Role of cytokines (interleukin 1, tumor necrosis factor, and transforming growth factor beta) in natural and lipopolysaccharide-enhanced radioresistance.

Studies of radioresistance and radioprotection provide an excellent in vivo model for dissection of the pathophysiological role of cytokines. The availability of neutralizing antibodies to cytokines has made it possible to assess the contribution of cytokines to host defense and repair processes involved in radioresistance and radioprotection. Administration of anti-interleukin 1 receptor (IL-1R) antibody (35F5) or anti-tumor necrosis factor (TNF) antibody (TN3 19.12) reduced survival of irradiated CD2F1 mice. These results demonstrate conclusively that natural levels of IL-1 and TNF contribute to radioresistance of normal mice. Furthermore, the radioprotective effect of administered IL-1 was blocked not only with anti-IL-1R antibody but also with anti-TNF antibody. Similarly, the radioprotective effect of TNF was reduced with anti-IL-1R antibody. These data suggest that cooperative interaction of both cytokines is necessary to achieve successful radioprotection. Finally, when LPS was used as a radioprotector, the combined administration of anti-IL-1R and anti-TNF not only blocked the radioprotection with LPS, but actually revealed LPS to have a radiosensitizing effect. This effect may be due to induction of TGF-beta, since administration of this cytokine results in reduced survival of irradiated mice.

Endotoxin and tumor necrosis factor challenges in dogs simulate the cardiovascular profile of human septic shock.

Survivors of both human and animal bacterial shock develop a characteristic pattern of progressive changes in cardiovascular function over a period of 7-10 d. In this present study, we examined whether endotoxin (a product of Gram-negative bacteria) or TNF (a cytokine released from macrophages) could reproduce the same complex cardiovascular changes observed in septic shock over a period of 7-10 d. To test this hypothesis, we implanted a thrombin-fibrin clot containing purified endotoxin from E. coli into the peritoneal cavity of eight dogs, and infused TNF into eight different dogs. Over the next 10 d, serial simultaneous heart scans and thermodilution cardiac outputs were performed in these awake nonsedated animals. By day 2 after challenge with either endotoxin or TNF, animals developed a decrease (p less than 0.05) in both mean arterial pressure and left ventricular ejection fraction. With fluid resuscitation, animals manifested left ventricular dilatation (increased [p less than 0.05] end diastolic volume index), increased or normal cardiac index, and decreased or normal systemic vascular resistance index. In surviving animals, these changes returned to normal with 7-10 d. The time course of these changes was concordant (p less than 0.05) with that previously described in a canine model of septic shock using viable bacteria. During the 10-d study, control animals receiving sterile clots or heat-inactivated TNF had not significant changes in hemodynamics. The results from this canine model demonstrate that either endotoxin or TNF alone can produce many of the same hemodynamic abnormalities seen in human septic shock and in a canine septic shock model induced by live bacteria. These findings support the hypothesis that the action of endogenous mediators (TNF) responding to bacterial products (endotoxin) is the common pathway that produces the serial cardiovascular changes found in septic shock.

Detection of gap junctions between the progeny of a canine macrophage colony-forming cell in vitro.

An in vitro monocyte-macrophage colony-forming cell (M-CFC) has been detected in canine bone marrow (BM). The colonies derived from these progenitor cells were similar to murine-derived M-CFC (MacVittie and Porvaznik, 1978, J. Cell Physiol. 97:305--314) colonies, since they showed a singular macrophage line of differentiation, a lag of 14--16 days before initiating colony formation, and they survived significantly longer in culture in the absence of colony-stimulating factor (CSF) than granulocyte-macrophage colony-forming cells (GM-CFC). Endotoxin (Salmonella typhosa lipopolysaccharide W)-stimulated dog serum was used as the CSF (7% vol/vol). Canine-derived M-CFC progeny were identified as macrophages on the basis of morphology, phagocytosis, and the presence of Fc receptors for IgG. Gap junctions were observed only in canine BM, M-CFC-derived colonies using freeze-fracture and lanthanum tracer techniques. They were not observed in any GM-CFC-derived colonies. The number of gap junctions observed in freeze-fracture replicas of BM, M-CFC-derived colonies (21 colonies from three different dogs) showed a significantly positive correlation (Kendall's tau = 0.70, P less than 0.001) with the size of the colony fracture plane area. Gap junctions were observed displaying hexagonal lattices of 9.3 nm +/- 0.08 (SE) particles with a center-to-center spacing of 10.4 nm +/- 1.0 (SE) on membrane P-fracture faces. On membrane E-fracture faces, highly ordered arrays of pits with 8.7 nm +/- 0.12 (SE) center-to-center spacing were observed. Arrays of both particles and pits were also observed in fracture-face breakthroughs within a gap junction. Thus, gap junctions can form in vitro between the cells of macrophage progeny of a canine M-CFC under appropriate growth conditions. The significance of this observation is that there may be a structural basis for cell-to-cell collaboration between BM macrophages and other capable cells that either pass into the tissue for modification or develop there into mature cell forms.

Combined administration of recombinant human megakaryocyte growth and development factor and granulocyte colony-stimulating factor enhances multilineage hematopoietic reconstitution in nonhuman primates after radiation-induced marrow aplasia.

This study compared the therapeutic potential of recombinant, native versus pegylated megakaryocyte growth and development factor (rMGDF and PEG-rMGDF, respectively), as well as that of the combined administration of PEG-rMGDF and r-methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF) on hematopoietic reconstitution after 700 cGy, 60Co gamma, total body irradiation in nonhuman primates. After total body irradiation, animals received either rMGDF, PEG-rMGDF, r-metHuG-CSF, PEG-rMGDF and r-metHuG-CSF or HSA. Cytokines in all MGDF protocols were administered for 21-23 d. Either rMGDF, PEG-rMGDF, or PEG-rMGDF and r-metHuG-CSF administration significantly diminished the thrombocytopenic duration (platelet count (PLT) < 20,000 per microliter)to o.25, 0, 0.5 d, respectively, and the severity of the PLT nadir (28,000, 43,000, and 30,000 per microliter, respectively) as compared with the controls (12.2 d duration, nadir 4,000 per microliter), and elicited an earlier PLT recovery. Neutrophil regeneration was augmented in all cytokine protocols and combined PEG-rMGDF and r-metHuG-CSF further decreased the duration of neutropenia compared with r-metHuG-CSF alone. These data demonstrated that the administration of PEG-rMGDF significantly induced bone marrow regeneration versus rMGDF, and when combined with r-metHuG-CSF significantly enhanced multilineage hematopoietic recovery with no evidence of lineage competition.

Gram-negative bacteremia produces both severe systolic and diastolic cardiac dysfunction in a canine model that simulates human septic shock.

A canine sepsis model that simulates the human cardiovascular response to septic shock was produced in 10 conscious unsedated dogs by implanting an Escherichia coli-infected clot into the peritoneum, resulting in bacteremia. By employing serial, simultaneous measurements of radionuclide scan-determined left ventricular (LV) ejection fraction (EF) and thermodilution cardiac index (CI), the end-diastolic volume index (EDVI) was calculated (EDVI = stroke volume index divided by EF). By using three different methods of quantifying serial ventricular performance (EF, shifts in the Starling ventricular function curve using EDVI vs. stroke work index, and the ventricular function curve response to volume infusion), this study provides evidence (P less than 0.01) that septic shock produces a profound, but reversible, decrease in systolic ventricular performance. This decreased performance was not seen in controls and was associated with ventricular dilatation (P less than 0.01); the latter response was dependent on an adequate volume infusion. Further studies of EDVI and pulmonary capillary wedge pressure during diastole revealed a significant, though reversible, shift (P less than 0.001) in the diastolic volume/pressure (or compliance) relationship during septic shock.

Role of endotoxemia in cardiovascular dysfunction and mortality. Escherichia coli and Staphylococcus aureus challenges in a canine model of human septic shock.

Using different types of bacteria and a canine model simulating human septic shock, we investigated the role of endotoxin in cardiovascular dysfunction and mortality. Either Escherichia coli (a microorganism with endotoxin) or Staphylococcus aureus (a microorganism without endotoxin) were placed in an intraperitoneal clot in doses of viable or formalin-killed bacteria. Cardiovascular function of conscious animals was studied using simultaneous radionuclide heart scans and thermodilution cardiac outputs. Serial plasma endotoxin levels were measured. S. aureus produced a pattern of reversible cardiovascular dysfunction over 7-10 d that was concordant (P less than 0.01) with that of E. coli. Although this cardiovascular pattern was not altered by formalin killing (S. aureus and E. coli), formalin-killed organisms produced a lower mortality and less myocardial depression (P less than 0.01). S. aureus, compared to E. coli, produced higher postmortem concentrations of microorganisms and higher mortality (P less than 0.025). E. coli produced significant endotoxemia (P less than 0.01), though viable organisms (versus nonviable) resulted in higher endotoxin blood concentrations (P less than 0.05). Significant endotoxemia did not occur with S. aureus. Thus, in the absence of endotoxemia, S. aureus induced the same cardiovascular abnormalities of septic shock as E. coli. These findings indicate that structurally and functionally distinct microorganisms, with or without endotoxin, can activate a common pathway resulting in similar cardiovascular injury and mortality.

Organ Doses Associated with Partial-Body Irradiation with 2.5% Bone Marrow Sparing of the Non-Human Primate: A Retrospective Study.

A partial-body irradiation model with approximately 2.5% bone marrow sparing (PBI/BM2.5) was established to determine the radiation dose-response relationships for the prolonged and delayed multi-organ effects of acute radiation exposure. Historically, doses reported to the entire body were assumed to be equal to the prescribed dose at some defined calculation point, and the dose-response relationship for multi-organ injury has been defined relative to the prescribed dose being delivered at this point, e.g., to a point at mid-depth at the level of the xiphoid of the non-human primate (NHP). In this retrospective-dose study, the true distribution of dose within the major organs of the NHP was evaluated, and these doses were related to that at the traditional dose-prescription point. Male rhesus macaques were exposed using the PBI/BM2.5 protocol to a prescribed dose of 10 Gy using 6-MV linear accelerator photons at a rate of 0.80 Gy/min. Point and organ doses were calculated for each NHP from computed tomography (CT) scans using heterogeneous density data. The prescribed dose of 10.0 Gy to a point at midline tissue assuming homogeneous media resulted in 10.28 Gy delivered to the prescription point when calculated using the heterogeneous CT volume of the NHP. Respective mean organ doses to the volumes of nine organs, including the heart, lung, bowel and kidney, were computed. With modern treatment planning systems, utilizing a three-dimensional reconstruction of the NHP's CT images to account for the variations in body shape and size, and using density corrections for each of the tissue types, bone, water, muscle and air, accurate determination of the differences in dose to the NHP can be achieved. Dose and volume statistics can be ascertained for any body structure or organ that has been defined using contouring tools in the planning system. Analysis of the dose delivered to critical organs relative to the total-body target dose will permit a more definitive analysis of organ-specific effects and their respective influence in multiple organ injury.

Rat monocytes in a model of combined injury express the OX8 antigen.

We have analyzed peripheral blood mononuclear cell preparations from a rat model of combined injury (CI) [whole-body irradiation (500 cGy 60Co) followed by a thermal injury (20% body surface area, dorsal, scald burn)] for the expression of OX8 antigens. Ficoll-separated mononuclear fractions were labeled with monoclonal antibodies MRC OX8, MRC OX19, W3/13 HLK, or W3/25 for flow cytometric analysis. Combined-injury trauma resulted in decreased mononuclear cells to 6% of normal. This effect was due to the rapid decrease in radiosensitive lymphocytes from 83% to 10%. The relative numbers of monocytes increased from a normal 13% to 70% at day 4 after CI. Labeling of cells with OX8 after CI shifted to a population which was significantly larger in volume than normal lymphocytes. At the same time the mean fluorescence intensity of OX8-positive cells was considerably reduced. With the use of a F(ab) fragment of OX8 as a probe, these results could be partially explained as unspecific binding of the whole molecule of OX8 to Fc receptors expressed by activated monocytes. But double-labeling and cell-sorting experiments also revealed the expression of OX8 antigens by a subset of OX8+/OX19- monocytes after CI.

Glucan: mechanisms involved in its "radioprotective" effect.

It has generally been accepted that most biologically derived agents that are radioprotective in the hemopoietic-syndrome dose range (eg, endotoxin, Bacillus Calmette Guerin, Corynebacterium parvum, etc) exert their beneficial properties by enhancing hemopoietic recovery and hence, by regenerating the host's ability to resist life-threatening opportunistic infections. However, using glucan as a hemopoietic stimulant/radioprotectant, we have demonstrated that host resistance to opportunistic infection is enhanced in these mice even prior to the detection of significant hemopoietic regeneration. This early enhanced resistance to microbial invasion in glucan-treated irradiated mice could be correlated with enhanced and/or prolonged macrophage (but not granulocyte) function. These results suggest that early after irradiation glucan may mediate its radioprotection by enhancing resistance to microbial invasion via mechanisms not necessarily predicated on hemopoietic recovery. In addition, preliminary evidence suggests that glucan can also function as an effective free-radical scavenger. Because macrophages have been shown to selectively phagocytize and sequester glucan, the possibility that these specific cells may be protected by virtue of glucan's scavenging ability is also suggested.

Population sizes of granulocyte-macrophage and monocyte-macrophage colony-forming cells in Sl/Sld.

The sizes of granulocyte-macrophage (CFU-c) and monocyte-macrophage (CFC) progenitor cell populations were measured and compared in Sl/Sld and +/+ mice. In the marrow, the CFU-c and CFC population sizes were 40% and 67%, respectively, of the +/+ marrow CFU-c and CFC population sizes. There was no difference in the size of these two-cell populations in the spleens or thymi of Sl/Sld mice as compared with +/+ mice. Furthermore, the marrow CFC population was found to be heterogeneous by velocity sedimentation. One of the marrow CFC subpopulations is characterized by a velocity sedimentation value of 5.05 mm/h while the other has a value of 6.30 mm/h.

Endotoxin-induced alterations in canine granulopoiesis: colony-stimulating factor, colony-forming cells in culture, and growth of cells in diffusion chambers.

Salmonella typhosa endotoxin injected into dogs produced elevated plasma CSF levels, transient leukopenia followed by leukocytosis, and stimulation of marrow granulopoiesis and mobilization of granulocyte-macrophage progenitors cells into the the peripheral circulation. The number of marrow CFU-c decreased to 65% of the control number within 6 h, returned to control levels by 24 h, and increased to 370% of the control number by 48h after endotoxin. The granulopoietic response was supported by a concomitant increase in the M:E ratio, an increased fraction of marrow-derived CFU-c susceptible to 3H-TdR suicide, and increased granulo-monocytopoietic activity of marrow- and peripheral blood-derived cells grown in diffusion chamber cultures. These results are consistent with the concept that endotoxin-induced CSF is a physiologic regulator of canine granulopoiesis, and that canine marrow responds to endotoxin with a significant increase in the concentration of marrow-derived granulocytic progenitors and with mobilization of granulocyte-macrophage progenitors into the peripheral circulation.

Canine granulopoiesis: alterations induced by suppression of gram-negative flora.

We investigated alterations in canine granulopoiesis following suppression of bacterial flora by antibiotic treatment. Beagles were decontaminated in sterile isolation with laminar air flow, by an antifungal and antibiotic regimen. Skin and fecal specimen cultures were usually negative after 4 days. The reduction of gram-negative bacteria resulted in a significant decrease in plasma colony-stimulating factor (CSF) levels, marrow CFU-c concentration, and the cytopoietic activity of marrow-derived diffusion chamber (DC) progenitor cells. The period of conventionalization was characterized by a significant although delayed increase in plasma CSF as well as marked increases in marrow CFU-c concentration and cytopoietic activity of marrow DC progenitors. There was also mobilization of marrow DC progenitors into the circulation. All parameters returned to control values within 7 days of conventionalization. These data supported the hypothesis that the gram-negative bacteria of the gut play a significant role in the regulation of normal canine granulopoiesis.

Hemopoiesis in the splenectomized-pregnant mouse following low-dose total-body irradiation.

The effect of splenectomy (SPLX) and total-body irradiation (TBI) (50-200 rad) on virgin and pregnant mouse hemopoiesis was studied, using peripheral blood hemogram values and femoral marrow hemopoietic progenitor cell activity (i.e., CFUE, BFUE, and GM-CFC). The SPLX-maternal red cell counts and hematocrit values were lower than those of SPLX-virgin mice, reflecting the anemia of pregnancy. But the white cell counts of both SPLX-virgin and SPLX-day-14.5 pregnant mice were significantly higher (P less than 0.005) than normal-virgin mice. Both nonirradiated and day-4 irradiated SPLX-maternal marrow Ep-independent and Ep-dependent CFUE were higher than the nonirradiated and day-4 irradiated SPLX-virgin values (respectively, for each TBI dose studied). On the other hand, nonirradiated and day-4 irradiated SPLX-maternal GM-CFC were lower than the nonirradiated and day-4 irradiated SPLX-virgin GM-CFC values. The data demonstrate the potential of the SPLX-maternal femoral marrow to respond to the stress of low-dose TBI with effective compensatory erythropoiesis, possibly at the expense of granulopoiesis.

Hematopoietic response of splenectomized C3HeB/FeJ and C3H/HeJ mice to lipopolysaccharide.

The paired, inbred mouse strains C3HeB/FeJ and C3H/HeJ differ in their extramedullary hemopoietic response to lipopolysaccharide (LPS). The C3H/HeJ exhibit reduced responses relative to the C3HeB/FeJ in terms of colony-stimulating factor, endogenous and exogenous spleen-derived stem cells (CFUS), and spleen-derived granulocyte-macrophage colony-forming cells (GM-CFC) whereas equivocal responses were noted for the marrow-derived CFUS and GM-CFC. In an effort to determine if the mutational defect was specifically limited to extramedullary expression, we utilized C3HeB/FeJ mice and C3H/HeJ mice at 6 weeks postsplenectomy. The splenectomy emphasizes the medullary response to an i.p. injection of 10 microgram Escherichia coli, LPS-W. Significant differences were revealed in the responses of C3HeB/FeJ and C3H/HeJ marrow-derived cells. Total cells, CFUS, GM-CFC, and the macrophage colony-forming cell (M-CFC) in the C3HeB/FeJ strain all decreased significantly from their saline-injected control values as well as from their counterpart C3H/HeJ values within 48 h after injection of LPS-W. The decline in C3HeB/FeJ CFUS, GM-CFC, and M-CFC was followed by an overshoot, with subsequent return to control values. The total nucleated cells, CFUs, GM-CFC, and M-CFC derived from the C3H/HeJ marrow were characteristically nonresponsive and never varied significantly from their saline-injected control values over the observation period. These results suggest that the phenotypic effect of the defective gene can extend to all hemopoietic tissue, medullary and extramedullary, that normally respond to the factors released through expression of the LPS locus.

CFUs kinetics observed in vivo by bromodeoxyuridine and near-UV light treatment.

Using continuous BrdUrd infusion in vivo and near-UV light irradiation in vitro, we have measured the time integrated S-phase fraction of CFUs derived from murine bone marrow. These measurements indicate a stochastic commitment to cycle of essentially the entire population of CFUs, consistent with the existence of a noncycling cell state G0. The data indicate that for B6D2F1 female mice the CFUs are committed to DNA synthesis approximately once every 52 h. Additionally, measurements of other CFUs parameters showed no significant effects of the BrdUrd infusion. Hydroxyurea challenges of BrdUrd-infused mice indicates that the capacity of the CFUs to respond to a proliferative stimulus during the infusion period is also unaffected by the BrdUrd.

Effects of combined administration of interleukin-6 and granulocyte colony-stimulating factor on recovery from radiation-induced hemopoietic aplasia.

Hemopoietic aplasia is the primary limitation of drug and radiation cancer therapies. We have previously demonstrated that, individually, both interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) can accelerate recovery from radiation-induced hemopoietic aplasia. In vitro studies suggest that IL-6 affects cells early in the hemopoietic hierarchy, while G-CSF affects more committed progenitor cells. Because these cytokines may also affect different cell populations in vivo, we hypothesized that the use of these agents in combination may further enhance recovery from hemopoietic aplasia. Female B6D2F1 mice were exposed to a high sublethal 7.75 Gy dose of 60Co radiation. Following irradiation, mice were administered subcutaneous injections of either saline, 500 micrograms/kg of recombinant human IL-6 once daily on days 1-6, 125 micrograms/kg of recombinant human G-CSF once daily on days 1-17, or both cytokines as described. Peripheral white blood cell (WBC), red blood cell (RBC), and platelet (PLT) counts, as well as femoral and splenic granulocyte-macrophage colony-forming cell (GM-CFC) and day-12 spleen colony-forming unit (CFU-S) contents were evaluated on days 7, 10, 14, 17 and 21 postirradiation. IL-6 treatment alone slightly accelerated postirradiation recovery of most hemopoietic parameters, while G-CSF treatment dramatically enhanced recovery of all hemopoietic parameters evaluated. Co-administration of IL-6 and G-CSF further enhanced the hemopoietic recovery. The most notable effects in combination-treated mice were on recoveries of bone marrow and splenic CFU-S, which were significantly enhanced above those in G-CSF-treated irradiated mice as early as day 10 postirradiation. Although by day 14 postirradiation, splenic GM-CFC and CFU-S recoveries in both G-CSF- and combination-treated mice had surpassed unirradiated control values, combination-treated mice exhibited a greater overshoot. These studies demonstrate the ability of IL-6 treatment to enhance G-CSF-mediated acceleration of multilineage recovery following radiation-induced hemopoietic aplasia.

Cell kinetics of GM-CFC in the steady state.

The kinetics of cell turnover for myeloid/monocyte cells that form colonies in agar (GM-CFC) were measured through the progressive increase in their sensitivity to 313-nm light during a period of cell labeling with BrdCyd. Two components of cell killing with distinctly separate labeling kinetics revealed both the presence of two generations within the GM-CFC compartment and the properties of the kinetics of the precursors of the GM-CFC. These precursors of the GM-CFC were not assayable in a routine GM-CFC assay when pregnant mouse uterus extract and mouse L-cell-conditioned medium were used to stimulate colony formation but were revealed by the labeling kinetics of the assayable GM-CFC. Further, these precursor cells appeared to enter the assayable GM-CFC population from a noncycling state. This was evidenced by the failure of the majority of these cells to incorporate BrdCyd during five days of infusion. The half-time for cell turnover within this precursor compartment was measured to be approximately 5.5 days. Further, these normally noncycling cells proliferated rapidly in response to endotoxin. High-proliferative-potential colony-forming cells (HPP-CFC) were tested as a candidate for this precursor population. The results of the determination of the kinetics for these cells showed that the HPP-CFC exist largely in a Go state, existing at an average rate of once every four days. The slow turnover time for these cells and their response to endotoxin challenge are consistent with a close relationship between the HPP-CFC and the Go pool of cells that is the direct precursor of the GM-CFC.

Recovery of hematopoietic colony-forming cells in irradiated mice pretreated with interleukin 1 (IL-1).

Data in this report determined the effect of a single injection of recombinant interleukin 1 alpha (rIL-1) prior to irradiation of B6D2F1 mice on the recovery of colony-forming cells (CFC) at early and late times after sublethal and lethal doses of radiation. Injection of rIL-1 promoted an earlier recovery of mature cells in the blood and CFC in the bone marrow and spleen. For example, 8 days after 6.5 Gy irradiation, the number of CFU-E (colony-forming units-erythroid), BFU-E (burst-forming units-erythroid), and GM-CFC (granulocyte-macrophage colony-forming cells) per femur was approximately 1.5-fold higher in rIL-1-injected mice than in saline-injected mice. Also, 5, 9, and 12 days after irradiation, the number of both day 8 and day 12 CFU-S (colony-forming units-spleen) was almost twofold greater in bone marrow from rIL-1-injected mice. The earlier recovery of CFU-S in rIL-1-injected mice was not associated with an increase in the number of CFU-S that survived immediately after irradiation. Also, 7 months after irradiation, the number of CFU-S per femur of both saline- and rIL-1-injected mice was still less than 50% of normal values. Data in this report demonstrate that a single injection of rIL-1 prior to irradiation accelerates early hematopoietic recovery in irradiated mice, but does not prevent expression of radiation-induced frontend damage or long-term damage to hematopoietic tissues.

Recovery from severe hematopoietic suppression using recombinant human granulocyte-macrophage colony-stimulating factor.

The ability of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) to enhance recovery of a radiation-suppressed hematopoietic system was evaluated in a nonuniform radiation exposure model using the rhesus monkey. Recombinant human GM-CSF treatment for 7 days after a lethal, nonuniform radiation exposure of 800 cGy was sufficient to enhance hematopoietic reconstitution, leading to an earlier recovery. Monkeys were treated with 72,000 U/kg/day of rhGM-CSF delivered continuously through an Alzet miniosmotic pump implanted subcutaneously on day 3. Treated monkeys demonstrated effective granulocyte and platelet levels in the peripheral blood, 4 and 7 days earlier, respectively, than control monkeys. Granulocyte-macrophage colony-forming unit (CFU-GM) activity in the bone marrow was monitored to evaluate the effect of rhGM-CSF on marrow recovery. Treatment with rhGM-CSF led to an early recovery of CFU-GM activity suggesting that rhGM-CSF acted on an earlier stem cell population to generate CFU-GM. Thus, the effect of rhGM-CSF on hematopoietic regeneration, granulocyte recovery, and platelet recovery are evaluated in this paper.

Stimulatory effects of products from inflammatory exudates upon stem cell production.

The present study is based on the hypothesis that a humoral agent produced in inflammatory exudates is transported to the bone marrow where it stimulates CFU-S to differentiate into leukocytic precursors. Inflammatory exudates were produced by the subcutaneous implantation of a sterile acrylic cup filled with bacteria-free Hanks' balanced salt solution (HBSS) into B6D2F1 mice. The cups were removed 24 h later and the cells were separated from the supernatant. Cells collected from individual mice were pooled, as was the supernatant. Thereafter, normal mice received via tail vein either exudate cells, supernatant, plasma from cup-implanted or from normal mice, bone marrow cells or splenic cells from normal animals or HBSS. Another 24 h later bone marrow was removed from all mice and injected into previously lethally irradiated mice or suspended in vitro in agar-supported media. Nine days later the mice were euthanatized, the spleens removed, and colonies (CFU-S) counted. At the same time, colonies (CFU-C) were enumerated from the agar plates. The results clearly indicate that the bone marrow obtained from mice stimulated with either exudate cells or with super-natant or plasma obtained from cup-implanted mice contained a significantly greater number of CFU-S and CFU-C than did any one of the four control groups. These findings appear to substantiate the hypothesis.