Stimulation of Hematopoiesis in Untreated and Cyclophosphamide Treated Mice by the Inhibition of Prostaglandin Synthesis

Abstract

Indomethacin (IN) was administered to untreated or to cyclophosphamide (CY) treated C57B1/6 mice to study the roles of prostaglandins in regulating hematopoiesis. The following hematopoietic parameters were quantitated: 1) peripheral blood leukocyte (PBL) count; 2) total nucleated cells per spleen; 3) total nucleated cells per femur; and 4) spleen weight. Assays were performed in vitro to measure the number of colony forming units (CFU) present in the bone marrow and spleen. Untreated mice administered IN had a transient rise in their PBL count. These animals also developed splenomegaly and had an increased number of nucleated cells in their spleen. All CY treated mice had a marked decrease in PBL count, spleen cellularity, bone marrow cellularity, and spleen size during the first 5 days after CY treatment. These observations were followed by hematopoietic recovery over the next 10 days. Cyclophosphamide treated mice exhibited a more rapid hematopoietic recovery when treated with IN than without IN treatment. Analysis of the CFU capacity of bone marrow and spleen cells in soft agar showed a larger number of CFU in the bone marrow and spleen of IN treated mice or of CY/IN treated mice than in animals not receiving IN. These results indicate that prostaglandins are involved in the regulation of hematopoiesis in untreated mice and that prostaglandins may limit the hematopoietic recovery of CY treated mice.     

Mechanisms of Immunological Response Induced in Cd2f1 Mice by Administration of Semisyngeneic L 1210 Leukemia Cells Treated with Cyclophosphamide

CD2F₁ mice were immunized against semisyngeneic L 1210 leukemia. Immunization was achieved by four i.p. injections, in weekly intervals, of L 1210 cells treated in vivo twice with 200 mg/kg of cyclophosphamide. The immunized animals survived i.p. challenge with 1000 untreated L 1210 cells that was lethal for nonimmunized mice. The immunity could be abrogated in vivo with anti-mouse thymocyte serum, carrageenan or reserpine, but not by anti-mouse IgG serum, suggesting participation of T lymphocytes and macrophages in the response. Moreover, lymphocytes and macrophages from the peritoneal cavity of immunized mice were cytotoxic in vitro for L 1210 cells. The immunity, at least partially, could be adoptively transferred with peritoneal exudate cells or splenocytes.     

Critical Differences in Hematopoiesis and Lymphoid Development between Humans and Mice

During the last five decades, elegant mouse models of hematopoiesis have yielded most of the seminal insights into this complex biological system of self-renewal and lineage commitment. More recent advances in assays to measure human stem and progenitor cells as well as high resolution RNA profiling have revealed that although the basic roadmap of blood development is generally conserved across mammals, evolutionary pressures have generated many differences between the species that have important biological and translational implications. To enhance the utility of the mouse as a model organism, it is more important than ever that research data are presented with regard to how they might be influenced by the species of origin as well as the developmental source of the hematopoietic tissue.     

Effect of Cyclophosphamide on Histoplasma capsulatum Infections in Mice

Mice were injected intraperitoneally (i.p.) with 300 mg of cyclophosphamide (CY)/kg of body weight, and 24 h later were injected i.p. with varying dosages of yeast-phase cells of Histoplasma capsulatum. At specific time intervals organs were removed, ground, and cultured to determine the number of viable organisms contained in the spleen, liver, and lungs. Injection of mice with CY was found to cause a dramatic increase in the numbers of parasites isolated from these organs when compared with non-drug-treated controls. Mice given 10(7) yeast cells showed the largest increase in colony numbers. A greater than fivefold increase in the numbers of organisms isolated from the spleens of CY and 10(3) yeast cell-treated mice, as compared with non-drug-treated animals, was observed at all time periods. The general trend for infected control animals was a decrease in colony numbers. All mice given CY plus 10(7) yeast cells intravenously (i.v.) died by day 20 postinfection. Mice given CY and 10(7) yeast cells i.p. showed no evidence of fatal Histoplasma infection. Deaths occurring by day 5 in CY-treated animals injected with H. capsulatum yeast cells i.v. or i.p. were considered due to bacterial infection or toxicity, or both. Hepatosplenomegaly was observed in mice treated with CY and 10(7) yeast cells of H. capsulatum. Enlarged lungs were also noted. CY control mouse spleens weighed 30% less than normal spleens. Organs of animals injected with H. capsulatum alone did not vary significantly from those of normal mice. Complete drug-induced suppression of humoral antibody response was achieved for 10 days, as determined by hemagglutination titrations.     

Compensation of Cyclophosphamide Immunosuppression by a Bacterial Immunostimulant (Broncho-Vaxom) in Mice

Abstract

The compensatory effect of a bacterial lysate, Broncho-Vaxom (BV) on the immunosuppressive action of cyclophosphamide (CY) was investigated. In CY immunosuppressed mice, BV treated animals recovered to normal levels of IgM and IgG in serum as well of IgA and IgG in gut secretions significantly earlier than controls. Furthermore, normal cell proliferation in thymus, as estimated by measuring the relative size of this organ was achieved earlier in BV treated mice than in control mice. Oral treatment with BV restores the number of IgM anti SRBC producing cells in spleen, in CY immunosuppressed mice. Since immunosuppression induced by CY increases the susceptibility to various infections, we tested in immunosuppressed animals the protective effect of BV towards IP challenge infections with Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae var ozaenae, Pseudomonas aeruginosa and Candida albicans. BV led to an enhanced resistance towards both pneumococci and staphylococci challenge infections but not to the other challenge microorganisms.     

Compensation of Cyclophosphamide Immunosuppression by a Bacterial Immunostimulant (Broncho-Vaxom) in Mice

The compensatory effect of a bacterial lysate, Broncho-Vaxom (BV) on the immunosuppressive action of cyclophosphamide (CY) was investigated. In CY immunosuppressed mice, BV treated animals recovered to normal levels of IgM and IgG in serum as well of IgA and IgG in gut secretions significantly earlier than controls. Furthermore, normal cell proliferation in thymus, as estimated by measuring the relative size of this organ was achieved earlier in BV treated mice than in control mice. Oral treatment with BV restores the number of IgM anti SRBC producing cells in spleen, in CY immunosuppressed mice. Since immunosuppression induced by CY increases the susceptibility to various infections, we tested in immunosuppressed animals the protective effect of BV towards IP challenge infections with Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae var ozaenae, Pseudomonas aeruginosa and Candida albicans. BV led to an enhanced resistance towards both pneumococci and staphylococci challenge infections but not to the other challenge microorganisms.     

Prostaglandin-mediated Inhibition of Noradrenaline Release: Its Resistance to Variations in Rate of Prostaglandin Synthesis

Isolated rabbit hearts were perfused according to Langendorff. The bilateral sympathetic nerve supply to the organ was stimulated at intervals, and the overflow of noradrenaline and of prostaglandins of the E series in the effluent was assayed, using fluorimetric and bioassay methods, respectively. The synthesis of prostaglandins in the organ was stimulated, either by perfusing the heart at a low pO₂, or by infusing nicotinic acid. Hypoxia increased the coronary flow, provided the prostaglandin synthesis was not inhibited, probably as a consequence of hypoxia stimulation of the endogenous formation of prostaglandins. The release of NA in response to nerve stimulation was, however, unaffected by hypoxia. Nicotinic acid also stimulated prostaglandin formation, doubling the overflow of the lipid in response to nerve stimulation. In this series, too, the release of NA induced by nerve stimulation was unaffected by stimulation of prostaglandin synthesis. It is concluded that local variations in the rate of prostaglandin synthesis are unable to change the degree to which the release of sympathetic neurotransmitter is inhibited. Furthermore, it is suggested that the prostaglandin synthesis in rabbit heart takes place in compartments, separated functionally or morphologically.     

Enhancement in Immunity of Tumor Bearing Mice by Immunization Against Prostaglandin E2

C57B1/6 mice hearing a palpable Lewis lung carcinoma (LLC) were immunized against prostaglandin E₂ (PGE₂) in order to prevent the immune suppression typical of tumor bearers. Proliferation in response to concanavalin A (Con A) by normal spleen cells cultured in the presence of PGE₂ or by spleen cells of LLC-bearing mice cultured with Con A alone was suppressed. However, the mitogenic response of spleen cells from PGE₂ immunized LLC-bearing mice was only partially suppressed. The random migration of normal macrophages cultured in the presence of PGE, or of macrophages from LLC-bearing mice cultured in medium alone was enhanced when compared to the random migration of normal macrophages cultured in the absence of PGE₂. In contrast, the random migration of macrophages obtained from PGE₂ immunized LLC-bearing mice was the same as that of normal macrophages cultured in the absence of PGE₂.     

Inhibition by Aspirin of the Enhanced Migration of Human Platelets in Response to Prostaglandin Precursors

Prostaglandins E₁ and E₂, and their biosynthetic precursors, 8,11,14-eicosatrienoic acid and arachadonic acid, respectively, enhance human platelet random migration in vitro by 100-200% as assessed in modified Boyden micropore filter chambers. Inhibitors of prostaglandin biosynthesis, such as 5,8,11,14-eicosatetraynoic acid, indomethacin, and aspirin blocked the enhancement of platelet migration by either precursor fatty acids or prostaglandins. Administration of 900 mg of aspirin to normal subjects 3-1/2 - 24 hr before harvesting their platelets also inhibited the capacity of precursors and prostaglandins to increase random migration in vitro.     

Generation and Maintenance of Immune Interferon-Producing Cells Induced by Allogeneic Stimulation in Mice

When spleen cells derived from C57BL/6 mice immunized with L cells 7 days previously were cocultured with antigenic cells, immune interferon appeared in the culture fluid. We analyzed the tissue distribution of the immune interferon-producing cells (IIPC) which appeared in various lymphoid organs after allogeneic stimulation. Although fluid from cocultures of L-cell-sensitized thymocytes and L-cells could not detect interferon activity consistently, small numbers of IIPC could be detected by using the enumeration method of IIPC. The generation, maintenance, and nature of IIPC emerging in the spleen were different depending on how the host mice were immunized. Multiple antigenic stimulations were more effective and induced longer-lasting immune interferon production than a single stimulation. IIPC induced by a single stimulation appeared to be sensitive to cortisone, vinblastine, and cyclophosphamide and were relatively short lived. In contrast, IIPC induced by multiple stimulations seemed to be partially resistant to these drugs and long lived. When mice were immunized with intact L-cells, carrageenan, a known antimacrophage agent, had no effect on immune interferon production. However, when mice were immunized with solubilized L-cell antigen, this drug displayed a suppressive effect on immune interferon production.     

Validation of Cytochemical Section Bioassay for Thyroid-Stimulating Immunoglobulins in Treated and Untreated Graves' Disease

The cytochemical section-bioassay of thyroid stimulating activity is described. Plasma of thyrotoxic patients as well as those on block-replace treatment with carbimazole and thyroid hormones was used. Linear parallel responses were obtained over the range 1.5 ± 10^?7 to 1.5 ± 10^?5 mU/1 MRC LATS-B standard. The index of precision was 0.19 × 0.028. The fiducial limits (p=0.95) of a sample tested on ten separate occasions were 52–192%. Specificity was investigated using time course studies, and the effect of anti-TSH or anti-IgG antisera. No effect of methimazole or a variety of other drugs was detected. The assay is accordingly validated for measurement of TSI in patients both untreated and on block-replace therapy.     

The Effects of Cyclophosphamide on Antibody Dependent Cell-Mediated Cytotoxicity in Mice

Abstract

The effect of cyclophosphamide (CY) on antibody dependent cell-mediated cytotoxicity (ADCC) was evaluated. The results indicated that a single injection of CY markedly increased ADCC activity of murine splenic cells. This effect was dose dependent and was maximal on day 7 after the drug injection. The number of lymphoid cells in treated mice decreased to approximatedly 50%; however the percentages of EA rosettes were similar to those of control animals suggesting that the augmented cytotoxic activity was not due to a relative increase in Fc receptor bearing cells. CY treatment also enhanced delayed type hypersensitivity, but suppressed antibody production to sheep red blood cells. The possibility that CY eliminates a suppressor cell subpopulation that normally regulates ADCC levels is discussed.     

The Effects of Cyclophosphamide on Antibody Dependent Cell-Mediated Cytotoxicity in Mice

The effect of cyclophosphamide (CY) on antibody dependent cell-mediated cytotoxicity (ADCC) was evaluated. The results indicated that a single injection of CY markedly increased ADCC activity of murine splenic cells. This effect was dose dependent and was maximal on day 7 after the drug injection. The number of lymphoid cells in treated mice decreased to approximatedly 50%; however the percentages of EA rosettes were similar to those of control animals suggesting that the augmented cytotoxic activity was not due to a relative increase in Fc receptor bearing cells. CY treatment also enhanced delayed type hypersensitivity, but suppressed antibody production to sheep red blood cells. The possibility that CY eliminates a suppressor cell subpopulation that normally regulates ADCC levels is discussed.     

Effect of Different Regimens of Co-Transplantation of Hemopoietic and Mesenchymal Hemopoietic Stem Cells on the Rates of Hemopoietic Recovery in Laboratory Mice Treated with Cyclophosphamide in Sublethal Doses

Bulletin of Experimental Biology and Medicine - We studied the effect of mesenchymal stem cells and hemopoietic stem cells co-transplanted in different regimens (sequence and intervals between the...     

Comparative Effects of Azathioprine, Cyclophosphamide and Frentizole on Humoral Immunity in Mice

Data is presented comparing the activities of three immunosuppressive agents, cyclophosphamide, frentizole and azathioprine in models of humoral immunity in mice. Cyclophosphamide and frentizole suppressed the primary and secondary plaque forming cell responses to sheep erythrocytes at lower doses than did azathioprine. Prolonged suppression of serum antibody titers occurred following short-term therapy with cyclophosphamide or frentizole, but not azathioprine. Azathioprine was also the least effective agent in suppressing a primary response to the T-independent antigen, trinitrophenylated lipopolysaccharide. All three agents were found to inhibit the induction and activity of suppressor cells at immunosuppressive doses.     

Comparative Effects of Azathioprine, Cyclophosphamide and Frentizole on Cellular Immunity in Mice

This report extends previous observations on the immunosuppressive properties of cyclophosphamide (CPA), azathioprine and frentizole (15) to include murine models of cellular immunity. Systemic and local graft vs host reactions (GVHR) were most effectively suppressed by CPA. In contrast to frentizole, both CPA and azathioprine were found to inhibit the proliferation of parental T-cells in a systemic GVHR. However, CPA was the only agent capable of inhibiting the proliferation of T-cells following contact sensitization with oxazolone.

Mice pretreated with a high dose of CPA or frentizole prior to inoculation with the murine sarcoma virus exhibited accelerated tumor growth. However, there was no accelerated growth of murine sarcoma virus induced tumors or an Sal spindle cell fibro-sarcoma during rather prolonged therapy with immunosuppressive doses of CPA, azathioprine or frentizole.

Normal mice treated with CPA showed a more drastic reduction in lymphoid elements of the spleen and thymus than mice treated with azathioprine or frentizole. Studies on the mitogenic responsiveness of spleen cells obtained from normal mice after an eight day course of therapy suggested that CPA has some selectivity of action on B-cells and azathioprine on T-cells.     

Enhancement of Immune Function and Tumor Growth Inhibition by Antibodies Against Prostaglandin E2

Mice bearing a Lewis lung carcinoma (LLC) were passively immunized against prostaglandin E₂ (PGE₂ by administration of rabbit serum containing anti-PGE₂ antibodies. The effect of PGE₂-inxnune serum on the suppressed imnunity of LLC-bearing mice was examined. Treatment of tumor-bearing mice with anti-PGE₂ prevented the suppression of lymphocyte mito-genesis and the alterations in macrophage migration which typically occur in LLC-bearing mice. Furthermore, tumor growth was reduced In LLC-bearing mice which received antibodies directed against PGE₂ rather than a placebo treatment.