Kinetics of haemopoietic recovery in endotoxin-treated mice.

Kinetics of mouse spleen colony forming units were studied after intra-peritoneal injection of 1 mug/blody weight bacterial endotoxin S. typhosa. When these mice were used as unirradiated and sublethally irradiated donors, it was possible to study the effect of the endotoxin injection upon the cells.Use of the treated mice as irradiated recipients of normal cells gave information about the host effect. In treated unirradiated mice, the total nucleated cell and the CFU counts were disturbed, and 2 days later a large fraction of the CFU were found in the DNA synthesis (S) phase. This meant that injection of endotoxin generated factors affecting the kinetics of the CFU and triggering the resting CFU into the proliferative cycle. If then the mice were given supralethal irradiation and used as recipients of normal bone marrow cells, more CFU seeded to the spleen as compared to normal recipients; but the dip and the growth rate of the CFU were not changed. Hence the endotoxin-generated factors had been eliminated in 2 days. A total body sublethal irradiation by 400 rad X-ray 2 days after endotoxin injection reduced the post-irradiation dip in the recovery curve of the CFU, indicating that though the factors affecting the cell kinetics had been eliminated, the cycling CFU behaved like a growing population. During the first week, the growth rate of the CFU remained the same as in control irradiated mice. The growth rate of the spleen CFU of the endotoxin-treated mice slowed down during the second week, and their self-replicating ability was low. Fluctuations in the DNA synthesizing fraction of the spleen CFU suggested a variability in the ratio of the length of the S phase and the cell generation time.     

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.     

THE ROLE OF BONE MARROW OF X-IRRADIATED MICE IN THYMIC RECOVERY

The influence of the bone marrow on the repopulation of the thymus in X-irradiated mice has been investigated.
It was observed that the thymus and a certain population of bone marrow lymphocytic cells were repopulated in parallel in a cyclic fashion. This occurred either after a single exposure of mice to 400 R or after serial weekly X-ray treatments with 170 R. Lethally irradiated recipients which were grafted with bone marrow cells obtained 12-24 days after four weekly irradiations of donor mice with 170 R also exhibited a cyclic repopulation of both the thymus and the bone marrow lymphocytic population. In contrast, mice which were transplanted with bone marrow cells from unirradiated donors, containing an equal number of stem cells (CFU), exhibited a continuous rather than a cyclic recovery of both cell populations. the bone marrow stem cells of mice recovering from X-irradiation were found to have a decreased proliferative activity, since they produced significantly smaller spleen colonies in lethally irradiated recipients than marrow cells from unirradiated mice.
The results were interpreted as indicating that the bone marrow lymphocytic cells may act as thymic precursor cells and that thymic lymphopoiesis is dependent on the presence of such cells. Evidently, the production of lymphocytic cells will decrease when the stimulus for granulocyte production increases due to the limited proliferative activity of the surviving bone marrow stem cells after irradiation. This may result in a cyclic variation of the production of bone marrow lymphocytic cells and it follows that thymic lymphopoiesis will run parallel.     

Factors influencing hematopoietic spleen colony formation in irradiated mice. IV. The effect of erythropoietic stimuli

A variety of erythropoietic stimuli influenced the number of endogenous spleen colonies in irradiated mice and the number of transplantable colony forming cells in the spleen and marrow of unirradiated mice. Bleeding was the most effective stimulus. Bleeding before irradiation resulted in a 30-fold increase in endogenous spleen colonies and in increases in spleen weight, spleen iron and iododeoxyuridine uptake and volume of packed red cells ten days after irradiation. Bleeding unirradiated mice produced a 10-fold increase in the number of transplantable colony forming cells in the spleen and a slight decrease in the total number in the humerus. Bleeding before irradiation resulted in a significant reduction in 30-day post irradiation deaths, an effect abolished by splenectomy. Plasma from bled mice induced an increase in endogenous colonies when injected before irradiation into normal mice. Injection of erythropoietin, testosterone or testosterone plus cobalt induced effects which were, in general, qualitatively similar to those of bleeding, although they were less effective quantitatively. Except for a slight effect induced by ten injections of erythropoietin, post-irradiation stimulation in normal mice proved ineffective. Erythropoietin increased colony numbers and spleen iron uptake when given after irradiation to hypertransfused mice. The results of these studies do not support the concept that the colony forming cell and the erythropoietin sensitive cell are separate entities.     

Genetically determined resistance to foreign bone marrow transplantation in mice: characterization of the effector cells

Mice of most strains show a genetically determined ability to reject a variety of foreign marrow grafts even after lethal irradiation. The phenomenon is both host strain and donor marrow graft-dependent. To characterize the effector cell responsible for graft rejection, attempts were made to 1) determine to what morphologic subclass it belongs; 2) determine its life span; and 3) establish whether genetically different host environments influence the functioning of the effector cell. Mice of the 129/J strain (normally nonresistant), C57BL/6 strain (made non-resistant), and the homozygous mutants of C57BL/6, i.e., C57BL/6 (bg/bg), were recipients of C57BL/6 marrow or spleen cells. After lethal irradiation, hosts were given marrow or spleen cells from normal, strongly resistant C57BL/6 donors pretreated with a) 950 R whole body irradiation or b) twice daily injections for 4 days of the cell cycle toxic drug hydroxyurea followed by 950 R. In other cases, hosts were recipients of the lymphoid cell-rich fraction of marrow from irradiated C57BL/6 donors or adherent cells taken from cultures of marrow cells of unirradiated C57BL/6 donors. Three hours after receiving C57BL/6 marrow or spleen cells, irradiated hosts were given allogeneic DBA/2 marrow (always strongly rejected by C57BL/6 mice and always accepted by 129/J strain mice). Seven days later, host spleens were removed and the numbers of microscopic colonies were counted from subserial sections. The results demonstrate that 1) mice either normally or rendered nonresistant to a marrow allograft can be made to develop resistance by the administration of either whole spleen cells or marrow lymphoid cells from lethally irradiated strongly resistant donors; 2) adherent cells from cultures of marrow from strongly resistant mice are ineffective in conferring resistance; 3) the cell effective in conferring resistance has a life span greater than 4 but less than 7 days; and 4) the effector cell can function in genetically different environments of nonresistant strains.     

RECOVERY IN THE MOUSE SPLEEN AFTER SUBLETHAL IRRADIATION

1. CBA and C57B1 mice were exposed to LD_50/30 doses of γ-radiation and at intervals after irradiation the numbers of haemopoietic colony-forming units (CFU) and of antibody-producing cells in the spleen were determined. 2. CFU repression was observed after transfer of non-irradiated cells to hybrid recipients. However, this repression did not appear until 60 days after irradiation of the donor. 3. The recovery after irradiation of the capacity to inactivate CFUs in spleen cell mixtures was studied. The results suggested that cells which produce 19S antibody are different from those which are reactive against foreign cells.     

Defective transient endogenous spleen colony formation in S1/S1d mice.

WCB6F1 mice of the genotype S1/S1d did not form transient 5-day endogenous spleen colonies following midlethal irradiation, either spontaneously or in response to postirradiation bleeding. Their hematologically normal (+/+) littermates produced colonies equivalent in number and morphologic type to a normal strain (D2B6F1), as evaluated by both macroscopic and microscopic criteria. Bone marrow cells from S1/S1d mice, when transplanted into lethally irradiated +/+ mice, were able to generate equivalent numbers of transient endogenous spleen colonies (TE-CFUs), as compared to that obtained when syngeneic +/+ marrow cells were injected into lethally irradiated +/+ recipients. A defective growth of an early class of hematopoietic progenitor cells, resulting in the clinical course of the S1/S1d anemia is suggested and confirms previous reports on the microenvironmental nature of this abnormality.     

Augmentation of transfer of experimental autoimmune thyroiditis (EAT) in mice by irradiation of recipients

Experimental autoimmune thyroiditis (EAT) can be adoptively transferred to normal syngeneic recipients using spleen cells from susceptible strains of mice primed in vivo with mouse thyroglobulin (MTg) and lipopolysaccharide (LPS) following in vitro activation of spleen cells by culture with MTg. Irradiation of recipient animals markedly augments the severity of thyroiditis induced in this system. Irradiation of recipients does not alter the time course of the development of thyroiditis, nor does it alter the requirement for both in vivo priming and in vitro activation of spleen cells for the development of EAT. Spleen cells from EAT-resistant strains of mice (e.g., Balb/c) do not induce EAT in irradiated recipients. Irradiated recipients develop significant levels of anti-MTg antibodies while unirradiated recipients have little detectable antibody response. The augmenting effect of irradiation can be substantially reversed by transferring naive spleen cells to recipients prior to the transfer of MTg/LPS-primed in vitro-activated spleen cells. In addition athymic CBA/Tufts nude mice develop more severe EAT than CBA/Tufts nude/+ littermates following transfer of activated CBA/J spleen cells. These data suggest that natural suppressor cells may regulate the development of EAT at the effector cell level.     

Radiation sensitivity of hemopoietic stroma: long-term partial recovery of hemopoietic stromal damage in mice treated during growth

We studied the ability of the hemopoietic organ stroma to recover from damage inflicted by 5 or 7 Gy gamma radiation administered during a period of stromal growth in 4-week-old mice. Irradiation resulted in an immediate depletion of femoral colony-forming fibroblastic progenitors (CFU-F) down to 10-20% of age-matched control values. A full recovery to normal numbers occurred between 120 and 240 days after irradiation and was followed by a secondary decrease 1 year after irradiation. This secondary decrease was accompanied by a decrease in the femoral CFU-S and CFU-C content. Femoral CFU-F attained normal numbers and it was demonstrated to occur from surviving CFU-F and could not be enhanced or prolonged following infusion of unirradiated bone marrow cells after irradiation. During the transient CFU-F recovery the hemopoietic stroma remained severely damaged as judged by the regenerative capacity of spleen and femur stroma after subcutaneous implantation, and the ability of the spleen to accumulate CFU-S in response to lipopolysaccharide injection. We have reported earlier that in similarly irradiated adult mice, no restoration of femoral CFU-F was observed. This difference between 4-week-old and adult mice could not be explained by a difference in in vitro radiosensitivity of CFU-F or in their in vivo regeneration kinetics following irradiation and subsequent lipopolysaccharide injection. We conclude from these observations that the recovery kinetics of the CFU-F population is different in young and adult irradiated mice, infused CFU-F do not contribute to CFU-F regeneration in an irradiated femur, CFU-F are not the sole determinants of stromal regeneration in femur and spleen following irradiation.     

In vitro reactivity of splenic lymphocytes from normal and UV-irradiated mice against syngeneic UV-induced tumors.

Skin tumors induced in mice by chronic ultraviolet (UV) irradiation are highly antigenic and are frequently immunologically rejected upon transplantation to normal syngeneic recipients. In this study we characterized this immune response with an in vitro microcytotoxicity test. Cytotoxic activity was present in the spleen cells of mice given a single injection of syngeneic UV-induced fibrosarcoma cells. After removal of adherent spleen cells, the remaining splenic lymphocytes were specifically cytotoxic for the immunizing tumor and showed no cross-reactivity with other syngeneic UV-induced or methylcholanthrene-induced tumors of similar histologic type. The level of cell-mediated reactivity against UV-induced tumors was quite high compared to that obtained with syngeneic tumors induced by methylcholanthrene, and the cytotoxicity was attributable to a population of theta antigen-bearing lymphocytes. With this in vitro test, we compared the response of normal mice, which reject a syngeneic tumor challenge, with that of UV-irradiated mice, in which the syngeneic UV-induced tumors grow progressively. After tumor cell inoculation, lymphocytes form the unirradiated (regressor) mice showed a high degree of cytotoxicity that reached a maximum level 8 days after injection. In contrast, no reactivity could be detected in the spleens of tumor-challenged UV-irradiated (progressor) mice.     

Long-term effects of in vitro sensitization on immune reactivity of lymphocytes: generation of suppressor and helper T cells.

Mouse spleen cells were cultured for 5 days with or without HRBC. Cultured cells were 'parked' in irradiated syngeneic recipients for 3 weeks and then tested for their immunologic reactivity in vitro. We found that spleen cells from recipients of HRBC-sensitized cells (S) as well as spleen cells from recipients of control unsensitized cells (U) possessed radiosensitive suppressor and radioresistant helper activities. Suppressor activity was observed by the capacity of unirradiated S and U spleen cells to inhibit the in vitro generation of IgM and IgG PFC by spleen cells primed in vivo to HRBC or to LacKLH. Helper activity was shown by the capacity of the irradiated S and U cells to restore IgM and IgG PFC responses of in vivo primed, T-depleted spleen cells to HRBC, LacHRBC, and LacCRBC. Both suppressor and helper activities were mediated by T cells. The possibilities that immunologically specific or nonspecific mechanisms account for these phenomena are discussed.     

Residual radiation effect in the murine spleen. In situ measurement by 125-iodo-deoxyuridine retention

Summary To investigate whether residual radiation damage in hematopoietic tissue is measurable in situ by a change in cell turnover, the retention of the thymidine analogue 5-(125-I)iodo-2-deoxyuridine (125-IUdR) following incorporation into DNA of cells in bone marrow and spleen of mice was measured 35 days after 0–500 rad whole body gamma irradiation.In the bone marrow a rapid and a slow turnover component of 125-IUdR retention were found. Both components were almost identical for unirradiated and irradiated mice. In the spleen the 125-IUdR retention curves exhibited three components with increasingly prolonged half-times. In the second component the half-time was longer in irradiated than in unirradiated mice. This was dose-dependent.The increased half-time of 125-IUdR retention in irradiated spleens may be caused by direct cellular damage of long-lived cells (lymphocytes, early hematopoietic progenitor cells) or/and by diminished stimulation of proliferation by microenvironmental or long-range factors.     

Studies of delayed hypersensitivity to L. Monocytogenes in mice: nature of cells involved in passive transfers

C3Hf/Umc mice were immunized by an intravenous injection of a sublethal dose of live Listeria monocytogenes. The animals developed delayed-type hypersensitivity (DH) concomitant with infectious immunity to this organism. Delayed hypersensitivity could be transferred to normal lethally irradiated mice with spleen cells from immune animals. The immune cells cells responsible for transfer of adoptive immunity were susceptible to in vitro cytolytic action of anti-theta iso-antibody and complement, since such treatment rendered these cells incapable of further passive transfer of specific immunity to Listeria. The acquired DH to Listeria persisted in mice after 900 R lethal irradiation, provided normal syngeneic bone marrow cells were also administered, thus indicating the persistance of a cell population in the immune irradiated mice, resistant to effects of radiation. The radio resistant nature of this immune cell population was further demonstrated by passive transfer with spleen cells, derived from preimmunized lethally irradiated mice to normal syngeneic mice or to lethally irradiated nonimmunized hosts reconstituted with normal bone marrow which then responded to antigenic challenge with DH.Treatment of the immune radio resistant spleen cells in vitro with anti-theta and complement eliminated passive transfers of DH by these cells; however, this effect was less obvious than similar treatment of the immune, nonirradiated, spleen cells.     

Deficits in T helper cells after total lymphoid irradiation (TLI): reduced IL-2 secretion and normal IL-2 receptor expression in the mixed leukocyte reaction (MLR)

Spleen cells from BALB/c mice treated with total lymphoid irradiation (TLI) and from normal, unirradiated mice were compared in the mixed leukocyte reaction (MLR). Although the percentage of CD4+ cells in the spleen was close to normal, 4 to 6 weeks after TLI, the MLR of unfractionated spleen cells from irradiated mice was more than 10-fold lower than controls. A similar reduction was observed when purified CD4+ cells were used as responders in the MLR. Secretion of IL-2 by cells from irradiated mice was also about 10-fold lower than controls. However, the percentage of CD4+ and CD8+ cells which expressed IL-2 surface receptors during the MLR was similar using spleen cells from irradiated and control mice. Addition of an exogenous source of IL-2 restored the proliferative capacity of the irradiated cells and suggests that the lack of IL-2 secretion is the likely explanation of the marked deficit in the MLR of CD4+ spleen cells after TLI.     

Graft-vs-host reactions (GVHR) across minor murine histocompatibility barriers. I. Impairment of mitogen responses and suppressor phenomena

In our laboratory, we have developed a murine model to examine GVHD across minor histocompatibility antigens. In our model, GVHD is induced by injecting B10.D2 spleen cells into irradiated BALB/c recipients. Seven to 10 days after irradiation and injection of cells, there are significant changes in cell function in the recipient spleens. In the B10.D2----BALB/c (600 rad) model, recipient spleen cells are profoundly unresponsive to Con A and LPS stimulation but show increased B cell activity measured by Staphylococcus aureus protein A plaque-forming activity. Spleen cells from such GVH mice profoundly suppress the mitogenic responses of normal BALB/c or B10.D2 spleen cells to Con A and LPS. The degree of impairment of the mitogenic response and the ability to suppress normal cells is proportional to the dose of cells used to induce GVH reactions. Both the inability to respond to mitogens and the capacity to suppress are also related to the dose of irradiation given to the recipients. In addition, immunosuppression across minor histocompatibility antigens shows an unevenhandedness. If we inject parental B10.D2 or BALB/c cells into F1 recipients (P----F1), there is greater inhibition of mitogenic responses when B10.D2 parental cells are given than when BALB/c cells are given to the irradiated F1 recipients. These experiments show that significant immunosuppression occurs during GVH reactions across minor histocompatibility barriers. The degree of suppression varies according to the dose of cells used to induce GVH, the dose of irradiation to the recipient and the "strength" of the GVH recognition system. Such experiments provide models for GVH disease seen in humans who receive treatment for leukemia or other diseases that involves recipient irradiation and infusion of HLA-identical bone marrow.     

Effect of erythrocyte breakdown products on mast cells and erythropoietin formation]

Experiments were conducted on CBA mice and albino rats. A study was made of the effect of erythrocyte destruction products (EDP) on the content of hemopoietic colony-forming units (CFU), differentiation of stem cells and the erythropoietin production. It was shown that 3 or 4 EDP injections to normal mice or to lethally irradiated (1000 rad) mice after the transplantation of bone marrow cells caused no changes in the CFU level of stem cells differentiation. In case of a daily (for 3 days) administration of EDP to mice before the irradiation (1000 rad) and bone marrow transplantation there was observed an increase of the colonies count in the recipients' spleen on account of the erythroid colonies. EDP injection caused no changes in the erythropoietic activity of the blood serum. A possible role of erythrocyte destruction products in the mechanism of erythropoiesis autoregulation is discussed.     

Further studies on the biological properties of Friend virus-induced leukemic cells differentiating along the erythrocytic pathway

When Friend virus-induced leukemic cell lines were injected into irradiated hosts after the second radiation dose, the colony-forming unit (CFU) in the recipient spleens per 10⁴ cells was found to be 7-fold higher than the CFU obtained when the second radiation dose had been given shortly after the inoculation of the cells. Serial passage of the cells from the spleen colonies to irradiated hosts resulted in a marked increase of the CFU value, indicating that this cell population was capable of both self-replication and erythroid differentiation. The “f” fraction, which indicates the percentage of the inoculated cells that reach the spleen in the irradiated recipients, was found to be approximately 15%. If the highest CFU value obtained from serial colony-to-colony passages is corrected by this factor, a final cloning efficiency of about 18% is demonstrated. Neither induced plethora nor the administration of erythropoietin (1 u/mouse/for 2 days) appeared to affect the spleen colony-forming ability of the leukemic cells. Erythroid differentiation is not detectable in the transplantable subcutaneous tumors which were used to initiate the tissue culture lines and which also are capable of inducing erythroid spleen clones in irradiated recipients. This lends support to the theory of the influence of “microenvironmental factors” on the fate of stem cells with potential for differentiation.     

Radioprotection of mice with interleukin-1: relationship to the number of spleen colony-forming units

Compared to saline-injected mice 9 days after 6.5 Gy irradiation, there were twofold more Day 8 spleen colony-forming units (CFU-S) per femur and per spleen from B6D2F1 mice administered a radioprotective dose of human recombinant interleukin-1-alpha (rIL-1) 20 h prior to their irradiation. Studies in the present report compared the numbers of CFU-S in nonirradiated mice 20 h after saline or rIL-1 injection. Prior to irradiation, the number of Day 8 CFU-S was not significantly different in the bone marrow or spleens from saline-injected mice and rIL-1-injected mice. Also, in the bone marrow, the number of Day 12 CFU-S was similar for both groups of mice. Similar seeding efficiencies for CFU-S and percentage of CFU-S in S phase of the cell cycle provided further evidence that rIL-1 injection did not increase the number of CFU-S prior to irradiation. In a marrow repopulation assay, cellularity as well as the number of erythroid colony-forming units, erythroid burst-forming units, and granulocyte-macrophage colony-forming cells per femur of lethally irradiated mice were not increased in recipient mice of donor cells from rIL-1-injected mice. These results demonstrated that a twofold increase in the number of CFU-S at the time of irradiation was not necessary for the earlier recovery of CFU-S observed in mice irradiated with sublethal doses of radiation 20 h after rIL-1 injection.     

MULTIPLICATION OF HAEMOPOIETIC COLONY FORMING UNITS (CFU) IN MICE AFTER X-IRRADIATION AND BONE MARROW TRANSPLANTATION

Kinetics of the multiplication of haemopoietic CFUs was studied in lethally irradiated mice receiving various numbers of syngeneic bone marrow cells. After transplantation of a small number of bone marrow cells, the growth rate of CFU in femoral bone marrow appeared to decrease after about 10 days after transplantation, before the normal level of CFU in the femur was attained. In the spleen it was found that the overshoot which was observed about 10 days after transplantation of a large number of bone marrow cells is smaller or absent when a small number of cells is transplanted. Experiments dealing with transplantation of 50 x 10⁶ bone marrow cells 0, 4 or 10 days after a lethal irradiation indicated that the decline in growth rate of CFUs about 10 days after irradiation could not be attributed to environmental changes in the host.
The results are explained by the hypothesis that a previous excessive proliferation of CFUs diminishes the growth rate thereafter. This hypothesis is supported by experiments in which 50 x 10⁶ bone marrow cells derived from normal mice or from syngeneic chimaeras were transplanted. The slowest growth rate was observed when bone marrow that had been subjected to the most excessive proliferation in the weeks preceding the experiment was transplanted.     

STUDY ON THE PROLIFERATIVE STATE OF HAEMOPOIETIC STEM CELLS (CFU)

Hydroxyurea was used to study the proliferation rate of haemopoietic stem cells (CFUJ in normal mice, after irradiation or transplantation into irradiated recipients. It was demonstrated that the proliferation rate of endogenous CFU_S (endo-CFU,) and exogenous CFU_S (exo-CFU_s) are identical. After irradiation (650 R) the surviving endo-CFU_s begin to proliferate immediately. By contrast exo-CFU, transplanted into the irradiated recipient mouse (850 R), begin to proliferate only after about 30 hr. However, injection of isoproterenol (which stimulates adenyl cyclase) or dibutyryl cyclic adenosine 3′,5′-monophosphate shortly after marrow cell graft, triggers the transplanted CFU_S into cell cycle as shown by an almost immediately increased sensitivity to hydroxyurea. Isoproterenol is capable of inducing DNA synthesis also in stem cells of normal mice but it takes about 20 hr before CFU, become to be increasingly sensitive to hydroxyurea.