Hemopoietic colony studies. VI. Increased eosinophil-containing colonies obtained by antigen pre-treatment of irradiated mice reconstituted with bone marrow cells

The cellular response to an intraperitoneal injection of antigen (tetanus toxoid) was studied in reconstituted animals in order to determine the mechanism of control of eosinophil granulocytopoiesis. Antigen treatment of the marrow cell donors did not consistently increase the number of spleen and bone marrow colonies in recipient animals or change the percentage of eosinophil or other hemopoietic colony types. Antigen pre-treatment of the irradiated recipients increased the percentage of eosinophil-containing colonies in the spleen and femoral bone marrow without significantly changing the total number of either spleen or marrow colonies. Antigen treatment of both the bone marrow cell donor and recipient produced a further increase in the percentage of eosinophil-containing colonies in the marrow cavity, but not in the spleen. Antigen treatment of the irradiated recipient increased the number of eosinophilic cells (but not the total number of cells) in both the peritoneal cavity and the bone marrow. Antigen treatment of both the marrow donor and recipient produced a further increase in the number of eosinophilic cells in the peritoneal cavity, but not in a single femur. Since antigen treatment of the marrow recipient, or recipient and donor, but not of the marrow donor alone, results in increased eosinophilic cell and colony numbers, the effect of antigen appears to be mediated through some host factor(s), perhaps the eosinophilic hemopoietic inducing microenvironment (HIM), rather than directly on the hemopoietic stem cells.     

Peritoneal exudate cells. I. Growth requirement of cells capable of forming colonies in soft agar

Mouse peritoneal exudate cells induced by thioglycollate medium can form colonies in soft agar with a plating efficiency of about 5% (0.6%–10%). Cells from an unstimulated peritoneal cavity form no colonies or have a plating efficiency of less than 0.001 %. These colony-forming cells from the peritoneal exudate are similar to bone marrow colony-forming cells in vitro in that they both require a substance(s) present in conditioned medium from L-cells or mouse embryo fibroblasts or the serum from endotoxin-treated mice for the initiation and the continuation of their growth. However, peritoneal exudate colony-forming cells have a much longer initial lag period (10–14 days) and can survive longer in the absence of L-cell conditioned medium than bone marrow colony-forming cells. Only mononuclear cells, presumably macrophages, are observed in peritoneal exudate colonies, whereas bone marrow cell colonies contain both polymorphonuclear cells and macrophages.     

Stimulated murine peritoneal macrophages in suspension and cell culture: enzyme determinations by biochemical and histochemical means

Summary Peritoneal exudate cells of mice were studied up to 14 days after i.p. injection of thioglycollate broth medium by means of conventional enzyme determinations and quantitative histochemical measurements of individual cells. Cells from the peritoneal cavity were either investigated immediately after harvesting or after culturing periods of up to six days for enzymic activities of aminopeptidase, esterase, lactate dehydrogenase and -galactosidase. A fairly good correlation exists between biochemical determinations of aminopeptidase and esterase activity and the mean of histochemical data. Following a sharp increase in the number of cells after stimulation, aminopeptidase, esterase and lactate dehydrogenase activities per cell were found to be increased. Moreover, the cells taken three or five days after stimulation synthesized large amounts of aminopeptidase and esterase as shown by culturing experiments. This capacity of the cells was subsequently lost in cells harvested seven day after stimulation but -galactosidase increased and lactate dehydrogenase was more readily released into culture supernatants. The increase in aminopeptidase and esterase was dependent on protein synthesis since it was abolished by cycloheximide. Thioglycollate broth medium provokes immigration of cells into the peritoneal cavity where cells apparently differentiate by increasing their aminopeptidase and esterase concentrations, and by raising their intracellular catabolism rates, which leads eventually to the decay of the cells. The different enzymic phenotypes and the large heterogeneity at any time point after stimulation presumably also reflect different functional properties during the inflammatory process.     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. III. Significance of hemopoietic stem cells for induction and maintenance of Mls tolerance by continuous supply of tolerance-inducing nonlymphocytes

The role of hemopoietic stem cells and other cell types in the induction and maintenance of immunologic tolerance in the thymus was investigated by intravenous injection of Mls-semi-allogeneic cells into newborn mice less than 24 hr after birth. Mls-specific tolerance was induced by inoculation of peritoneal cells and thymus cells, and the tolerant state was compared with that induced by bone marrow cells which had hemopoietic stem cell activity and were able to create a stable chimera in both central and peripheral lymphoid organs. When peritoneal or thymus cells were injected, the level of tolerance attained was proportional to the number of cells injected, though peritoneal cells were 20 times as effective as thymus cells. In vivo functions of tolerance-inducing cells and their immediate precursors were radiosensitive and belonged to a Thy-1-, nylon-wool-nonadherent (probably non-B), weakly Sephadex G-10-adherent cell population. Tolerance induced by peritoneal cell injections was transient, starting to terminate within the first 2 weeks of life, while tolerance caused by bone marrow cell injections persisted through more than 6 weeks. Such transient tolerance induced by the former became long-lasting when followed by an additional injection of bone marrow cells, which did not cause thymic lymphocyte chimerism. All data indicated that bone marrow stem cells were engaged in tolerance induction and maintenance by continuously supplying tolerance-inducing nonlymphocytes.     

The number of the rat peritoneal cells in normal condition, blood loss, and under colchicine injection

The modification of peritoneal cells derivation method proposed for the peritoneum rat, and the absolute amount of peritoneal lymphocytes, monocytes, macrophages, stem and polymorphonuclear neutrophyles, mat cells was determined. The procedure of acute 2% blood loss was found to reduce the total amount of the peritoneal cells due to depletion of lymphocytes accompanied by an increase in amount and proliferative activity of monocyte peritoneal cells, stem neutrophiles appearance in the peritoneal cavity. The subcutaneous injection of colchicine solution reduces the number of the peritoneal macrophages in normal rats, decreases the number of the monocytes, polymorphonuclear neutrophiles and led to disappearance of the stem neutrophiles on the second day of blood loss.     

The oxidative metabolism of thioglycollate-elicited mouse peritoneal macrophages: the relationship between oxygen, superoxide and hydrogen peroxide and the effect of monolayer formation

The oxygen and glucose metabolism of peritoneal macrophages harvested from untreated mice (resident cells) and mice given an i.p. injection of thioglycollate broth (thioglycollate cells) were examined. Thioglycollate cells consumed approximately 3 times as much O2 at rest and during phagocytosis as resident cells, but oxygen reduction products (superoxide and hydrogen peroxide) could be recovered in only minimal amounts despite triggering by phagocytosis or exposure to PMA. Indirect evidence for the formation of oxygen reduction products such as O2- by thioglycollate cells was obtained by observation of the major pathways for glucose oxidation and NBT dye reduction. When thioglycollate cells were allowed to adhere to a glass surface O2- and H2O2 were easily recovered in the extracellular medium with a 20-fold increase above cells in suspension exposed to PMA. This study suggests that thioglycollate-elicited macrophages have a vigorous oxidative metabolism but that recovery, and perhaps utilization, of O2 reduction products formed will depend on the conditions of incubation. These events may be significant both for the study of parameters of macrophage "activation" in vitro as well as the function of these cells in vivo.     

Experimental research on the cellular interactions during hemopoiesis

We present a review of experimental studies performed at the Laboratory of Histogenesis of the Institute of Developmental Biology, Russian Academy of Sciences, on the problem of cell interactions during hemopoiesis. Special attention has been given to original experimental models, such as production of hemopoietic foci on underlayers of fibroblasts encapsulating a foreign body in the peritoneal cavity of rodents (after intraperitoneal transplantation of hemopoietic cells) and repopulation of ectopic hemopoietic territories under the kidney capsule of mice by syngeneic or xenogeneic hemopoietic cells. We describe the competitive interactions of genetically different hemopoietic cells after the transplantation of their mixtures to irradiated mice (multicomponent radiation chimeras). Xenogeneic and multicomponent chimeras have also been obtained in long-term bone marrow culture. We have examined characteristics of hemopoiesis on stromal cell underlayers produced by cells of various origins in vitro and then transplanted into the peritoneal cavity of irradiated mice. We discuss the results obtained and possible mechanisms of these phenomena.     

Regulation of bone marrow cell survival in short-term cultures: a new macrophage function

The involvement of macrophages (M phi) in the regulation of bone marrow (BM) cell survival in short-term cultures was studied. We developed a system to measure the survival of fresh BM cells in vitro, by evaluating 111indium (111In) release from prelabeled BM cells. 111In release was proportional to cell death and inversely related to the number of trypan blue excluding cells. Upon 24 hr of culture in conventional medium, more than 50% of BM cells died. In order to investigate whether BM cell death could be reduced by coculture with other cell types, 111In-labeled BM cells were incubated for 24 hr with peritoneal M phi, thymocytes (THY), or polymorphonuclear cells (PMN) and then assayed for their survival. We found that coculture of BM cells with M phi dramatically increased BM survival, whereas THY or PMN consistently failed to enhance BM survival. The ability to promote BM cell survival, here designated nurse activity, represented a novel function of M phi and was further characterized. The stage of activation of M phi did not influence their nurse activity, since M phi elicited in vivo by proteose-peptone, thioglycollate, or Bacillus Calmette-Guérin, as well as resident M phi unstimulated or activated in vitro with lipopolysaccharide, equally sustained survival of BM cells. BM-derived M phi (adherent cells from BM cultures maintained in 20% L-cell-conditioned medium for 14 days) were equally effective in exerting nurse activity. Moreover, nurse activity was also exerted across the histocompatibility barriers. Supernatants from M phi cultures or killed M phi were ineffective. We propose that the nurse effect of M phi on BM is a primitive function that may play an important role in the development of the hemopoietic system.     

The comparison of lysosomal enzymes activities in alveolar and peritoneal macrophages of rat

Studies were carried out on macrophages isolated from control and thioglycollate injected rats. Intraperitoneal injection of thioglycollate had no effect on the number of harvested alveolar macrophages but caused a marked increase in the number of peritoneal macrophages. The specific activities of all the investigated enzymes were significantly lower in peritoneal macrophages from control rats in comparison to alveolar macrophages. Thioglycollate stimulation of peritoneal macrophages caused increase in activities of all lysosomal hydrolases studied. Cathepsin B, N-acetylglucosaminidase and esterase showed the highest level of stimulation.     

Ontogenic development of the reactivity of macrophages to antigenic stimulation

Experiments were performed to test the ability of macrophages from newborn mice to participate in immune reactions. It was found that peritoneal cells from 4-day-old mice injected at birth with thioglycollate did not reconstitute reactivity to Shigella in adult, irradiated mice, while normal adult macrophages did.The total yield of peritoneal exudate cells (PEC) was relatively low, yet the number of precursor cells of macrophages in the newborn spleen was found in significantly higher concentrations than in the adult. The number of precursor cells in the spleens of 0–3-day-old mice did not increase in response to antigenic stimulation, indicating that they too are unable at this stage to develop reactivity to immunological signals.     

Protection of mice against herpes simplex virus infection by a Lactobacillus casei preparation (LC 9018) in combination with inactivated viral antigen

Heat-killed Lactobacillus casei YIT 9018 (LC 9018) cells enhanced the resistance to herpes simplex virus type 1 (HSV-1) in adult mice, but not significantly. The protection of mice against HSV-1 infection and the production of neutralizing antibodies were significantly enhanced by the administration of LC 9018 in combination with inactivated HSV-1 antigen. The optimal enhancement of resistance was seen in mice 14 days after the simultaneous administration of these substances. The resistance to HSV-1 infection in mice could be transferred with peritoneal exudate cells from syngeneic mice previously treated with LC 9018 alone and LC 9018 in combination with inactivated HSV-1 antigen or with thioglycollate broth, whereas the transfer of peritoneal exudate cells induced by thioglycollate broth alone and of spleen cells induced by LC 9018 in combination with thioglycollate broth or by thioglycollate broth alone was not effective. These results suggest that mouse peritoneal macrophages induced by the administration of LC 9018 in combination with inactivated HSV-1 antigen may play an important role in host defense mechanisms against HSV-1 infection.     

Different effect of testosterone on polypotential stem hematopoietic stem cells and immunocompetent B-lymphocytes]

A research was made to study the dynamics of the proliferative, colony-forming and migration capacity of stem hemopoietic cells in (CBA X C57Bl) F1 hybrid mice under the influence of testosterone propionate, 10 mg/100 g, as well as the migration of immunocompetent B lymphocytes from the bone marrow to the spleen and the accumlation of their progeny, antibody-producing cells, in the spleen. The immunodepressive effect of testosterone was manifested by a decrease in the migration of B cells and the number of antibody-producing cells in the spleen. On the contrary, testosterone had a stimulating effect on the functional activity of stem hemopoietic cells, increasing their proliferation and migration. Under conditions of the suppressed erythropoietic differentiation of multipotent stem hemopoietic cells the injection of testosterone resulted in an increase in the number of antibody-producing cells in the spleen. This suggests that the stimulation of erythropoiesis and immunosuppression, induced by testosterone, are interconnected and determined by the direct action of the hormone on the cellular cycle of the stem cells, as well as by their prevailing differentiation towards the erythroid series, resulting in the decrease of their differentiation into B cells.     

Rat peritoneal macrophage procoagulant and fibrinolytic activities. An expression of the local inflammatory response.

1. Lysates of rat (Rattus norvegicus, Wistar strain) peritoneal macrophages selected by adherence were analysed for procoagulant activity (PCA) and plasminogen activator activity (PAA). 2. PCA is expressed following in vitro stimulation by lipopolysaccharide and in vivo (respectively 118 +/- 19.1 and 147.2 +/- 45.2 mUnits/10(6) M phi) very early after the intraperitoneal injection of thioglycollate. 3. PAA present in 24 hr thioglycollate stimulated cells (79.5 +/- 26.1 mI.U./10(6) M phi), disappears in a time dependent fashion after in vitro lipopolysaccharide stimulation. 4. Our findings indicate that rat peritoneal macrophages regulate their coagulolytic activities in a specific manner. 5. Rat peritoneal cavity may be proposed as a model for the study of inflammatory reaction with PCA and PAA as its indexes.     

The Effect of Superoxide Dismutase on the Recovery of Human Bone Marrow Hemopoietic Stem Cells Stored at 4°C

The effect of superoxide dismutase (SOD) on the recovery of human hemopoietic stem cells stored at 4°C has been studied. Human bone marrow cells from cancer patients without hemopoietic diseases were stored at 4°C for 1–7 days in RPMI 1640 medium containing 10% human AB serum with or without 6 U/ml of SOD. The recovery of colony-forming units of the following types were counted: granulocyte and macrophage type; megakaryocyte type; mixed granulocyte, erythrocyte, megakaryocyte, and macrophage type; and burst-forming units of erythrocytic type. The SOD groups recovered, respectively, 5.6, 5.3, 4.6, and 4.4 times as many colonies as the control group. This study demonstrates that the addition of SOD to hypothermic storage media may increase the recovery of hemopoietic stem cells.     

Expansion of umbilical cord blood for clinical transplantation

Since the first successful cord blood transplant was performed in 1988 there has been a gradual increase in the use of cord blood for hemopoietic stem cell transplantation. Worldwide, over 8,000 unrelated cord blood transplants have been performed with the majority being for children with hemopoietic malignancies. Transplantation for adults has increased but is limited by the low number of nucleated cells and CD34(+) cells within a single cord blood collection. Cord blood hemopoietic stem cells are more primitive than their adult counterparts and have high proliferative potential. Cord blood ex vivo expansion is designed to improve transplant outcomes by increasing the number of hemopoietic stem cells with long term repopulating potential and their differentiated progeny. However, despite a large amount of research activity during the last decade, this aim has not been realized. Herein we discuss the rationale for this approach; culture methods for ex vivo expansion, ways to assess the functional capacity of ex vivo generated hemopoietic stem cells and clinical outcomes following transplantation with ex vivo expanded cord blood.     

Helianthus tuberosus agglutinin directly induces neutrophil migration, which can be modulated/inhibited by resident mast cells.

We investigated the effect of Helianthus tuberosus agglutinin (HTA) on neutrophil migration in vivo and in vitro. The role of resident cells in this effect was analyzed. Peritonitis was induced by injecting stimuli into rat (150-200 g) peritoneal cavities, and in vitro neutrophil chemotaxis was performed using a Boyden microchamber. HTA (80, 200, or 500 microg/mL per cavity) induced significant in vivo neutrophil migration (p < 0.05); in vitro assays showed that this lectin also induced neutrophil chemotaxis, an effect inhibited by the incubation of lectin associated with alpha-D(+)-mannose, its specific binding sugar. Depletion of the resident-cell population by peritoneal lavage did not alter HTA-induced neutrophil migration (200 microg/mL per cavity). The opposite strategy, increasing peritoneal macrophages by intraperitoneally injecting rats with thioglycollate, did not enhance the neutrophil migration produced by HTA (200 microg/mL per cavity). In addition, injection of supernatant from HTA-stimulated macrophage culture (300 microg/mL) into rat peritoneal cavities did not induce neutrophil migration. However, reduction of the peritoneal mast-cell population potentiated the neutrophil migration (p < 0.05) induced by HTA (200 microg/mL per cavity). Lectin from H. tuberosus has a direct neutrophil chemotatic effect that is modulated by mast cells.     

Comparison of direct and indirect radiation effects on osteoclast formation from progenitor cells derived from different hemopoietic sources

Hemopoietic stem and progenitor cells from different sources differ in radiosensitivity. Recently, we have demonstrated that the multinucleated cell responsible for bone resorption and marrow cavity formation, the osteoclast, is in fact of hemopoietic lineage. In this investigation we have studied the radiosensitivity of osteoclast formation from two different hemopoietic tissues: fetal liver and adult bone marrow. Development of osteoclasts from hemopoietic progenitors was induced by coculture of hemopoietic cell populations with fetal mouse long bones depleted of their own osteoclast precursor pool. During culture, osteoclasts developed from the exogenous cell population and invaded the calcified hypertrophic cartilage of the long bone model, thereby giving rise to the formation of a primitive marrow cavity. To analyze the radiosensitivity of osteoclast formation, either the hemopoietic cells or the bone rudiments were irradiated before coculture. Fetal liver cells were found to be less radiosensitive than bone marrow cells. The D0, Dq values and extrapolation numbers were 1.69 Gy, 5.30 Gy, and 24.40 for fetal liver cells and 1.01 Gy, 1.85 Gy, and 6.02 for bone marrow cells. Irradiation of the (pre)osteoclast-free long bone rudiments instead of the hemopoietic sources resulted in a significant inhibition of osteoclast formation at doses of 4 Gy or more. This indirect effect appeared to be more prominent in the cocultures with fetal than with adult hemopoietic cells. Furthermore, radiation doses of 8.0-10.0 Gy indirectly affected the appearance of other cell types (e.g., granulocytes) in the newly formed but underdeveloped marrow cavity. The results indicate that osteoclast progenitors from different hemopoietic sources exhibit a distinct sensitivity to ionizing irradiation. Radiation injury to long bone rudiments disturbs the osteoclast-forming capacity as well as the hemopoietic microenvironment.     

Effect of the interferon inducer tilorone in inbred CBA mice.

Tilorone hydrochloride (200 mg/kg) was administered intragastrically to inbred CBA mice. After 5, 18 and 48 hr the number of circulating leucocytes and peritoneal cells as well as the migration of unstimulated peritoneal cells, the blood corticosteroid level and interferon production were investigated. In spite of the considerable decrease of the number of mononuclear cells in the blood and polynuclear ones in the peritoneal exudate, the drug induced production of circulating interferon and stimulated its synthesis by peritoneal cells. The blood corticosteroid level and the mast cell count in the peritoneal cavity were significantly elevated, but the migration of peritoneal cells in antigen-free medium decreased.     

In vivo inflammatory stimulation induces a transient change in the binding of thrombin to rat peritoneal macrophages.

The binding of 125I-labeled thrombin to rat peritoneal macrophages isolated 20 h after the ip injection of thioglycollate broth or lipopolysaccharide decreased to 20% of the value found in resident macrophages due to a decrease in the number of receptors. The binding returned to normal values within a week after the injection. The decline parallelled more or less the Vmax for the 5'-nucleotidase activity. This decrease in the binding of thrombin could not be explained by an immigration of monocytes into the peritoneal cavity, since the binding of 125I-labeled alpha 2-macroglobulin-trypsin complex increased 4.5-fold in the same cell population due to an increase in the number of receptors, and blood monocytes do not bind alpha 2-macroglobulin-trypsin complex. The increase in the binding of alpha 2-macroglobulin-protease complex parallelled an increase in the incorporation of glucosamine, although the latter did not increase to the same extent. Engulfment of plasma membrane after phagocytosis did not result in a decreased binding of thrombin, but preincubation at 37 degrees C with concanavalin A caused a minor reduction in the binding. There was a positive correlation between the binding of alpha 2-macroglobulin-trypsin complex and the fraction of polymorphonuclear leukocytes in the peritoneal exudate and a negative correlation between the binding of thrombin and the fraction of polymorphonuclear leukocytes in the exudate, when the inflammation was induced by a milder stimulus, sterile NaCl, indicating a common signal for the polymorphonuclear leukocyte chemotaxis and the macrophage differentiation.     

B-1 cell (CD5+/CD11b+) numbers and nIgM levels are elevated in physically active vs. sedentary rats.

Habitual, moderate exercise is associated with improved health, including reductions in illness. These benefits may stem, in part, from immune function improvements. We have previously reported that daily wheel running increases serum and peritoneal natural IgM (nIgM) in pathogen-free Sprague-Dawely rats. B-1 cells, which primarily reside in the peritoneal cavity, produce nIgM in the absence of antigen stimulation. This study examined whether physical activity would also increase B-1 cell numbers in the peritoneal cavity, mesenteric lymph nodes, and spleen. Male, pathogen-free Fischer 344 rats were sedentary (standard cages) or physically active (running wheel access) for 6-7 wk. Peritoneal cavity, mesenteric lymph nodes, and spleen cells were taken, and the number of CD5+/CD11b+ (B-1) cells were measured by using two-color flow cytometry. The results were that physically active animals had increased numbers of CD5+/CD11b+ cells in the peritoneal cavity. In addition, physically active animals had increased serum and peritoneal nIgM, thus replicating our previous observations. These results indicate that voluntary running selectively increases the B-1 cell population, which is most likely responsible for the elevated serum and peritoneal nIgM in active rats. Because B-1 cells are important in host defense, these changes may contribute to the health benefits of exercise.