Adherent cells (macrophages?) in tumor-bearing mice suppress MLC responses

The spleens of mice bearing transplanted methylcholanthrene (MCA)-induced fibrosarcomas (MCA-1425 and MCA-1460) were shown to contain cells capable of suppressing the generation of cytolytic T lymphocytes (CTL) in mixed leukocyte cultures (MLC). The suppressive activity was first detected 21 days after tumor transplantation. No suppression was seen with lymph node cells taken at the same time as the spleen cells. The cells responsible for the suppressive activity were adherent to nylon wool and plastic dishes and they were not lysed by anti-T-cell serum plus complement. The suppressor cells were phagocytic and were resistant to irradiation (3000 rads) in vitro. Spleen cells from tumor-bearing nude mice were as suppressive as were spleen cells from tumor-bearing conventional mice. We conclude from these findings that T cells were not involved either as inducers or as effectors of the suppression observed, although the responsible adherent cells may have exerted their effect by interacting with a T-suppressor cell population in the MLC mixtures. While spleen cells of tumor-bearing mice were suppressive when added at any time during the first 4 days of a 5-day MLC, they showed no effect on the cytotoxicity of fully differentiated CTL. Indomethacin reversed suppression, suggesting that prostaglandins may have been involved.     

Diffusion chamber and spleen colony assay of murine haematopoietic stem cells

Mouse bone marrow cells have been cultured in diffusion chambers and their capacity to form spleen colonies in irradiated mice investigated after different culture periods. The number of spleen colony-forming units (CFU) in the chambers decreased during the first day of culture. The number then increased rapidly to a level significantly above the original chamber value on the third to fifth day of culture. By that time large numbers of granulocytes and macrophages had also appeared. Histological examination of spleen colonies showed that prior culturing did not alter the ratio between the different types of colonies. Cultured bone marrow cells which were transferred to new chambers retained granulopoietic capacity. This capacity increased between the first and second day of primary culturing. At this time hydroxyurea injections to chamber hosts revealed that the progenitor cells were proliferating. The results show that the granulopoietic progenitor cells of the chambers are stem cells, and that one progenitor cell type is identical with the CFU.     

Quantitative study of regression and regeneration of the thymic cell population after X-irradiation in the newtPleurodeles waltlii Michah

Summary The changes in cell numbers of different thymic cell populations and the conditions governing the regeneration of these populations and the thymus itself were examined after X-irradiation (700 rads) of different parts of the body. The general effects of the irradiation were studied in each experimental group in terms of mortality and growth rate. The particular effects on each thymic cell population were studied by the measurement of mitotic activity and of evaluation of the changes in numbers among these populations in the thymus itself, and were compared with the effects in the granulopoietic layer of the liver and in the spleen. The great reduction in the number of lymphocytes after irradiation demonstrates that they are more radiosensitive than other cell types; this reduction can be compensated for by the arrival of new lymphoid cells originating from other lymphoid organs (if they have been protected from irradiation) and by allowing thymic regeneration. Thus, irradiation has indirect effects on non-irradiated areas, and demonstrates that the lymphoid cell population has a high potential for multidirectional migration.     

Merocyanine 540 as a fluorescent probe of membranes: selective staining of leukemic and immature hemopoietic cells.

We have reported (Easton, Valinsky and Reich, 1978) that merocyanine 540 (MC 540) specifically stains a variety of living excitable cells, but not nonexcitable cells. This paper describes the exceptional permeability to MC 540 of leukemic leukocytes and immature hemopoietic precursor cells. We have used fluorescence microscopy and uptake of radioactive dye to study MC 540 staining of peripheral blood leukocytes from 80 leukemic and 34 normal individuals; leukemic leukocytes stain, whereas normal leukcytes do not. The leukocyte staining reaction differs from that previously described for excitable cells since it is independent of the ionic composition of the staining medium, kinetically complex, enhanced by light, enhanced by oxygen and essentially irreversible. Virtually all circulating nucleated cells from leukemic individuals are stained to approximately the same extent, and there is no qualitative or quantitative distinction between the various forms of leukemia. We have also found that MC 540 interacts with granulopoietic colony-forming cells (CFU-C) and with spleen colony-forming cells derived from mouse bone marrow (CFU-S). We cannot as yet identify a specific property of leukocyte plasma membranes that determines MC 540 permeability; since changes in MC 540 uptake appear to be correlated with cellular maturation during normal hemopoiesis, the retention of staining by leukemic cells, some of which appear morphologically normal, may indicate of failure in membrane maturation during leukemic blood cell development.     

Erythropoietic progenitors capable of colony formation in culture: state of differentiation

The differentiated state of mouse erythropoietic progenitor cells (CFU-E), detected by their ability to form erythropoietin-dependent colonies in vitro, has been investigated. Transfusion-induced plethora was found to reduce the population size of CFU-E in both spleen and femoral marrow, which indicates that a significant number of CFU-E arise by differentiation processes that are themselves erythropoietin-dependent. Individual spleen colonies were found to be heterogeneous in their content of CFU-E, and the numbers of CFU-E per colony were not correlated either positively or negatively with the numbers of granulocyte-macrophage progenitors (CFU-C) present in the same colonies. The absence of a negative correlation between CFU-E and CFU-C indicates that the erythropoietic and granulopoietic pathways of differentiation are not mutually exclusive within individual spleen colonies. The numbers of CFU-E per spleen colony were also found to vary independently of the numbers of pluripotent stem cells (CFU-S) per colony; in contrast, as found previously, the numbers of CFU-C and CFU-S per colony were positively correlated. These results indicate that more randomizing events separate CFU-E from CFU-S than separate CFU-C from CFU-S, and are consistent with the view that CFU-E occupy a position on the erythropoietic pathway of differentiation that is more remote from the pluripotent stem cells than is the corresponding position of CFU-C on the granulopoietic pathway.     

Hypergravity-induced immunomodulation in a rodent model: lymphocytes and lymphoid organs

The major goal of this study was to quantify changes in lymphoid organs and cells over time due to centrifugation-induced hypergravity. C57BL/6 mice were exposed to 1, 2 and 3 G and the following assays were performed on days 1, 4, 7, 10, and 21: spleen, thymus, lung, and liver masses; total leukocyte, lymphocyte, monocyte/macrophage, and granulocyte counts; level of splenocyte apoptosis; enumeration of CD3+ T, CD3+/CD4+ T helper, CD3+/CD8+ T cytotoxic, B220+ B, and NK1.1+ natural killer cells; and quantification of cells expressing CD25, CD69, and CD71 activation markers. The data show that increased gravity resulted in decreased body, spleen, thymus, and liver, but not lung, mass. Significant reductions were noted in all three major leukocyte populations (lymphocytes, granulocytes, monocyte/macrophages) [correction of macrphages] with increased gravity; persistent depletion was noted in blood but not spleen. Among the various lymphocyte populations, the CD3+/CD8+ T cells and B220+ B cells were the most affected and NK1.1+ NK cells the least affected. Overall, the changes were most evident during the first week, with a greater influence noted for cells in the spleen. A linear relationship was found between some of the measurements and the level of gravity, especially on day 4. These findings indicate that hypergravity profoundly alters leukocyte number and distribution in a mammalian model and that some aberrations persisted throughout the three weeks of the study. In certain cases, the detected changes were similar to those observed after whole-body irradiation. In future investigations we hope to combine hypergravity with low-dose rate irradiation and immune challenge.     

Studies on the Longevity,Sequestration and Release of the Leukocytes in Chronic Myelogenous Leukemia

The intravascular life-span of leukocytes labelled in vitro with radioactive di-isopropylfluorophosphate was studied in 12 patients with chronic myelogenous leukemia (CML). In relapse, leukocyte specific activity (LSA) disappeared slowly; in remission, LSA curves approached normal and only a small proportion of LSA disappeared slowly. The level of maturation of the leukocytes that persisted in the blood was investigated by a leukocyte fractionation technique which excluded immature myeloid cells from leukocyte samples. The influence of extracorpuscular factors upon the pattern of disappearance of LSA was investigated by means of cross-transfusion experiments, and LSA curves obtained with in vitro and in vivo labelling were compared. The results suggest that: (1) the intravascular life-span of the mature leukemic neutrophil is prolonged in relapse and in remission; (2) intrinsically abnormal leukocytes are sequestered in an extravascular pool(s) but recycling occurs; (3) extracorpuscular factors modify the LSA curves; (4) exchange of leukocytes between intravascular and extravascular pools may not occur in relapse; and (5) the intravascular and extravascular pools constitute a self-sustaining pool(s) not replenished from a non-miscible precursor pool.     

Differing time courses of spleen leukocyte MIF synthesis and cytotoxicity during rejection of a murine lymphoma allograft

In vitro spleen leukocyte MIF synthesis and direct cytotoxicity were studied during growth and rejection of the EL-4 murine lymphoma in allogeneic tumor-bearing BALB/c mice to compare the time courses of these events. Rejection of EL-4 tumors occurred between 6 and 8 flays after intraperitoneal inoculation. Spleen leukocyte cytotoxicity measured by isotope release from chromium-51 labeled EL-4 target cells was first detected on day 6 and was maximal between days 11 and 18. In contrast, spleen leukocyte MIF synthesis stimulated by intact EL-4 cells was sometimes observed on day 11 and was maximal between 18 and 25 days after tumor challenge. These results show that maximal spleen cytotoxicity and MIF synthesis occur after completion of, rather than during ip tumor rejection and, in addition, that these two in vitro lymphocyte responses follow independent, significantly different time courses (P < 0.05). This asynchrony of MIF synthesis and cytotoxicity suggests that these in vitro correlates are mediated by distinct lymphocyte subpopulations.     

Remission of acute myeloid leukemia: studies of in vitro colony formation by bone marrow cells

Human bone marrow contains cells which form leukocyte colonies in semisolid culture media. Each leukocyte colony arises from a single colony-forming cell which is thought to be a unipotential stem cell, and which is subject to regulation in vitro by colony-stimulating factor. In acute myelogenous leukemia variable abnormalities in colony formation by marrow cells occur. Usually colony formation either fails to occur or the colonies that are formed are small and contain fewer than 50 cells. Similar abnormalities have been described in bone marrow dysfunction preceding overt leukemia. Usually remission of leukemia is accompanied by improved cloning by marrow cells. In this study three patients are reported in whom remission was associated with impaired cloning, and one of these patients has remained in continuous remission for a further 18 months. These observations suggest that remission status is not necessarily associated with repopulation of the bone marrow by normal hematopoietic cells.     

Suppression of the mixed leukocyte response and of graft-vs-host disease by spleen cells following total lymphoid irradiation (TLI)

Total lymphoid irradiation (TLI) was administered to mice as 17 fractions of 200 rads delivered to the major lymphoid organs. Spleen cells capable of suppressing the in vitro mixed leukocyte response (MLR) and in vivo graft-vs-host disease (GVHD) were found in mice after treatment with TLI. Suppression was not antigen specific and was markedly reduced by treatment of the spleen cells with anti-Thy-1.2 antiserum and complement. Suppressor activity declined with time after irradiation and disappeared within 30 to 40 days. The evidence suggests that the suppressor cells may prevent initial BM rejection and acute GVHD in allogeneic BM transplant recipients prepared with TLI.     

Splenic granulopoietic morphology in laboratory rats after experimental induction of marrow hypoplasia

Within the granulopoietic recovery phase the proportion of neutrophils and their precursors in the spleen was elevated from 1.7 +/- 0.3% (controls) to 14.7 +/- 2.2% (on day 14 after cyclophosphamide). Splenic granulopoiesis persisted for a time after the appearance of marrow recovery. These data suggest that the rat adult spleen can become a markedly granulopoietic organ during the regeneration of granulopoiesis which follows experimentally induced myelosuppression.     

Spleen cells from adult mice given total lymphoid irradiation (TLI) or from newborn mice have similar regulatory effects in the mixed leukocyte reaction (MLR). II. Generation of antigen-specific suppressor cells in the MLR after the addition of spleen cells from newborn mice

Spleen cells from newborn BALB/c mice were added to the mixed leukocyte reaction (MLR) between a variety of responder and stimulator cells. The newborn cells nonspecifically suppressed the uptake of (3H)-thymidine and the generation of cytolytic cells regardless of the responder-stimulator combination used. Suppressor cell activity fell rapidly during the first 4 days after birth, and could not be detected by day 20. Newborn spleen cells inhibited the generation of nonspecific suppressor cells during the MLR but did not inhibit the generation of antigen-specific suppressor cells. Thus, newborn spleen cells exhibit a pattern of regulation of the MLR similar to that reported previously for spleen cells from adult mice given total lymphoid irradiation (TLI). These regulatory interactions provide a model that explains the ease of induction of transplantation tolerance in vivo in newborn mice and in TLI-treated adult mice.     

Effect of Myleran On Murine Hemopoiesis

Time- and dose-dependent patterns of depletion and regeneration of hemopoietic progenitor cells in mouse femora and spleens following treatment with the antileukemic agent Myleran (Busulphan, MY) were studied using the murine spleen colony system and the agar gel in vitro colony system. MY was found to depress granulopoiesis selectively, as manifested by the development of marked prolonged neutropenia, hypoplasia of the bone marrow and (to a lesser degree) of the spleen, reduction of the incidence of multipotential hemopoietic progenitor cells (CFU-S) and of granulocytic progenitor cells (CFU-C) in both femora and spleens, and impairment of the capacity of CFU-S from either tissue to generate granulocytic colonies in the spleens of irradiated hosts. the severity and duration was greatest at high dose levels of MY (800 μ). the action of MY on CFU-S was more pronounced than that on CFU-C, suggesting that MY is a cycle-independent agent. Repopulation of the CFU-C pool preceded that of the CFU-S pool. Development of neutropenia and maximal marrow hypoplasia followed the onset of depression of CFU-S and CFU-C incidence, while recovery of normal nucleated cellularity in the blood, femur and spleen preceded repopulation of the CFU-S and CFU-C pools. MY treatment resulted in transitory stimulation of colony stimulating factor (CSF) generation by the femur but had no effect on serum CSF levels. the peak of femoral CSF generation coincided with the nadir of CFU-C depression. These findings indicated that the prolonged neutropenia following MY treatment was secondary to depletion of the progenitor cell pools, that during recovery granulopoietic repopulation took precedence over self-maintenance of the hemopoietic progenitor cell pools, and that increased generation of CSF may play a role in the early phase of granulopoietic recovery.     

Regulation of cell-mediated cytotoxicity. II. Synergism of two types of thymus dependent cells in vivo.

Sublethal (500 rads) doses of radiation given to mice before the intravenous injection of allogeneic spleen cells induced the development of an increased cell-mediated cytotoxicity (CMC) of the recipients' spleen cells. The effector cells from the irradiated animals were shown to carry the θ alloantigenic marker and to be capable of transferring adoptive immunity in vivo. On the other hand, irradiation of mice with the same dose before the administration of skin or tumor allografts induced a suppression of CMC. The response of irradiated mice treated with tumor allografts was restored with small numbers of spleen or lymph node cells from syngeneic or semi-allogeneic F₁ hybrid donors. With the use of the appropriate cytotoxic alloantisera, it was demonstrated that the majority of the effector cells generated in the spleens of mice restored with semiallogeneic cells were of host origin. These results demonstrate that the precursors of the cytotoxic lymphocytes are radioresistant and indicate that for their stimulation some radiosensitive T cells are necessary to amplify their reaction to nonlymphoid allografts. Allogeneic lymphoid cells, on the other hand, supply a stimulus which does not require the intervention of such amplifier cells. In this case, irradiation induces a stronger CMC response probably by inactivating radiosensitive cells with suppressor activity.     

A cytological study of the capacity for differentiation of normal hemopoietic colony-forming cells

A two-stage procedure has been used to obtain hemopoietic spleen colonies derived from single precursor cells containing radiation-induced chromosomal markers. Of a total of 46 colonies examined, 17 were found to contain cells with abnormal karyotypes. In each of the 17 marked colonies, 90% or more of the dividing cells in the colony carried the same marker. Cell suspensions prepared from each of the individual colonies were tested for their content of dividing cells possessing recognizable differentiated functions. Metaphase cells with peroxidase-positive granules in their cytoplasm were considered to be members of the granulopoietic series, while metaphase cells which contained Fe⁵⁵ were considered to be members of the erythropoietic series. Results were obtained for 12 of the marked colonies, and in nine of these, the percentage of metaphases lacking the marker was less than the percentage of metaphases which were scored as erythropoietic, and also was less than the percentage of metaphases scored as granulopoietic. This is the result which would be expected if the marker were present in both erythropoietic and granulopoietic cells. These results provide support for the view that colony forming hemopoietic stem cells are multipotent, and that differentiation along more than one pathway can occur during the formation of macroscopic splenic colonies.     

Treatment of NZB/NZW mice with total lymphoid irradiation: long-lasting suppression of disease without generalized immune suppression

We used total lymphoid irradiation (TLI; total dose = 3400 rad) to treat the lupus-like renal disease of 6-mo-old female NZB/NZW mice. Similar to our past studies, this treatment resulted in a marked prolongation of survival, decrease in proteinuria, and decrease in serum anti-DNA antibodies compared with untreated littermate controls. Although there was no evidence of disease recurrence in TLI-treated mice until after 12 mo of age, the in vitro proliferative response to phytohemagglutinin by NZB/NZW spleen cells recovered within 6 wk such that responses were greater than control NZB/NZW animals. A similar recovery and overshoot after TLI were evident in the primary antibody response to the T cell-dependent antigen sheep red blood cells (SRBC). Both the total and IgG anti-SRBC antibody responses after TLI were greater than those of untreated NZB/NZW controls, and were comparable with those of untreated non-autoimmune mice. Despite this increased response to mitogens and antigens after TLI, we noted a decrease in spontaneous splenic IgG-secreting cells and a decrease in IgG but not IgM antinuclear antibody production. Nonspecific suppressor cells of the mixed leukocyte response were detectable in the spleens of NZB/NZW mice early after TLI. However, the disappearance of suppressor cells was not associated with recrudescence of disease activity. Furthermore, transfer of large numbers of spleen cells from TLI-treated NZB/NZW mice did not result in disease suppression in untreated age-matched recipients. In summary, treatment of NZB/NZW mice with TLI results in a prolonged remission in autoimmune disease, which is achieved in the absence of generalized immunosuppression.     

Effect of low-dose whole-body irradiation on granulopoietic progenitor cell subpopulations: implications for CFUc release

Abstract. The reaction of the granulopoietic system to whole body irradiation with 0.80 and 1.60 Gy was studied in dogs by means of colony formation assays in combination with velocity sedimentation and tritiated thymidine cytocidal techniques. Depression of circulating CFUc was associated with a marked shift in the size distribution of granulopoietic progenitors in the bone marrow. This effect lasted much longer in those animals receiving the lower X-ray dose. We conclude that circulating CFUc are not a random proportion of the bone marrow but a subpopulation of cells which are smaller in size. These cells are in an equilibrium with the larger marrow CFUc, which is very sensitive to perturbations.     

Evaluation of the marrow culture on the macrophage layer covering intraperitoneally implanted membrane as an assay for hemopoietic progenitor cells

When cellulose acetate membranes are implanted into abdominal cavity of mice they turn into a foreign body overgrown with macrophages. Such macrophage layer has been shown by other authors to be able to support the growth of hemopoietic colonies formed by intraperitoneally injected hemopoietic cells. This study confirms and extends this observation by showing that both granulopoietic and erythropoietic colonies may be observed. The number of colonies grown is in linear correlation with that of injected hemopoietic cells. The frequency of erythropoietic colonies was greatly enhanced by blood letting of the host mice. Colony forming cells were most numerous in the bone marrow then in the spleen and peripheral blood and hardly in the thymus. Prior irradiation of the host mice was essential for obtaining colony growth and the optimal dose was determined to be 6.0 Gy. This technique opens the way to studies into hemopoietic progenitor cells for laboratories having no sophisticated tissue culture equipment and where necessary reagents are easily available.     

Effect of myleran on murine hemopoiesis. I. Granulocytic cell line specificity of action on progenitor cells.

Time- and dose-dependent patterns of depletion and regeneration of hemopoietic progenitor cells in mouse femora and spleens following treatment with the antileukemic agent Myleran (Busulphan, MY) were studied using the murine spleen colony system and the agar gel in vitro colony system. MY was found to depress granulopoiesis selectively, as manifested by the development of marked prolonged neutropenia, hypoplasia of the bone marrow and (to a lesser degree) of the spleen, reduction of the incidence of multipotential hemopoietic progenitor cells (CFU-S) and of granulocytic progenitor cells (CFU-C) in both femora and spleens, and impairment of the capacity of CFU-S from either tissue to generate granulocytic colonies in the spleens of irradiated hosts. The severity and duration was greatest at high dose levels of MY (800 microgram). The action of MY on CFU-S was more pronounced than that on CFU-C, suggesting that MY is a cycle-independent agent. Repopulation of the CFU-C pool preceded that of the CFU-S pool. Development of neutropenia and maximal marrow hypoplasia followed the onset of depression of CFU-S and CFU-C incidence, while recovery of normal nucleated cellularity in the blood, femur and spleen preceded repopulation of the CFU-S and CFU-C pools. MY treatment resulted in transitory stimulation of colony stimulating factor (CSF) generation by the femur but had no effect on serum CSF levels. The peak of femoral CSF generation coincided with the nadir of CFU-C depression. These findings indicated that the prolonged neutropenia following MY treatment was secondary to depletion of the progenitor cell pools, that during recovery granulopoietic repopulation took precedence over self-maintenance of the hemopoietic progenitor cell pools, and that increased generation of CSF may play a role in the early phase of granulopoietic recovery.     

Molecular cloning,chromosome mapping and characterization of the mouse CRTH2 gene,a putative member of the leukocyte chemoattractant receptor family

We have cloned the mouse CRTH2 (chemoattractant receptor-homologous molecule expressed on TH2 cells) gene encoding a putative leukocyte chemoattractant receptor, of which human homologue is expressed selectively in Th2 but not in Th1 clones among T cell clones. The deduced amino-acid sequence of mouse CRTH2 bears 77% identity with its human homologue. Phylogenetic analysis suggested that both mouse and human CRTH2 are closely related to the N-formyl peptide receptor and the C5a receptor among leukocyte chemoattractant receptors. The mouse CRTH2 gene was mapped on chromosome 19c with FISH, where no other genes for leukocyte chemoattractant receptors are mapped. RT–PCR analysis revealed that mouse CRTH2 mRNA is expressed in various cell lineages, including both hematopoietic and non-hematopoietic cell lines. Expression was also observed in liver, lung, kidney, brain, heart, thymus, and spleen. These results suggest that mouse CRTH2 functions in a variety of cells, making the effects of CRTH2 pleiotropic.