadelta T cells during the acute phase of T. cruzi infection restored the capacity of these mice to mount a humoral immune response to a non-related antigen such as ovalbumin. These results indicate that gammadelta T cells of extrathymic origin cannot mediate suppression and that the thymus has a role in the regulation of suppression during the acute phase of T. cruzi infection.

Suppressor macrophages mediate depressed lymphoproliferation in chickens infected with avian reovirus

A previous study indicated that spleens from reovirus-infected chickens contained macrophages that were primed to produce nitric oxide (NO). The presence of these primed macrophages correlated with depressed in vitro T cell mitogenesis. The current studies indicated that splenic adherent macrophages from virus-exposed chickens inhibited concanavalin A (ConA) induced proliferation of normal spleen cells. ConA-stimulated spleen cells from uninfected chickens, but not virus-exposed chickens, produced large quantities of interleukin-2 (IL-2) and a factor that induced NO production. This factor was tentatively named NO inducing factor (NOIF). The removal of macrophages from the spleens of virus-exposed chickens by plastic adherence resulted in partial recovery of ConA-induced proliferation and the production of normal levels of IL-2 and increased levels of NOIF, although these remained below normal. However, nonadherent spleen cells produced substantial quantities of NO, which indicated an incomplete removal of macrophages. Because removal by plastic adherence did not result in the depletion of all macrophages, spleen cells were panned with anti-CD3 antibody to obtain an almost pure population of T cells. Fractionated T cells from virus-exposed chickens proliferated vigorously to ConA and produced normal levels of IL-2 and NOIF. When splenic adherent cells from virus-exposed chickens were added to purified T cells, the T cells failed to respond to ConA. Addition of splenic adherent cells from virus-free chickens did not induce mitogenic inhibition. Further, the addition of purified T cells from the spleens of reovirus-infected chickens to T cells from virus-free birds did not adversely affect T cell mitogenesis. These data indicated that reovirus infection in chickens does not compromise the functional capabilities of T cells but induces suppressor macrophages that inhibit T cell functions.

Differential effects of viable and killed bacteria on IL-12 expression of macrophages.

Listeria monocytogenes-specific, IFN-gamma-producing CD4+ T cells are induced in 5-day in vitro cultures of naive spleen cells with viable L. monocytogenes (VLM) but are not induced in cultures with heat killed L. monocytogenes (HKLM). Anti-IL-12 Abs abrogated the induction of the IFN-gamma-producing CD4+ T cells by VLM, suggesting an importance of IL-12 in the induction of IFN-gamma-producing CD4+ T cells. Accordingly, IL-12p40 was expressed by macrophages (M phi) from the cultures of whole spleen cells with VLM but not with HKLM, although HKLM induced IL-12p40 expression on M phi when nonadherent cells were removed before culture. In vivo analysis also showed that VLM induced IL-12p40 expression by M phi, whereas HKLM failed to induce it. On the other hand, the addition of anti-IL-10 mAb into cultures of whole spleen cells and HKLM resulted in an increase of IL-12p40 expression by M phi. Furthermore, we detected an expression of IFN-gamma by both adherent and nonadherent spleen cells at the early stage of stimulation with VLM before the appearance of IFN-gamma-producing CD4+ T cells. These data suggest that HKLM induce nonadherent spleen cells to produce IL-10 which may down-regulate IFN-gamma-producing CD4+ T cells by blocking IL-12 production by M phi. In contrast, VLM support IFN-gamma production by CD4+ T cells by stimulating M phi to produce IL-12 and IFN-gamma.

Cell-mediated immunity in host resistance against infection caused by Penicillium marneffei.

Penicillium marneffei is one of the most important opportunistic infectious pathogens in AIDS patients in Thailand and Southeast Asia. However, very little is known about the host defence mechanisms against P. marneffei infection. In the present study, we established the first experimental murine model of chronic pulmonary and disseminated infection using P. marneffei, and examined the immunological response to such infection in euthymic and athymic mice. In this model, micro-organisms inoculated intratracheally multiplied progressively in the lungs and disseminated to the liver and spleen. However, the number of organisms decreased gradually in these organs. In contrast, congenitally athymic mice developed severe pulmonary and disseminated systemic mycosis. Pulmonary penicilliosis marneffei was associated with a marked cellular inflammatory response as evident by histological abnormalities and increased intraparenchymal leucocyte count. To confirm the importance of cell-mediated immunity in host resistance to P. marneffei infection, we transferred nylon wool non-adherent spleen cells into the athymic mice. Such treatment significantly reduced the number of yeasts in the organs of athymic mice. Taken together, our results demonstrate that the cell-mediated immunity play a central role in a host defence mechanism against infection with P. marneffei, and suggest that our new model may be a useful approach for studying the pathogenesis of this fungal disease.

Immunoprophylactic potential of a 120 kDa Brugia malayi adult antigen fraction, BmA-2, in lymphatic filariasis.

A 120 kDa antigen containing SDS-PAGE fraction BmA-2 isolated from Brugia malayi adult parasite was highly reactive with normal sera from filarial endemic area. BmA-2 was analysed for its prophylactic potential in in vitro and in vivo. Sera collected from BmA-2 immunized jirds induced a significant level (80 to 90%) of protection against infective larvae and microfilariae in in vitro ADCC assay as well as in in situ micropore chamber implantation studies. Mastomys natalensis immunized with BmA-2 showed a significant level of protective response against circulating microfilariae by clearing 90% of them from circulation by fifth day after challenge infection. Immunization of jirds with BmA-2 resulted in an enhanced level of antibody response against BmA-2 and 88% reduction in the development of the parasites to the adult stage. Passive transfer of immunesera from jirds immunized with BmA-2 to naive jirds resulted in 71% reduction in adult worm recovery as observed 90 days after challenge infection with B. malayi. On the other hand the passive transfer of non-adherent spleen cells from immune jirds did not show any significant effect on the development of parasite. Administration of jirds anti BmA-2 serum to microfilaraemic jirds showed a temporary decrease in microfilarial count which was increased to pretherapeutic level within 100 days and there was no effect on the adult worms. This implies that the immune protective effect of BmA-2 is mainly antibody dependent and active immunization with BmA-2 is effective against filarial infection.

Enhancement of Nitric Oxide Production from Activated Macrophages by a Purified Form of Ginsenoside (Rg1)

We studied the actions of purified ginsenosides Rg1 and Rb1 on nitric oxide production from macrophages and a macrophage cell line RAW264-7. Although neither Rg1 nor Rb1 induced nitric oxide from resting macrophages, Rg1 enhanced the production of nitric oxide from IFN-gamma activated-macrophages or RAW cells. Rg1 also enhanced the production of nitric oxide from macrophages cocultured with nonadherent spleen cells stimulated by conA, LPS or anti-CD3. Rb1, however, did not significantly enhance nitric oxide production from stimulated macrophages or RAW cells. Rg1 enhanced the tumor cell killing by nitric oxide produced from IFN-gamma-activated macrophages.

Nitric oxide production from a macrophage cell line: interaction with autologous and allogeneic lymphocytes.

The indirect stimulation of macrophages to produce nitrite was examined by using the macrophage cell line J774.J774 spontaneously produced nitrite, when cultured at high concentration. J774 cultured in low concentration (< 10(4) cells in 100 microliters) barely produced nitrite. J774 cultured in low concentration produced a large amount of nitrite by the co-culture of nonadherent spleen cells or nonadherent peritoneal exudate cells, which were stimulated with con A, anti-CD3, or staphylococcal enterotoxin A. J774 (BALB/c derived: H-2d) cultured with either syngeneic (BALB/c) or allogeneic (B6; H-2b B10BR; H-2k) nonadherent lymphocytes, which were stimulated with conA or anti-CD3, produced nitric oxide. However, J774 produced nitric oxide by stimulation with SEA only when co-cultured with SEA-reactive T lymphocytes. Peritoneal exudate cells from mice, which did not proliferate by the stimulation of conA or anti-CD3, proliferated well by the addition of L-arginine homologue, NG-monomethyl-L-arginine. The proliferation of nonadherent peritoneal exudate cells stimulated with conA or anti-CD3 was suppressed by the addition of peritoneal macrophages. This suppression was abolished by the addition of NG-monomethyl-L-arginine.

The generation of macrophages from precursor cells incubated with brain endothelial cells--a release of CSF-1 like factor from endothelial cells.

The perivascular macrophages in the brain are thought to be derived from bone marrow precursor cells. Results presented here demonstrate that immature macrophages obtained from nonadherent spleen cell populations can adhere to cerebrovascular endothelial cell (EC) monolayers and proliferate. The proliferation of these cells can be stimulated by either purified murine granulocyte-macrophage colony-stimulating factor (GM-CSF) or EC conditioned medium but not by IL-3. No proliferation of the GM-CSF- or IL-3-dependent murine cell line IC-2 was observed in the presence of EC conditioned medium. Macrophage-like cells could also be derived from murine bone marrow cells but inhibited by anti-macrophage-CSF (M-CSF or CSF-1). The capacity of EC conditioned medium to induce the proliferation of macrophage-like cells from the spleen is therefore not due to the release of GM-CSF but CSF-1. In concomitant experiments using mature macrophages incubated on EC monolayers, no proliferation was observed. These findings suggest that the growth of perivascular macrophages in the brain may be stimulated by cerebrovascular EC.

Role of adherent spleen cells in the induction of cytotoxic activity by Toxoplasma lysate antigen.

In order to identify mechanisms responsible for the anti-tumor effects of Toxoplasma lysate antigen (TLA), we used an in vitro 51Cr release assay to study the functional properties of plastic-adherent cells during induction of splenic cytotoxic activity by TLA. Cytotoxic activity of non-adherent cells was measured in all experiments after a 6 days incubation. Induction of cytotoxic non-adherent cells by TLA required the presence of plastic-adherent spleen cells. In contrast, rhIL-2 alone was able to induce transformation of cytotoxic non-adherent cells from non-adherent spleen cells. Contact between adherent and non-adherent spleen cells was necessary for successful induction of cytotoxic non-adherent cells by TLA. Treatment of spleen cells with anti-macrophage serum prevented induction of cytotoxic activity by TLA. Biologically active IL-2 was not detected in culture supernatants of spleen cells exposed to TLA. These findings suggest that contact between TLA-sensitized non-adherent cells and macrophages is necessary for induction of cytotoxic cells in the presence of TLA. This contact, however, is not necessary for generation of IL-2-induced killer cells.