Further studies on mobilization of CFUs.

Mobilization of CFUs from haemopoietic tissues into circulation was studied after injection of different bacterial lipopolysaccharides (LPS), zymosan, phytohaemagglutinin (PHA), concanavalin A (Con A), trypsin and di-isopropyl-fluorophosphate-inhibited trypsin. All bacterial LPS used gave an increase of CFUs in the peripheral blood at 1 h after i.v. injection. Some variation in activity could not be excluded. As with Salmonella typhosa LPS, zymosan gave an increase in circulating CFUs during the first few hr and a second peak a few days later. After injection of zymosan as well as S. typhosa LPS the second peak in the blood was accompanied by a large increase in CFUs numbers in the spleen. PHA gave an immediate mobilization of CFUs, but the mobilization after injection of Con A during the first few hr occurred more slowly. After injection of S. typhosa LPS, zymosan and PHA the blood C3 level was found to be depressed considerably. This might indicate that the complement system is involved in the early mobilization of CFUs. Dexamethasone, a synthetic hormone which has been reported to give sequestration of several cell types in the bone marrow, did not inhibit the early and late mobilization of CFUs which normally occurs after injection of S. typhosa LPS.     

STUDIES ON THE MECHANISM OF HAEMOPOIETIC STEM CELL (CFUs) MOBILIZATION

A variety of substances can mobilize haemopoietic stem cells (CFUs) into the peripheral blood. In this study the involvement of the complement system in the mobilization process was investigated. Pretreatment of mice with the complement-activating factor of cobra venom (CoF), which lowered the serum C3 levels to 10–25% of the normal value, could completely prevent CFUs mobilization induced by high doses of CoF, endotoxin (ET) from Salmonella typhosa, inulin, zymosan and the proteolytic enzymes proteinase and trypsin. On the other hand, mobilization induced by the polyanions dextran sulphate and the copolymer of polymethacrylic acid and styrene could not be prevented, or at least affected only slightly. There appears to be a relationship between the extent of decomplementation by CoF and the extent of CFUs mobilization induced by ET. The results indicate that certain agents mobilize CFUs via the complement system, whereas other agents induce CFUs mobilization independent of the availability of complement components.     

Genetic Control of Lipopolysaccharide-Induced Mobilization of Cfus Dissociation Between Early and Delayed Mobilization of Cfus In Complement C5-Deficient Mice and Lps Non-Responder Mice

Lipopolysaccharide (LPS)-induced mobilization of CFUs from haemopoietic tissues into the circulation has a biphasic pattern. the first rise occurs within 30 min of LPS injection, the second 4–7 days later. This second rise coincides with an increase of the CFUs number in the spleen from about 3000 to about 50,000. We have investigated the relationship between the two peaks by making use of complement C5-deficient mouse strains and the LPS non-responder mouse strains C3H/HeJ and C57BL/10ScCr. These latter two strains lack a serologically identifiable structure (‘LPS-receptor’) which is present in all LPS-responder strains. After injection of eleven different mouse strains with LPS, the numbers of circulating CFUs increased rapidly in all strains, except in the C5-deficient A/J, AKR/J, DBA/2J and B10.D2/oSn mice. On the other hand, the delayed LPS-induced accumulation of CFUs in blood and spleen occurred in all mouse strains tested, including the C5-deficient strains, but not in the LPS non-responder strains C3H/HeJ and C57BL/10ScCr. These results show that (a) early LPS-induced mobilization of CFUs is dependent on the availability of C5, in contrast to the delayed CFUs accumulation in blood and spleen, (b) the presence of the LPS receptor is not required for early CFUs mobilization by LPS and (c) recognition of the mobilizing agent by a specific receptor is required for the delayed accumulation of CFUs in blood and spleen.     

Modulation of lymphocyte functions by group A streptococcal membrane

Protoplast membranes isolated from group A streptococci suppress functions of mouse B cells in vivo and in vitro. Intraperitoneal injection 24 or 72 hr (but not 12 hr) before collection of lymphoid cells results in a selective decrease in the mitogenic response of bone marrow cells to dextran sulfate (DS). The response of bone marrow cells to lipopolysaccharide (LPS), and spleen cells to both DS and LPS, is unaltered. In vitro exposure of lymphocytes to membranes concomitantly with mitogen reduces the response to both DS and LPS, however, the DS response is more susceptible to low doses of membrane. Suppression of the response to DS in vitro is not mediated by cells bearing Thy 1.2 antigen. Neither the phytohemagglutinin (PHA)-responsive cells nor the adherent cells participate in suppression of the LPS response in vitro. In contrast to the suppression of B-cell functions neither the PHA nor concanavalin A (Con A) response of mouse bone marrow, spleen, or thymus cells is altered by streptococcal protoplast membranes injected 24 hr before collection of cells. In vitro exposure of spleen cells to a limited range of concentrations of membrane results in an enhanced proliferative response of spleen cells stimulated by suboptimal doses of PHA. This synergism is not mediated by the adherent cells. Addition of membranes to spleen cell cultures in vitro has no effect upon the response of spleen cells to suboptimal doses of Con A or to optimal doses of either Con A or PHA. Higher concentrations of membranes reduce the proliferative response of both control and mitogen-stimulated cells. This nonselective suppression by high doses of membranes is not due to toxicity. Delayed hypersensitivity to sheep erythrocytes is potentiated by injection of membranes. These studies suggest that streptococcal membranes preferentially suppress the immature B cells and enhance certain T-cell functions.     

Studies on the mechanism of haemopoietic stem cell (CFUs) mobilization. A role of the complement system.

A variety of substances can mobilize haemopoietic stem cells (CFUs) into the peripheral blood. In this study the involvement of the complement system in the mobilization process was investigated. Pretreatment of mice with the complement-activating factor of cobra venom (CoF), which lowered the serum C3 levels to 10-25% of the normal value, could completely prevent CFUs mobilization induced by high doses of CoF, endotoxin (ET) from Salmonella typhosa, inulin, zymosan and the proteolytic enzymes proteinase and trypsin. On the other hand, mobilization induced by the polyanions dextran sulphate and the copolymer of polymethacrylic acid and styrene could not be prevented, or at least affected only slightly. There appears to be a relationship between the extent of decomplementation by CoF and the extent of CFUs mobilization induced by ET. The results indicate that certain agents mobilize CFUs via the complement system, whereas other agents induce CFUs mobilization independent of the availability of complement components.     

MOBILIZATION OF HAEMOPOIETIC STEM CELLS (CFU) INTO THE PERIPHERAL BLOOD OF THE MOUSE; EFFECTS OF ENDOTOXIN AND OTHER COMPOUNDS

Factors affecting the circulation of haemopoietic stem cells (CFU) in the peripheral blood of mice were investigated. I.v. injection of sublethal doses of endotoxin, trypsin and proteinase appeared to raise the number of CFU per ml blood from about 30–40 to about 300–400 or more within 10 min. The effect was smaller when smaller doses of the substances were injected. After this initial rise the number of circulating cells returned to normal in a few hours. Following endotoxin there was a second rise which started 2–3 days after injection and attained a peak on the 6th–7th day. The first rise is explained as a mobilization of stem cells from their normal microenvironments into the blood stream; the second rise is considered to reflect proliferation of CFUs in the haemopoietic tissues. The spleen seems to be acting as an organ capturing CFUs from the blood and not as a source adding stem cells to the blood.
The early mobilization of CFU after endotoxin injection did not coincide with a mobilization of neutrophils. The number of circulating band cells was increased during the first hours.
The importance of 'open sites'in the haemopoietic tissue for capturing CFUs was studied by emptying these sites through a lethal X-irradiation and injecting normal bone marrow cells. When a greater number of syngeneic bone marrow cells was injected intravenously, the level of circulating CFU in irradiated mice was slightly lower than the level in unirradiated mice during the first hours.     

Differential effects of concanavalin A and phytohemagglutinin on murine immunity. Suppression and enhancement of humoral immunity.

The abilities of concanavalin A (Con A) and phytohemagglutinin P (PHA) to selectively induce different T-cell activities affecting humoral immunity were evaluated. The mitogens were intravenously injected before, with, or after injection of sheep red blood cells (SRBC) into mice, and the 3 to 6-day plaque-forming cell (PFC) responses were assessed. Mitogenic treatment differentially influenced the resultant in vivo PFC responses to SRBC. The in vivo suppressive effects induced by Con A were shown to be temporary; only the Day 4 PFC response was inhibited. Con A given 3 hr before, with, or after the antigenic challenge enhanced the PFC response. In contrast, PHA given at all intervals inhibited both the 4- and 5-day PFC response. Neither mitogen appeared to affect the kinetics of the in vivo PFC response to SRBC. Both mitogens enhanced in vivo DNA synthesis by the splenic cells, and Con A appeared biphasic in its stimulation. Con A-induced effects on the humoral immune response were short-lived and transient, while PHA induced a longer-lasting effect on humoral immunity.     

Some Mechanisms of Disturbances and Recovery of T-Lymphocyte Migratory Properties In Irradiated Mice

Abstract Migration of ⁵¹Cr-labelled T cells from irradiated mice into lymph nodes of syngeneic unirradiated recipients decreased in a dose-dependent fashion. Influx of labelled T cells between 4 and 24 hr after injection (secondary migration) is more radiosensitive than lymph-node migration of T cells in the first 4 hr (primary migration). Treatment of T cells from irradiated mice in vitro with Con A or with trypsin does not enhance radiation-induced alteration of their migratory properties, but irradiation enhances the effects of Con A and trypsin on T-cell migration. Recovery of primary migration of irradiated T cells is completed 3 months after irradiation; it is probably caused by T-cell renewal. the defect of T-cell secondary migration is more stable: it remains 6 months after irradiation in a dose of 4 Gy. Post-irradiation defects of the T-cell differentiation process as a cause of long-lasting alteration of T-cell secondary migration are discussed.     

Stimulation of chicken lymphocytes by T- and B-cell mitogens.

Cultures of chicken spleen, peripheral blood, thymus, and bursal lymphocytes were tested for mitogenic stimulation by phytohaemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM), bacterial lipopolysaccharide (LPS), trypsin, and insulin. Spleen and blood leukocytes were stimulated by both the lectins and LPS, and also to some degree by trypsin and insulin as judged by increased incorporation of [³H]thymidine into acid-insoluble material. This was observed in cultures incubated in serum-free medium as well as in the presence of foetal bovine serum or autologous plasma. Thymus cells were reproducibly stimulated by high concentrations of PHA. No significant responses were obtained in bursal cell cultures with any of the compounds tested. Removal of cotton wool-adherent cells from the spleen cell suspensions resulted in a subpopulation of cells which were stimulated by PHA but showed little response to ConA, PWM, or LPS. This procedure did not remove surface immunoglobulin-bearing cells from the original suspension. Both these enriched spleen lymphocytes and the unfractionated spleen, blood and thymus leukocyte cultures were effectively stimulated by a partially purified PHA but with a highly purified PHA preparation only at very high concentrations. These and other results suggest that the mitogenic components in crude PHA preparations are different for chicken and human or mouse cells.     

The effect of a ribosomal Shigella sonnei vaccine on cellular immune reactions]

The influence of S. sonnei ribosomal vaccine on hematopoiesis, T- and B-cell-mediated immune reactions has been studied in the course of the development of experimental vaccinal process. The vaccine stimulated hematopoiesis, that was characterized by a dose-dependent increase in colony-forming units in the spleen (CFUs), a rise in CFUs in the blood and bone marrow and an increase in the pool of proliferating stem cells in bone marrow, shortly after injection. A pronounced immunostimulating effect of the vaccine on the formation of antibody-producing cells (APC) to heterologous antigen (sheep red blood cells) in the spleen has been established, and the vaccine has also been found to stimulate, though to a lesser extent, APC synthetizing specific antibodies to S. sonnei LPS. The injection of S. sonnei ribosomal vaccine influences the functional activity of effector T-cells; in its turn this phenomenon produces phasic changes in the migration activity of spleen cells in the presence of specific LPS and surface polysaccharide antigen of S. sonnei in phase I.     

Production of an antiviral factor by murine spleen cells after treatment with Corynebacterium parvum.

We have previously shown that injection of Corynebacterium parvum (CP) in mice protected them against lethal encephalitis induced by herpes simplex virus, (HSV). It is shown here that spleen cells of CP-injected mice in vitro produce a factor capable of inhibiting the replication of HSV in mouse embryo fibroblasts (MEF). A similar activity was produced after in vitro exposure of spleen cells from untreated mice to CP. CP was only slightly mitogenic in contrast to phytohemagglutinin (PHA), concanavalin A (Con A), and bacterial lipopolysaccharide (LPS) which were strongly mitogenic but did not induce antiviral activity high enough to be detected in the HSV-MEF system. The activity produced by CP-treated spleen cells appeared to be interferon since it was trypsin sensitive and species specific and not virus specific, and since preincubation of the cells was required to demonstrate an antiviral effect. However, the identity of CP-induced interferon with any of the previously described subclasses of interferon remains to be determined.     

Mitogen Synergism in low-responding CBA/CaJ mice.

Although neither phytohemagglutinin (PHA) nor concanavalin A (Con A) stimulated blood cultures in vitro from low-responding CBA/CaJ mice effectively, a mixture of PHA and Con A over a range of concentrations stimulated a response from CBA/CaJ mouse blood that was greater than the sum of the responses produced by using PHA or Con A individually. This synergistic effect was expressed as the percentage by which the responses to the PHA and Con A mixture exceeded the sum of the responses to PHA alone and Con A alone. When the mitogen concentrations that gave maximum responses individually were used, the synergistic effect averaged 319% in cultures of blood from low-responding CBA/CaJ mice. Apparently simultaneous exposure to PHA and Con A stimulates DNA synthesis in white blood cells of CBA/CaJ mice that fail to respond to either mitogen alone.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

Changes in the number of cells and the weight of various lymphoid organs of mice, such as the regional lymph node (right inguinal node), spleen, thymus, bone marrow, and peripheral blood, were followed after the subcutaneous injection of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K). For comparison, the changes after injection of various polyclonal lymphocyte activators (PLA) including various preparations of bacterial lipopolysaccharide (LPS) were concurrently studied. The number of cells of all of the lymphoid organs tested and that of nucleated cells in the peripheral blood decreased significantly within a few days after injection of CPS-K, and increased later. Above all, the increase in the number of cells and in the weight of the regional lymph node was most prominent (about 10 times larger than that of the normal control). Such a marked increase in the number of cells of the regional lymph node was not induced by the injection of any preparation of LPS or any other PLA tested. The initial decrease in the number of cells after CPS-K injection was most marked and long lasting in the thymus. Although LPS prepared by Westphal's method from Escherichia coli O55 or Salmonella enteritidis exhibited a stronger decreasing effect on the number of cells of the thymus, the effect of LPS prepared by Westphal's method from E. coli O111 or that by Boivin's method from E. coli O55 was similar to that of CPS-K. It is concluded therefore that CPS-K has the ability to decrease the number of cells of various lymphoid organs, especially that of the thymus, initially after injection, which is a property in common with LPS, and CPS-K has a unique ability to increase markedly the cells of various lymphoid organs, especially those of the regional lymph node, at later stages after injection. Considering that CPS-K exhibits a much stronger adjuvant effect on the antibody response than does LPS or other polyclonal lymphocyte activators, it is suggested that this extraordinarily potent activity of CPS-K in increasing the number of cells of the regional lymph node is closely related to its strong adjuvant action.     

Mechanism of histamine-induced inhibition of lymphocyte response to mitogens in mice

Histamine induced, in mice, an inhibition of lymphocyte response to PHA and LPS, at molar concentrations ranging from 10^?3 to 10^?9M. This inhibition occurs as a specific interaction between histamine and T lymphocytes bearing H2-type receptors for this hormone (H + cells) and Ly 2 membrane antigens. Two features of the suppressive activity of this T-cell subpopulation were observed: (i) when histamine is added at the beginning of the culture period with PHA or LPS, it activates the suppressor activity of H + cells which act on the lymphocyte population responding to PHA and LPS; (ii) preincubation of spleen lymphocytes with histamine for 24 hr induces suppressor cells which inhibit the response to PHA, but not to LPS, of syngeneic lymphocytes in a coculture system, and which are radiosensitive. The role of PHA as a second stimulus of histamine-induced suppressor cells, and the relation between these cells and PHA or Con A-induced suppressor cells, are discussed.     

Immunosuppressive Effect of Bacterial Lipopolysaccharide on Antibody Response

Injection of endotoxins (bacterial lipopolysaccharide: LPS) several days prior to immunization causes the suppression of antibody response. The suppressive effects of several kinds of LPS preparations on the plaque-forming cell (PFC) antibody response in the spleen of mice were examined after immunization with sheep red blood cells (SRBC). Glycolipids obtained from heptoseless mutants (Re form) of salmonella or its lipid A preparation coupled artificially with bovine serum albumin (BSA) are capable, like LPS obtained from a wild type (S form) strain, of inducing suppression of the PFC response, while alkaline-detoxified LPS can not. The refractory periods of the PFC response induced by LPS injection last only a few days. However, the use of cyclophosphamide (CY) together with LPS can extend the refractory periods of antigenic stimulation for several weeks. Injections of LPS and CY can also induce unresponsive states of OH agglutinin antibody response to antigenic stimulation with formalin-killed organisms of Escherichia coli or Salmonella enteritidis (presumably both thymus-independent antigens). These unresponsive states induced by LPS and CY are easily terminated by a transfer of syngeneic bone marrow cells but not by thymocyte transfer.     

Effect of Escherichia coli Lipopolysaccharide on Bacteroides fragilis Abscess Formation and Mortality in Mice

To study the mechanism of synergism between Bacteroides fragilis and Escherichia coli, the effect of sublethal dose of E. coli lipopolysaccharide (LPS) (25μg/mouse) was checked on B. fragilis abscess formation. LPS was administered prior or after inoculum injection. No significant difference in the abscess size was observed at necropsy on day 6. However, all the groups receiving LPS showed higher incidence of recovery of additional intestinal bacteria (23.5–45.5%) from the abscess pus. When LPS was given 4 hr prior to inoculum administration, 83–100% mortality was observed. Detailed investigation showed autoclaved cecal contents alone could also cause similar mortality. Studies with stimulation of endogenous cytokines by E. coli LPS demonstrated induction of all of them within 3 hr in the blood stream with TNF-α demonstrating peak at 1 hr, IL-1α and IL-6 at 4 hr and IFN-γ between 6–9 hr with moderately high levels at 4 hr. This E. coli LPS-triggered cytokine cascade possibly gets further stimulated by injection of autoclaved cecal contents containing high concentration of endotoxins (1.6 × 10⁵ EU/ml) contributed by dead bacteria and lead to the mortality of animals.     

EFFECTS OF BACTERIAL ENDOTOXIN ON HEMOPOIETIC COLONY-FORMING CELLS IN THE SPLEENS OF NORMAL MICE AND MICE OF GENOTYPE S1/S1

Multiple doses of S. typhosa endotoxin caused an increase in the number of hemopoietic stem cells present in mouse marrow and spleen that could be detected using the spleen-colony assay. This increase was inhibited by Colcemid, and by the genetically-determined defect in hemopoiesis in mice of genotype S1/S1^d. However, the defect in S1/S1^d hosts did not prevent an endotoxin-induced increase in the number of cells capable of forming colonies in cell culture. The results support the view that bacterial endotoxin acts, via a genetically-controlled regulatory mechanism, to stimulate the proliferation of hemopoietic stem cells in the spleen.     

Differential effects of concanavalin A and phytohemagglutinin on murine immunity. Suppression and enhancement of cell-mediated immunity.

To assess the effects of the selective T-cell mitogens concanavalin A (Con A) and phytohemagglutinin P (PHA) on cell-mediated immunity (CMI), the mitogens were injected before, with, or after intravenous (iv) challenge of mice with Listeria monocytogenes. Mitogenic treatment differentially influenced the CMI response to Listeria. Con A enhanced listericidal activity when given before or with Listeria challenge, but Con A suppressed the CMI response when given after infection with Listeria. In contrast, PHA enhanced listericidal activity at all intervals. Since Con A, but not PHA, affected the growth of Listeria in the spleens of mice 24 and 48 hr after infection, Con A was shown to have an immediate effect on the development of listericidal activity and PHA was shown to have a delayed effect. In addition, Con A induction of immediate nonspecific listericidal activity was short-lived, while PHA induced a longer-lasting effect on resistance to Listeria. The mitogen-induced effects in the CMI response to Listeria were shown to be dependent upon the activities of activated T cells. The enhancement and suppression of listericidal activity appears to result from the activation of different T-cell subpopulations, known to be stimulated preferentially by Con A or PHA.     

Stimulatory effect of Bestatin,a new specific inhibitor of aminopeptidases,on the blastogenesis of guinea pig lymphocytes

A potent protease-inhibitor of Actinomycetes origin, Bestatin. which is of dipeptide nature and inhibits aminopeptidase B and leucine-aminopeptidase competitively, strongly stimulates blastogenesis of small lymphocytes triggered with polyclonal mitogen. such as phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and lipopolysaccharide of Escherichiae coli (LPS), whereas it inhibits DNA synthesis of normal resting lymphocytes. The stimulatory effect is non-selective with respect to the category of small lymphocytes, i.e. T- and B-lymphocytes, but strikingly selective with respect to the stage of blastogenesis: the stimulation is greatest at a relatively early stage, diminishes as mitogen-activation proceeds, and is not appreciable at a later stage of lymphocyte blastogenesis.The pattern of Bestatin stimulation on lymphocyte blastogenesis is specific for the mitogen used: in T-lymphocyte activation with PHA or Con A, the stimulation first increases and then decreases with increase in mitogen concentrations, whereas in B-lymphocyte activation with LPS, with increasing concentrations of the mitogen, the stimulation increases to a plateau at approximately 100 μg/ml of mitogen. The optimum concentration of Bestatin was found to be approximately 50 μg/ml (0.16 mM) for either PHA or Con A activation, and 50 to 75 μg/ml for B-cell activation with LPS. Bestatin must remain in cultures of T- and B-lymphocytes with polyclonal mitogens for at least about 24 and 16 hr, respectively, to exert its stimulatory effect on blastogenesis.Biochemical results, together with those from autoradiographic analyses, indicate that Bestatin increases the number of blastoid-transformed lymphocytes with polyclonal stimulants. It is suggested that aminopeptidases, possibly located at the cell surface, may play a role in the control of lymphocyte activation during immune responses.     

Induction of lysozymelike activity in the hemolymph and hemocytes of an insect, Spodoptera eridania

Lysozymelike activity is present in the hemocytes and cell-free hemolymph of Spodoptera eridania. Its level remains essentially constant during larval development and can be induced by injection of various foreign materials. Serum bacteriolytic activity rises 24 hr after injection of saline, BSA, bacteria, bacterial endotoxin (LPS), latex particles, or sham injection. However, the magnitude and subsequent duration of the response depends on the nature of the injected material. The response is transient following sham injection or injection of soluble substances, such as saline and BSA, as compared to treatment with latex or bacteria. Both soluble and insoluble fractions of bacterial LPS preparations stimulated the lysozyme response. The response to a single injection of E. coli LPS was dose dependent and persisted for at least 5 days; however, additional injections had no effect on serum lysozyme level. The basal intracellular lysozyme level was significantly increased by E. coli LPS injection. Lysozyme release by hemocytes was proportional to intracellular concentration and did not increase after phagocytic stimulation of hemocytes.