Effects of lymphokines and immune complexes on murine placental cell growth in vitro

Isolated murine placental cells obtained at Day 16 of allogeneic gestation (C3H x DBA/2J) were cultured for 3 days alone or in coculture with irradiated mouse splenocytes at the end of which 3H-thymidine was added for an additional 18-h culture to assess cell proliferation. Placental cell proliferation was significantly enhanced at spleen cell:placental cell ratios of 10:1 and 25:1 above that observed in the absence of added spleen cells. The stimulatory effect of irradiated allogeneic (C3H plus Balb/cJ) spleen cell cultures was significantly greater (approximately 2-fold) than that of isogeneic spleen cells (C3H alone). Conditioned medium from murine spleen cells cultured with concanavalin A (ConA) to induce lymphokine production had dose-dependent inhibitory effects on proliferation when added to placental cell cultures over a range of concentrations from 10 to 40% (vol:vol). Addition of pseudo "immune complexes" in the form of heat-aggregated human gamma globulin (AHGG) to culture medium failed to alter placental cell proliferation over a range of concentrations from 2 to 200 micrograms/ml either in the absence or presence of ConA-conditioned medium. In contrast to late-gestational stage placental cells, cell suspensions obtained from Days 8-9 murine ectoplacental cone (EPC) outgrowths, or from earlier stage placentas (Days 12-14) responded to low concentrations of conditioned medium from ConA-stimulated splenocytes with increased proliferation. The effect was less impressive on placental cells at gestational ages later than 12 days than on earlier stage preparations. On all placental cell suspensions tested, as well as EPC cells, a clear-cut inhibition of growth was observed at high doses of conditioned medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of cytotoxic T lymphocytes requires at least two spleen cell-derived helper factors besides interleukin 2

The dependency of induction of T cell cytotoxicity on lymphokines was studied. 1 X 10(5) nylon wool-purified thymic lymphocytes or 10(4) spleen cells were cultured with TNP-haptenated syngeneic UV-irradiated spleen cells in the presence of a variety of lymphokine preparations. Concanavalin A-induced spleen cell supernatants mediated strong cytotoxic responses in this system. Three other preparations, namely, a partially purified IL 2 preparation from PMA-stimulated EL-4 thymoma cells, a Con A-induced spleen cell supernatant that was absorbed with an IL 2-dependent cell line, and a Con A-induced supernatant that was dialyzed at pH 2 were all ineffective in mediating a cytotoxic response. In reconstitution experiments, cytotoxic responses were only obtained when either the absorbed preparation or the pH 2-treated preparation was mixed with the IL 2 preparation from EL-4 cells. No reconstitution occurred after mixing of the absorbed with the pH 2-treated preparation. pH 2 treatment of the absorbed preparation did not abolish its synergistic effect when added to the IL 2 preparation from EL-4 cells. These results led to the conclusion that activation of cytotoxic lymphocyte precursors requires at least two other lymphokines in addition to IL 2. One T cell cytotoxicity-inducing factor (TCF1) remained in Con A-induced supernatants after absorption with IL 2 receptor-bearing T cell line cells. It was pH 2-resistant and was not found in EL-4 supernatants. A second T cell cytotoxicity-inducing factor (TCF2) was pH 2-sensitive and was found in Con A-induced spleen cell supernatants as well as in interferon-free supernatants of PMA-stimulated EL-4 cells. This activity co-purified with IL 2. It was absorbed by the IL 2-dependent T cell line together with IL 2. IL 2 differs from TCF2 since it is pH 2-resistant.     

Astrocytes of the brain synthesize interleukin 3-like factors

Interleukin 3 (IL 3) is produced by T lymphocytes and T cell lines, as well as by a myelomonocytic cell line (WEHI-3), and it activates lymphocytes and mast cells, as well as macrophages. Recently we have demonstrated that astrocytes act as immune accessory cells through the secretion of interleukin 1 and the presentation of antigens to T lymphocytes. Here we show that cultured astrocytes from newborn mice release a 30,000 m.w. factor that induces the expression of 20-alpha-hydroxysteroid dehydrogenase in nu/nu spleen cells and the proliferation of the IL 3-dependent cell line 32DCL. An analogous biological activity was detected in supernatant of cultured rat C6 glioma cells. Production of IL 3-like factors by astrocytes of the central nervous system may be essential for development and maintenance of hemo and lymphopoietic cells within inflammatory brain lesions.     

Interleukin 2 deficiency in murine Leishmaniasis donovani and its relationship to depressed spleen cell responses to phytohemagglutinin

This study examined the kinetics and mechanisms of depressed spleen cell responses to phytohemagglutinin (PHA) that occur during Leishmania donovani infection of BALB/c mice. In co-culture experiments, neither spleen cells from infected animals nor parasite-infected macrophages suppressed PHA responses of normal spleen cells. In addition, parasite-mediated suppression of PHA-stimulated spleen cell proliferation could not be demonstrated. Mice with 2 wk of infection did manifest an impairment in spleen cell production of interleukin 2 (IL 2) and by 8 wk IL 2 activity in supernatants from these cells was reduced by approximately 95%. This finding was not explained by an alteration in the kinetics of IL 2 production. Furthermore, diminished IL 2 activity in supernatants of PHA-activated spleen cells from infected animals was not caused by suppressive factors in these fluids as shown by their inability to suppress IL 2 stimulation of IL 2-dependent T cells. When spleen cells from mice with 8 wk of infection were cultured with PHA and supplemented with exogenous IL 2, there was an approximately 48% increase in mitogenesis. These data indicate that abnormal PHA-induced spleen cell activation in BALB/c mice with L. donovani infection is associated with impaired production of IL 2. In addition, the observation that supplementation of spleen cells from infected mice with IL 2 resulted in partial reconstitution of the PHA response is consistent with a defect in IL 2 responsiveness.     

Antigen-specific immune-suppressor factor in herpes simplex virus type 2 infections of UV B-irradiated mice.

UV B-irradiation (280 to 320 nm) of mice at the site of cutaneous infection with herpes simplex virus type 2 (HSV-2) induced suppressor T-cell circuits that decreased HSV-2-induced proliferative responses of HSV-2-immune lymph node cells. Adoptive transfer experiments indicated that splenocytes from UV B-irradiated HSV-2-infected animals contain L3T4+ cells that suppress proliferative responses in vivo, consistent with suppressor inducer cells. However, following in vitro culture of the splenocytes with HSV-2 antigen, the proliferation of immune lymph node cells was inhibited by Lyt2+ suppressor T cells, consistent with antigen-induced suppressor effector cells. Antigen-specific and nonspecific suppressor factors were fractionated from supernatants of HSV-2-stimulated spleen cells by molecular-sieve chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the Sephadex fraction that contained the antigen-specific suppressor factor, in the presence or absence of 2-mercaptoethanol, defined a 115-kilodalton protein consisting of two disulfide-bound components with molecular sizes of 70 and 52 kilodaltons. The implications of these results with respect to the regulation of HSV-induced cell-mediated immunity following UV B-irradiation are discussed.     

Lymphokines and aging: interleukin-2 production and activity in aged animals

The capacity of aged animals to produce and respond to the T cell-replacing factor, interleukin-2 (IL-2), has been examined. IL-2 activity in the supernatants of concanavalin A-activated aged spleen cells is 5- to 10-fold lower than comparable supernatants prepared using young spleen cells. This lesion in IL-2 synthesis may limit antibody production to T-dependent antigens, because supplementation with purified IL-2 markedly enhances the number of anti-SRBC plaques generated by aged spleen cells. The response of aged splenocytes can be fully restored to that obtained using young adult cells. However, there appears to be a defect in the ability of aged cells to effectively translate the IL-2 signal into B cell helper activity, in the absence of T lymphocytes. That is, although young adult, nylon wool-purified T cells can interact with aged T-depleted spleen cells, producing a normal high level anti-SRBC response, IL-2 is incapable of reconstituting the response in aged animals to this level. On the other hand, both young adult T cells and IL-2 can interact with young adult T-depleted splenic lymphocytes to produce a normal, high level anti-SRBC response.     

Nematode infection enhances survival of activated T cells by modulating accessory cell function.

The type of immune response generated following exposure to Ag depends on a variety of factors, including the nature of the Ag, the type of adjuvant used, the site of antigenic entry, and the immune status of the host. We have previously shown that infection of rodents with Nippostrongylus brasiliensis (Nb) shifts the development of type 1 allo-specific responses toward type 2 immunity, suggesting nematode modulation of T cell activation. In this report we explore the immunomodulatory effects of Nb on T cell activation. We found that spleen cells from Nb-infected mice exhibited dramatically increased proliferation in response to Con A and anti-CD3. This hyperproliferation could be transferred in vitro to naive splenocytes by coculture with mitomycin C-treated cells from Nb-infected animals. The transfer was mediated by non-T accessory cells and supernatants derived from Con A-activated non-T cells, suggesting the involvement of a soluble factor secreted by accessory cells. The accessory cells secreted high levels of IL-6, and anti-IL-6 treatment abrogated the supernatant-induced hyperproliferation, thus confirming that IL-6 was mediating the effect. Further, spleen cells from Nb-infected mice were more resistant to activation-induced cell death (AICD) following mitogenic stimulation. Reduced AICD was also transferable and IL-6 dependent. Thus, the hyperproliferation was in part due to enhanced activated T cell survival. These phenomena mediated by accessory cells may contribute to the powerful polyclonal activation of type 2 immunity caused by nematode infection.     

Suppressor cell regulation of encephalitogenic T cell lines: generation of suppressor macrophages with cyclosporin A and myelin basic protein.

Chronic relapsing experimental allergic encephalomyelitis (CR-EAE) can be adoptively transferred using myelin basic protein (BP)-specific helper T cell lines, and suppressor cells may be important in recovery from EAE. In order to generate suppressor cells, spleen cells obtained from BP-complete Freund's adjuvant (CFA) inoculated SJL/J mice and from normal mice were cultured for 7 days with medium, with cyclosporin A (CsA), or with CsA and antigen (BP or purified protein derivative of mycobacterium (PPD)). Cultured spleen cells were assayed for suppressor activity in vitro by coculture with BP-specific and PPD-specific helper T cell lines derived from SJL/J mice. Immunized donor spleen cells cultured with cyclosporin A (CsA) and BP were potent inhibitors of T cell line proliferation, and suppressor activity was increased 17-fold compared with control splenocytes. The number of suppressor cells required to suppress PPD-specific line proliferation by 50% (I50) was significantly higher than the number required to suppress BP-specific line proliferation, suggesting an antigen-specific component to the suppression. The major effector cell required for suppression was a large granular Mac-1+ cell with the functional characteristics of a macrophage. Suppressor activity persisted after depletion of Thy 1.2+ cells, but suppression was no longer antigen-specific, suggesting that culture of spleen cells with CsA and BP may generate suppressor macrophages which are antigen-nonspecific and Thy 1.2+ suppressor cells which are antigen-specific. These suppressor cells may be important in the regulation of CR-EAE and the techniques described for their generation may prove useful for treatment and prevention of disease.     

Eimeria tenella and E. acervulina: lymphokines secreted by an avian T cell lymphoma or by sporozoite-stimulated immune T lymphocytes protect chickens against avian coccidiosis

The role of avian lymphokines as nonspecific immunomodulators of host immunity against the intracellular parasite Eimeria was investigated. Prophylactic treatment of normal chickens with crude cell-free supernatants obtained from JMV-1 culture, concanavalin A (Con A)-stimulated normal spleen cells, or sporozoite-stimulated immune T cells prior to inoculation with E. tenella or E. acervulina conferred significant protection. These crude cell-free culture supernatants also inhibited intracellular development of eimerian parasites in vitro. Avian macrophages pretreated with these supernatant preparations showed inhibitory activity against Eimeria. This inhibitory activity could not be ascribed to anti-Eimeria antibody, complement, or cell-free Marek's disease virus and was therefore considered to be due to immunomodulating lymphokines present in the culture supernatants. These results suggest that JMV-1-transformed T lymphoblastoid cells, immune T lymphocytes, and Con A-stimulated normal spleen cells secrete lymphokines that can enhance host immunity in a nonspecific manner and implicate cell-mediated immunity as a major mechanism of the protective host immune response against eimerian infections.     

IFN-beta production by macrophages obtained from mice undergoing graft vs host disease

We have previously shown that when splenocytes are obtained from mice undergoing graft vs host disease (GVHD), and then placed in culture, IFN-beta is produced spontaneously in supernatants without any stimulus. Thus, when B10.D2 spleen cells are injected into sublethally irradiated BALB/c recipient mice, in vitro splenic IFN-beta production is readily apparent between days 10 to 20 post-transplantation. In the present study, experiments were carried out to characterize the cell population(s) responsible for spontaneous IFN-beta production from GVHD spleen cells. Specific antibody and C lysis of selected cell types, including T cells, B cells, and NK cells, failed to abrogate in vitro IFN production. However, IFN-producing cells from GVHD splenocytes were nylon wool adherent and could be isolated using discontinuous Percoll gradient centrifugation from the upper most large cell fractions. IFN production was also partially resistant to irradiation. Using indirect immunofluorescence and flow cytometry, IFN production was shown to be associated with MAC-1 positive cells. MAC-1 negative splenocytes demonstrated no spontaneous IFN production. Treatment of GVHD spleen cells with silica, a selective toxin for phagocytic cells, resulted in a complete inhibition of IFN production. Thus, in vitro IFN production from GVHD splenocytes appeared to come from cells having a phenotype associated with macrophages. This in vitro IFN production by macrophages represents a unique aspect of GVHD which is not typically found in tissues of normal mice.     

Generation and characterization of hamster-mouse hybridomas secreting monoclonal antibodies with specificity for lipopolysaccharide receptor.

Experiments are described for the partial purification of the 80-kDa LPS binding protein expressed on macrophages and lymphocytes. This partially purified Ag was used to immunize adult Armenian hamsters and splenocytes from immunized animals were fused with murine myeloma cell lines. Hybridoma cell culture supernatants containing mAb were screened by ELISA for positive binding to the immunizing Ag, murine splenocytes and the murine 70Z/3 pre B cell and for an absence of binding to sheep E. Positive clones were further screened for reciprocal competitive binding with LPS on spleen cells and ability to modulate B lymphocyte mitogenic activity. Two hybridoma cell lines secreting IgM monoclonals, termed mAb3D7 and mAb5D3, were identified that satisfied all of the selection criteria. These hybridoma cell lines were subcloned and expanded. Binding of one (mAb3D7) was abrogated by treatment of Ag with mild periodate; binding of the second (mAb5D3) was destroyed by digestion of Ag with proteinase K. Binding specificity for mAb5D3 has been confirmed by ELISA using highly purified 80-kDa protein. These mAb have been of value in establishing that the 80-kDa LPS binding protein previously identified may serve as a specific functional receptor for LPS.     

Production of BSF-1 during an in vivo, T-dependent immune response

BSF-1, a cytokine produced by some T lymphocyte tumors, has been shown to act with anti-Ig antibodies to stimulate B lymphocyte proliferation, to independently induce resting B lymphocytes to increase their expression of surface Ia antigen, and to induce some activated B lymphocytes to differentiate into IgG1- or IgE-secreting cells. To determine whether BSF-1 might be secreted by normal lymphoid cells in the course of a physiologic immune response, BALB/c mice were injected with an affinity-purified goat antibody to mouse IgD (GaM delta), which induces the generation of a large, polyclonal T-dependent IgG1 response; 4-hr culture supernatants of spleen cells from these mice were prepared, and these supernatants were assayed for BSF-1 activity by analyzing their ability to induce BALB/c nu/nu spleen cells to increase their expression of cell surface Ia in vitro. Culture supernatants of unfractionated spleen cells removed from mice 4 to 8 days after GaM delta antibody injection induced substantial increases in B lymphocyte surface Ia expression; these increases were blocked by a monoclonal anti-BSF-1 antibody. Culture supernatants of spleen cells from untreated BALB/c mice or from untreated or GaM delta antibody-treated BALB/c nu/nu mice induced small to moderate increases in B cell surface Ia expression, and GaM delta antibody itself induced large increases in B cell surface Ia expression; however, these increases were not significantly blocked by a monoclonal anti-BSF-1 antibody. A culture supernatant of T cell-enriched spleen cells from untreated mice induced small increases in B cell surface Ia expression that were inhibited by anti-BSF-1 antibody, as was the larger increase in B cell Ia expression induced by a culture supernatant of T cell-enriched spleen cells from mice sacrificed 3 days after GaM delta injection. On the other hand, T cell-depleted spleen cells from BALB/c mice injected with GaM delta antibody 7 days before sacrifice failed to generate culture supernatants with BSF-1 activity. Supernatants prepared from spleen cells taken from untreated mice or mice treated with GaM delta antibody 1 to 3 days before sacrifice did not block the ability of purified BSF-1 to induce an increase in B cell surface Ia expression, and thus did not contain inhibitors of BSF-1 activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

CD4+ Leu-8+ T cell supernatant activity that inhibits Ig production.

The subpopulation of CD4+ T cells that expresses the Leu-8 peripheral lymph node homing receptor suppresses PWM-stimulated Ig synthesis. To determine the mechanism of this suppression, the immunoregulatory activity of culture supernatants obtained from peripheral blood CD4+ Leu-8+ T cells cultured with anti-CD3 mAb and PMA (Leu-8+ supernatant) was determined. Leu-8+ supernatant suppressed PWM-stimulated Ig synthesis in cultures containing non-T cells and CD4+ Leu-8- T cells. In contrast, the supernatant from CD4+ Leu-8- T cells did not suppress Ig synthesis. The inhibitory activity of CD4+ Leu-8+ T cell supernatants could not be accounted for by a deficiency or excess of IL-2, IL-4, IFN-gamma, IL-6, or PGE2. In studies examining the effect of CD4+ Leu-8+ supernatant on T cells, the supernatant did not alter either mitogen-induced proliferation or the helper function of CD4+ Leu-8- T cells. In studies examining the effect of CD4+ Leu-8+ supernatant on B cells, the supernatant inhibited Staphylococcus aureus Cowan I strain-induced B cell Ig secretion but not B cell proliferation. The suppressor activity of Leu-8+ supernatant was eliminated by protease treatment and was eluted by HPLC in two main peaks, with molecular sizes of 44 and 12 kDa. In summary, these studies indicate that supernatants from activated CD4+ Leu-8+ T cells directly suppress B cell Ig production.     

Prevention of human PC-346C prostate cancer growth in mice by a xenogeneic tissue vaccine

Vaccination, as an approach to prostate cancer, has largely focused on immunotherapy utilizing specific molecules or allogeneic cells. Such methods are limited by the focused antigenic menu presented to the immune system and by immunotolerance to antigens recognized as “self”. To examine if a xenogeneic tissue vaccine could stimulate protective immunity in a human prostate cancer cell line, a vaccine was produced by glutaraldehyde fixation of harvested PAIII prostate cancer cells tumors (GFT cell vaccine) from Lobund-Wistar rats. Immunocompetent Ncr-Foxn1<nu> mice were vaccinated with the GFT cell vaccine four times, 7 days apart. The control animals were either not vaccinated or vaccinated with media or glutaraldehyde-fixed PC346C human prostate cancer cells and adjuvant. About 8 days after the final boost, serum and spleens were harvested. The splenocytes were co-incubated with PC346C cells and then transplanted orthotopically into sygneneic immunodeficient nude mice. About 10 weeks later, the prostates were weighed and sampled for histolologic examination. The spleens were harvested from additional mice, and the splenocytes were cultured, either with or without pulsing by GFT cells, and the supernatants harvested 72 h later for cytokine analysis. Results showed that vaccination with GFT cells resulted in increased serum antibody to a PAIII cell lysate; reduced weight of the prostate/seminal vesicle complex and reduced incidence of prostate cancer in nude mice; increased splenocyte supernatant levels of TNF-α, IL-2, IFN-γ and IL-12, cytokines associated with Th1 immunity; and increased splenocyte supernatant levels of IL-4 and IL-10, cytokines associated with Th2 immunity. In summary, the results suggest that use of a xenogeneic tissue vaccine can stimulate protective immunity against human prostate cancer cells.     

Differing macrophage and lymphocyte roles in resistance to Legionella pneumophila infection.

Similar to guinea pig macrophages and human monocytes, macrophages from the peritoneal cavity of thioglycolate pretreated A/J mice are permissive for growth of Legionella pneumophila. In contrast, macrophages from BDF1 mice are not permissive for L. pneumophila. Lymphocytes from A/J and BDF1 mice proliferated in response to Legionella Ag but guinea pig lymphocytes did not. Also, splenocyte cultures from A/J mice treated with either Con A or Legionella vaccine produced supernatants which induced A/J macrophages to restrict Legionella growth, but guinea pig splenocyte culture supernatants obtained after stimulation with L. pneumophila vaccine did not induce Legionella growth restriction activity by guinea pig macrophages. Murine rIFN-gamma but not rIFN-alpha markedly inhibited growth of Legionella in A/J mouse macrophages and monoclonal anti-IFN-gamma antibody neutralized the anti-Legionella activity of culture supernatants from A/J mouse splenocytes responding to Legionella Ag. From these data, IFN-gamma appears to be an important factor in anti-Legionella activity of Ag-activated mouse splenocyte culture supernatants. Cyclosporin A, when given to either A/J or BDF1 mice, reduced the proliferation responses of splenocytes to T cell mitogens and also decreased the IFN production of A/J spleen cells to Legionella Ag. In addition, drug treatment decreased the resistance of A/J mice to Legionella infection as shown by an increase in the number of viable bacteria in the liver. However, injection of drug treated mice with lymphokine-rich splenocyte culture supernatant reconstituted the resistance of these animals. These results suggest an important role for lymphocyte activation and lymphokine production in the resistance of A/J mice to Legionella infection. The greater resistance of BDF1 mice, however, may result from nonpermissive macrophages and responsive lymphocytes. In the case of guinea pigs, susceptibility to Legionella infections may result from both the permissive nature of the macrophages and the relatively unresponsive nature of the lymphocytes in these animals.     

Trichinella spiralis: the influence of short chain fatty acids on the proliferation of lymphocytes, the goblet cell count and apoptosis in the mouse intestine

This study was carried out to determine the influence of short chain fatty acids (SCFA) on spleen and mesenteric lymph node lymphocyte proliferation, goblet cells and apoptosis in the mouse small intestine during invasion by Trichinella spiralis. BALB/c mice were infected with 250 larvae of T. spiralis. An SCFA water solution containing acetic, propionic and butyric acids (30:15:20 mM) was administered orally starting 5 days before infection and ending 20 days post infection (dpi). Fragments of the jejunum were collected by dissection 7 and 10 dpi, and were examined for apoptotic cells in the lamina propria of the intestinal mucosa, and for goblet cells. The proliferation index of the cultured spleen and mesenteric lymph node lymphocytes with MTT test was also determined. The orally administered SCFA solution decreased the proliferation of mesenteric lymph node lymphocytes in the mice infected with T. spiralis at both examination times, but did not influence the proliferative activity of the spleen cells. Seven dpi, both in the spleen and mesenteric lymph nodes, the highest proliferation index of concanavalin A (Con A)-stimulated lymphocytes was found in the group of uninfected animals receiving SCFA animals. This tendency could still be seen 10 dpi in the mesenteric lymph nodes but not in the spleen, where the proliferation index in this group had significantly decreased. In vitro studies revealed, that butyric and propionic acids added to the cell cultures suppressed the proliferation of Con A-stimulated mesenteric lymph nodes and spleen lymphocytes taken from uninfected and T. spiralis-infected mice. Acetic acid stimulated proliferation of splenocytes taken from uninfected mice but did not affect lymphocyte proliferation in mesenteric lymph nodes from uninfected or infected mice. Orally administered SCFA increased the number of goblet cells found in the epithelium of the jejunum 7 dpi, but this number had decreased 10 dpi. The number of apoptotic cells in the lamina propria of the intestinal mucosa of animals infected with the T. spiralis and receiving SCFA was also lower, particularly 10 dpi. The above results show that SCFA can participate in the immune response during the course of trichinellosis in mice.     

Large quantity of ribosomal RNA exists extracellularly in mouse spleen

When BALB/c mouse spleens were gently homogenized in saline, the resultant supernatant (without cells and tissue debris) contained significant amount of 28S and 18S ribosomal RNA, reaching up to 70% of the total spleen RNA. Haemoglobin assays indicated that less than 15% of the spleen cells were lysed during the homogenization process, indicating that the majority of the spleen `supernatant RNA' was from the extracellular space of the organ rather than released by the splenocytes as a consequence of grinding. Quantitative RNA analysis showed that the ratio of spleen supernatant RNA/total RNA of BALB/c mice was inversely correlated with age (from approximately 70% at 3 weeks to 45% at 6 months), but that of BXSB mice (an animal model for systemic lupus erythematosus) remained at about 70% irrespective of age. Methyl Green–Pyronin Y staining of paraffin sections of mouse spleen revealed that extracellular RNA was distributed mainly in the sinuses of the organ. Culture supernatants of apoptotic splenocytes contained significant amounts of RNA, suggesting that the extracellular RNA in the spleen might have come from apoptotic lymphocytes. This is supported by the fact that `thymus supernatant' also contained significant amount of RNA. A possible correlation between spleen extracellular RNA and autoimmune diseases is discussed.     

Enhancement of proliferation and differentiation in bone marrow hematopoietic cells by Spirulina (Arthrospira) platensis in mice

This study evaluates whether Spirulina, including its components such as phycocyanin, enhances or sustains immune functions by promoting immune competent-cell proliferation or differentiation. The effects of Spirulina of a hot-water extract (SpHW), phycocyanin (Phyc), and cell-wall component extract (SpCW) on proliferation of bone marrow cells and induction of colony-forming activity in mice were investigated. The Spirulina extracts, SpHW, Phyc, and SpCW, enhanced proliferation of bone-marrow cells and induced colony-forming activity in the spleen-cell culture supernatant. Granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) were detected in the culture supernatant of the spleen cells stimulated with the Spirulina extracts. Bone marrow-cell colony formation in soft-agar assay was also significantly induced by the blood samples and the culture supernatants of the spleen and Peyer's patch cells of the mice which ingested Spirulina extracts orally for 5 weeks in in vivo study. Ratios of neutrophils and lymphocytes in the peripheral blood and bone marrow, consequently, increased in the mice. Spirulina may have potential therapeutic benefits for improvement of weakened immune functions caused by, for example, the use of anticancer drugs.     

NZB serum factor (NZB-SF): B precursor cell maturation factor identified in murine lupus. I. Identification of 60-kDa glycoprotein as the major component from both spleen cell supernatant and serum

NZB serum factor (NZB-SF), initially identified in sera of very young NZB mice, can enhance maturation and proliferation of sIg- pre-B cells in marrow. In the present study, spleen cell supernatant from young NZB mice was used as a source of NZB-SF. NZB mice were treated with Corynebacterium parvum 2 weeks prior to sacrifice, and harvested spleen cells obtained at sacrifice were cultured for 24 hr in serum-free medium. One liter of spleen cell supernatant prepared in this way contained NZB-SF-like activity equivalent to that present in 10 ml of serum collected from young NZB mice. NZB-SF was purified on an affinity chromatographic column conjugated with mouse IgG1 monoclonal antibody (mAb) against NZB-SF. The purified NZB-SF had pI 7.8 and showed one major band of 60 kDa and a faintly stained 35-kDa band upon SDS-PAGE under nonreducing conditions. The 60-kDa NZB-SF extracted from the gel slice was also more potent in dot blot ELISA (greater than 100 times) than the 35 kDa NZB-SF and was biologically active. After endoglycosidase F treatment, but not after treatment with a reducing agent (2-ME), the two bands merged into a single band at the 15-kDa position. Amino acid sequence analysis of endo-F treated NZB-SF indicated that the N-terminus of this protein is blocked. Serological and functional studies of affinity-purified NZB-SF have revealed that NZB-SF is distinguishable from IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, CSF-GM, IFN-gamma, and TNF alpha. Therefore, a major component of NZB-SF(s) in the spleen cell supernatant may be an apparently novel 60-kDa glycoprotein with a single amino acid backbone. Sera and spleen cell supernatants from normal strains of mice (DBA/2, B6, or BALB/c) were also applied to the immuno-affinity column used to purify NZB-SF. It was found that trace amounts of NZB-SF are present also in serum of normal strains of mice and that spleen cells of these mice can also produce NZB-SF in vitro following stimulation with C. parvum. In SDS-PAGE, the 60-kDa NZB-SF is also the major component of NZB-SF in normal strains of mice. These results suggest that the 60-kDa NZB-SF may be of physiological importance in B cell differentiation and that this physiological factor is autoimmune-prone NZB mice.     

Immunosuppressive properties of murine trophoblast

The modification of the immunological response by murine trophoblast cells of different sources was investigated using the mixed lymphocyte reaction (MLR) and the cell mediated lympholysis (CML) test. MLR between C57BL (H-2b) stimulator splenocytes (mitomycin C treated in the unidirectional MLR) and BALB/c (H-2d) responder lymph node cells were markedly suppressed by trophoblast of ectoplacental cone (EPC) and placental origin. The same in vitro effect was observed with supernatants (SN) of trophoblast cells and with supernatants of blastocysts. Addition of anti-progesterone serum (APS), anti-testosterone serum (RAT), and anti-immunoglobulin serum (RAHIg) in serial dilutions to the trophoblast-MLR system revealed that the immunosuppressive effect of trophoblast giant cells and trophoblast giant cell culture supernatants can be abolished with APS. Identical results were obtained with APS added to immunosuppressive doses of progesterone. CML between C57BL responder lymph node cells and mitomycin C-treated BALB/c stimulator spleen cells was also markedly suppressed when trophoblast of EPC origin was added. A similar suppression of cytotoxic T-cell induction was seen when progesterone was added to the system. The immunosuppressive action of trophoblast as detected in vitro is likely to play an important role in the maintainance of pregnancy by protecting the semiallogeneic conceptus against immune aggression by the maternal immune system.