Pulmonary neutrophil kinetics after thrombin-induced intravascular coagulation

Previous studies have indicated that neutrophils are required for the development of increased lung vascular permeability after thrombin-induced pulmonary microembolization. In this study, we examined neutrophil kinetics and uptake in the sheep lung before and after lung vascular injury. Sheep neutrophils were isolated by a Percoll-gradient method and labeled with indium-111 oxine. A maximum lung activity of 40% of the injected indium-111 neutrophil activity was attained 8-12 min after the injection. The calculated half-lives of both circulating and pulmonary neutrophils were 700 min. The rate of washout of labeled neutrophils from the lungs was the same as the loss of the peripheral blood activity, indicating removal of neutrophils from the lung and blood by a common pathway (e.g., liver and spleen). Intravenous infusion of alpha-thrombin resulted in an immediate uptake of neutrophils of 14% above the base-line activity. The increased uptake was associated with an immediate decrease in the blood activity, indicating sequestration of the neutrophils in the pulmonary circulation. The neutrophil uptake after alpha-thrombin was transient, reaching a maximum 15 min after infusion. Neutrophil uptake did not occur with alpha-thrombin (which lacks the fibrinogen recognition site), suggesting that the uptake was secondary to intravascular coagulation. An increase in the pulmonary blood volume cannot explain the increased neutrophil sequestration because pulmonary blood volume determined by [99mTc]pertechnetate-labeled erythrocytes did not increase after the alpha-thrombin infusion. Therefore, alpha-thrombin results in a transient neutrophil sequestration in the lung, and the response is secondary to the intravascular coagulation induced by the alpha-thrombin.     

Partial recovery of neutrophil functions during prolonged hypothermia in pigs

The changes in circulation and migration of mature and immature neutrophils during 12 h of hypothermia have been studied using an experimental pig model. At 29 degrees C the number of circulating neutrophils fell from 5 +/- 1.1 at 37 degrees C to 3.5 +/- 0.6 X 10(9)/l and then remained unchanged while hypothermia was maintained. The number of circulating immature neutrophils did not fall during hypothermia. During hypothermia, hydrocortisone failed to stimulate the release of mature and immature neutrophils from the bone marrow. In contrast, endotoxin caused a profound neutropenia followed by a gradual increase in the number of circulating mature neutrophils, which by 6 h, was similar to the number circulating before endotoxin administration. At 29 degrees C the number of circulating immature neutrophils also fell following endotoxin but then increased over the number circulating before endotoxin administration by approximately 10-fold. Compared with neutrophil migration at 37 degrees C, very few mature or immature neutrophils migrated to an inflammatory site during the 12 h of hypothermia (29 degrees C). Unlike hypothermia in vitro, where neutrophil function may improve with time in vivo, neutrophil function remains compromised.     

Recombinant granulocyte colony-stimulating factor induces production of human neutrophil peptides in lung cancer patients with neutropenia

Human neutrophil peptides (HNPs) 1, 2 and 3 are antimicrobial peptides localized in the azurophil granules of neutrophils. We investigated the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the biosynthesis of HNPs 1-3 using a sensitive radioimmunoassay and Northern blot analysis. Seven patients with lung cancer were first treated with various anticancer agents for 3 days (days 1-3) followed by treatment with rhG-CSF (2 microgram/kg weight/day) for 7 days (days 8-14). Chemotherapy caused neutropenia but the neutrophil count increased biphasically between days 8 and 14. Chemotherapy did not change the baseline plasma concentration of HNPs 1-3 (74.1+/-2.1 pmol/ml) but the concentration increased from day 12, 5 days after commencement of rhG-CSF therapy, to reach a peak value of 430.8+/-57.0 pmol/ml on day 15, 1 day after the last administration of rhG-CSF. Baseline HNPs 1-3 content per neutrophil was 0.59+/-0.02 fmol, decreased to 0.30+/-0.07 fmol on day 9, then increased to 0.78+/-0.07 fmol on day 15. Analyses of peripheral blood neutrophils by Northern blot and reverse-phase high-performance liquid chromatography showed that the amounts of HNPs 1-3 mRNA and precursors of HNPs 1-3 markedly increased in response to rhG-CSF. Our results indicate that recombinant hG-CSF does not only increase neutrophil count but stimulates HNPs 1-3 biosynthesis in neutrophils, thus enhancing the host defense system of compromised hosts with neutropenia.     

Neutrophil chemotactic factors promote leukocytosis. A common mechanism for cellular recruitment from bone marrow.

We investigated cellular responses in a rabbit to i.v. administration of five established chemotactic factors (leukotriene B4 (LTB4), platelet-activating factor (PAF), C5a, N-Formyl-Met-Leu-Phe (F-MLF), and IL-8), and each exerted a characteristic effect on circulating white blood cell levels. All five factors induced a rapid and transient leukopenia. The blood was nearly devoid of circulating neutrophils 5 min after administration of each chemotactic factor. Other leukocytes were also variably depleted during the leukopenic phase, including eosinophils, basophils, monocytes, and lymphocytes. The lymphocyte numbers remained significantly depressed (approximately 30%) for as long as 3 h after administration of PAF or f-MLF. Each chemotactic factor produced a marked neutrophilia (i.e., 250-400% of baseline levels) after the initial leukopenia. Eosinophil numbers were elevated along with the neutrophil response in the C5a- and LTB4-treated animals. Basophil levels were significantly elevated only in LTB4-treated animals. The cellular response to PAF, f-MLF, and IL-8 appeared to be specific for the neutrophils. The kinetic profiles of the neutrophilia induced by PAF (10 micrograms/kg) or f-MLF (2.5 micrograms/kg) were similar, with maximal responses occurring 3 to 4 h after administration. In contrast, LTB4 (10 micrograms/kg), IL-8 (2.5 micrograms/kg), and C5a (5 micrograms/kg) induced a more rapid neutrophilia, with peak responses occurring 1 to 1.5 h after injection, and remaining elevated for 3 to 4 h. In all animals the neutrophilia was accompanied by a relative increase in the number of nonsegmented neutrophils (bands), suggesting that a major component of leukocytosis is caused by the release of bone marrow reserves. Phenidone (10 mg/kg), a dual cyclooxygenase/5-lipoxygenase inhibitor, affected neither the neutropenia nor the neutrophilia induced by C5a, f-MLF, or PAF. The protein synthesis inhibitor actinomycin D also failed to suppress neutrophil responses induced by either C5a or PAF. These results suggest that leukocytosis is a common response induced by all neutrophil chemotactic factors. Leukocytosis appears to be a direct result of the dynamic adaptive response of neutrophils to chemotactic factor stimulation without involvement of a secondary mediator system.     

Methotrexate inhibits superoxide production and chemotaxis in neutrophils activated by granulocyte colony-stimulating factor

Treatment of circulating human neutrophils with recombinant human granulocyte colony-stimulating factor (rhG-CSF) for 30 min augmented superoxide generation and chemotaxis induced by N-formylmethionyl-leucyl-phenylalanine (fMLP) in a dose dependent manner. When neutrophils were treated with 1 μM of methotrexate (MTX) for 60 min after incubation with rhG-CSF (10 ng/ml), the effects of rhG-CSF on superoxide generation and chemotaxis were inhibited by approximately 49 and 29%, respectively. Although inhibitory effects of MTX were also seen in neutrophils not pretreated with rhG-CSF, the degree of inhibition was much less. The addition of either hypoxanthine or guanosine at a concentration of 100 μM to the culture medium significantly attenuated the effects of MTX. However, in neutrophils obtained from a patient with Lesch-Nyhan syndrome, which lacked hypoxanthine-guanine phosphoribosyl transferase activity, neither hypoxanthine nor guanosine had any rescue effect. These results suggest that MTX inhibits superoxide generation and chemotaxis in rhG-CSF-activated neutrophils, at least in part, by disturbing purine nucleotide biosynthesis. © 1996 Wiley-Liss, Inc.     

Systemic complement activation and acute lung injury

Experimental studies of rats have provided significant evidence that intravascular complement activation after i.v. injection of cobra venom factor (CVF) or thermal injury of skin can result in acute lung injury. This has been determined by morphological changes in lung and increases in lung vascular permeability. Systemic complement activation is associated with an early appearance of C5-derived chemotactic activity in the circulation coincident with the development of transient neutropenia, followed by extensive granulocytosis and sequestration of neutrophils in lung interstitial capillaries. The acute pulmonary injury depends on availability of complement and neutrophils. Depletion of either complement or blood neutrophils before CVF injection or thermal injury will prevent development of lung injury. Interventional studies with catalase, scavengers of hydroxyl radical OH., and iron chelators have revealed that the acute pulmonary injury is related to production of oxygen-derived free radicals by activated neutrophils. OH. appears to be the key mediator involved in the acute lung microvascular injury.     

Kinetics and mechanisms of recombinant human interleukin 1 and tumor necrosis factor-alpha-induced changes in circulating numbers of neutrophils and lymphocytes

Human recombinant interleukins 1 alpha and 1 beta (rIL-1 alpha and -1 beta) both induced monophasic peripheral neutrophilia and lymphopenia in Lewis rats 1.5 hr after i.v. injection. The kinetics of rIL-1 alpha- and -1 beta-induced neutrophilia were similar to those induced by human monocyte-derived IL-1, IL-1 alpha, and IL-1 beta, and the peripheral neutrophilia was accompanied by a marked decrease in marrow neutrophils. Arachidonic acid metabolites are implicated as biochemical intermediates in the production of the neutrophilia but not lymphopenia, since indomethacin and dexamethasone both completely abrogated IL-1-induced neutrophilia but did not affect the IL-1-induced lymphopenia. Acetylsalicylic acid, a cyclooxygenase inhibitor, did not inhibit IL-1-induced neutrophilia, suggesting that products of the lipoxygenase rather than the cyclooxygenase pathway of arachidonate metabolism may contribute to the neutrophilia. Human recombinant tumor necrosis factor-alpha (rTNF) administered i.v. to Lewis rats induced peripheral neutropenia, two peaks of neutrophilia, and lymphopenia. A wide range of doses of rTNF resulted in an initial neutropenia at 0.5 hr after injection followed by a first peak of neutrophilia at 1.5 hr and a second peak of neutrophilia at 6 hr. The initial neutropenia and the first peak of neutrophilia were not inhibited by pretreatment of rats with dexamethasone, indomethacin, or aspirin. The second peak of neutrophilia was inhibited by both dexamethasone and indomethacin, but was not at all inhibited by aspirin, suggesting that the second peak of neutrophilia is mediated by the release of endogenous cytokines, especially by IL-1, since exogenous IL-1-induced neutrophilia is also completely inhibited by dexamethasone and indomethacin but not by aspirin. The TNF-induced peripheral neutrophilia is also accompanied by a significant depletion of bone marrow neutrophils, indicating that the source of increased circulating neutrophils is, at least in part, via recruitment of marrow neutrophils. Systemic blood pressure was not affected by IL-1 or rTNF at the dosages employed, showing that the changes in circulating leukocyte subsets were not attributable to hemodynamic changes nor to the hemodynamic change-related release of adrenal hormones. Adrenalectomy did not alter the IL-1- or rTNF-induced neutrophilia or lymphopenia, also demonstrating that neither monokine mediates its hematologic effects on peripheral blood leukocytes via the release of adrenal hormones.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Leukotriene B4 increases pulmonary transvascular filtration by a neutrophil-independent mechanism

We examined the role of circulating granulocytes in the pulmonary microvascular response to leukotriene B4 (LTB4) by prior depletion of circulating granulocytes using hydroxyurea. LTB4 (2 micrograms/kg injection followed by infusion of 2 micrograms/kg over 15 min) produced transient increases in pulmonary arterial pressure and pulmonary vascular resistance, indicating that neutrophils were not required for the pulmonary hemodynamic effects of LTB4. Infusion of LTB4 in granulocyte-depleted sheep also resulted in transient increases in pulmonary lymph flow (QL) with no significant change in the lymph-to-plasma protein concentration ratio (L/P), findings similar to those in control animals. In vitro studies indicated that LTB4 (10(-7) or 10(-9) M) produced a transient adherence of neutrophils to cultured pulmonary artery endothelial monolayers. Maximal responses occurred at 10 min after the addition of LTB4 to the endothelial cell-neutrophil coculture system, and the adherence decreased to base line within 60 min. LTB4 infusion in sheep also produced a transient uptake of autologous 111In-oxine-labeled neutrophils. The results indicate that LTB4-mediated increase in pulmonary transvascular protein clearance (QL x L/P) is independent of circulating granulocytes.     

Effects of IL-1 on hematopoietic progenitors after myelosuppressive chemoradiotherapy

Recently it has been recognized that IL-1 plays an important role in hematopoietic regulation. Administration of 5-fluorouracil (5-FU) to mice causes prolonged neutropenia. rHIL-1 injected to mice after 5-FU, accelerated the recovery of hematopoietic progenitors and blood neutrophils. The combination of rhIL-1 and rhG-CSF reduced the neutropenic period significantly. Sublethal irradiation of mice induced profound neutropenia for 3 weeks which was associated with 80% mortality. Administration of rhIL-1 20 hours prior to or 2 hours post irradiation resulted in a significantly improved survival and rapid recovery of the neutrophil count. IL-1 administered alone or in combination with other colony stimulating factors to spontaneous breast tumor bearing mice following 5-FU therapy resulted in a rapid recovery of neutrophils, improved survival, and markedly reduced the tumor mass. Experiments in primates demonstrated that rhIL-1 administered to 5-FU treated animals shortened the neutropenic period from 30 to 17 days and increased the number of marrow progenitors responsive to other CSFs. Prolonged administration of IL-1 (14 days) to these animals resulted in a delayed neutrophil recovery as compared to animals receiving short courses of IL-1. rhIL-1 administered to primates receiving marrow grafts after lethal irradiation, did not result in rapid hematopoietic recovery. In humans, studies with CD-34 positive marrow cells showed that IL-1 had a radioprotective effect on a committed and early marrow progenitors. These data show the therapeutic potential of IL-1 in the treatment of chemoradiotherapy induced myelosuppression.     

Leucokinetic study. Morphology of the bone marrow and blood after experimental induction of marrow hypoplasia by cyclophosphamide in laboratory rats

Experimental hypoplasia of femoral bone marrow in rats was induced by cyclophosphamide, injected i.p. at various clock hours (08.00, 12.00, 16.00, 20.00, 24.00, and 04.00). Cyclophosphamide-induced neutropenia persisted for six days and was followed by transitory granulocytosis with subsequent decrease in the cont of circulating mature granulocytes. The absolute counts of circulating segmented neutrophils changed in parallel with the absolute counts of segmented neutrophils in the bone marrow. The count of circulating neutrophils was not essentially influenced by the clock hour of cyclophosphamide injection. The toxic action of cyclophosphamide upon the bone marrow exhibited a circadian rhythmicity: the greatest decrease in the count of nucleated marrow cells was found in the morning, and the least, in the evening. The minimal compartment transit times of the development stages of bone-marrow neutrophils, and the daily granulocyte production in the rat were calculated.     

IL-6 plays an essential role in neutrophilia under inflammation

In the present study, we explored the involvement of interleukin-6 (IL-6) in neutrophilia under inflammatory conditions. The neutrophil count in the peripheral blood was high in arthritic monkeys, and anti-IL-6 receptor antibody reduced neutrophil counts to normal levels. IL-6 injection into normal monkeys significantly increased neutrophil counts in the blood 3h after injection. The expression of cluster of differentiation (CD) 162 on circulating neutrophils was reduced by IL-6 injection. IL-6 treatment in vitro did not affect CD162 expression on neutrophils from human blood. In IL-6-treated monkeys, IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels in plasma were clearly elevated. IL-8 and GM-CSF treatment in vitro reduced cell-surface CD162 expression on human neutrophils, and moreover, increased soluble CD162 expression in the cell supernatant. The addition of IL-6 into human whole peripheral blood induced IL-8 production and reduced CD162 expression on neutrophils. Furthermore, IL-8 and GM-CSF augmented mRNA expression of a disintegrin and metalloprotease like domain 10 (ADAM10) in neutrophils. Knock-down of ADAM10 by siRNA in neutrophil-like HL-60 cells partially reversed the expression of CD162 reduced by GM-CSF and IL-8 on HL-60 cells. In conclusion, IL-6 induced neutrophilia and reduced CD162 expression on neutrophils in inflammation.     

Platelet-activating factor (PAF) mediation of rat anaphylactic responses to soluble immune complexes. Studies with PAF receptor antagonist L-652,731

A new synthetic compound, L-652,731 (trans-2,5-(3,4,5-trimethoxyphenyl) tetrahydrofuran), which has been demonstrated by Hwang et al. to be a potent and specific platelet-activating factor (PAF) receptor antagonist causes 100% inhibition of 1 microM PAF-induced neutrophil degranulation at 50 microM, but has no effect on neutrophil degranulation induced by precipitating immune complexes (323 micrograms/ml), fMet-Leu-Phe (10(-7) M), or the calcium ionophore A23187 (10(-5) M). Intravenous infusion of 1 mumol L-652,731 results in almost 100% inhibition of hypotension induced by PAF but not that induced by isoproterenol, histamine, bradykinin, or acetylcholine. With the use of this novel PAF receptor antagonist, the in vivo mediator role of PAF in the soluble immune complex-induced hypotension, extravasation, vascular lysosomal hydrolase secretion, and neutropenia in rats was determined. The hypotension, extravasation, and lysosomal hydrolase release induced by immune complex infusion take 2 to 10 min longer to occur than the same responses elicited by PAF infusion. The neutropenia response is immediate with both stimuli. L-652,731 when orally administered to rats (20 mg/kg, 1.5 hr before PAF infusion) inhibited PAF-induced hypotension (69%), extravasation (76%), vascular lysosomal hydrolase release (79%), and neutropenia (73%). The same L-652,731-dosing regimen inhibited immune complex-stimulated hypotension (87%), extravasation (77%), and vascular lysosomal hydrolase release (31%). The initial and complete neutropenia induced by immune complex infusion was not inhibited in L-652,731-pretreated rats, but the rate of return of neutrophils to the blood was faster in the latter rats. Rats with blocked circulation to the liver still exhibited extensive extravasation and vascular lysosomal hydrolase release in response to PAF, but there was no extravasation and greatly reduced hydrolase release in response to immune complexes. Thus PAF is indicated to be a major mediator of soluble immune complex-induced hypotension and vascular permeability and a minor mediator of immune complex-induced lysosomal hydrolase release in rats. PAF probably does not mediate the initial and complete neutropenia stimulated by immune complexes. The liver is probably the major site for PAF production in response to circulating immune complexes.     

Leukocyte and platelet margination within microvasculature of rabbit lungs

These studies compare the behavior of radiolabeled neutrophils, monocytes, lymphocytes, and platelets during their first pass through the pulmonary circulation after a central venous injection and their distribution within the circulation 10 min later. Their first pass through the pulmonary circulation was compared with erythrocytes (RBCs) using the indicator-dilution technique, and their recovery within the circulation of the lung and other organs was determined at 10 min by counting the radioisotopes in each organ. The extraction of each cell relative to RBCs during the first pass through the lung correlated with cell size in that the neutrophils (volume 107-140 fl) showed 97.6 +/- 0.6% extraction, monocytes (volume 80-105 fl) showed 91.4 +/- 1.7% extraction, lymphocytes (volume 36-75 fl) showed 80.1 +/- 4.4% extraction, and platelets (volume 4-7 fl) showed 33.1 +/- 3.9% extraction. After 10 min of circulation, the proportion of injected cells remaining in the lung was similar for neutrophils and monocytes (27.4 +/- 1.8 vs. 31.4 +/- 1.6%) but lower for lymphocytes (18.6 +/- 2.9%) and platelets (3.1 +/- 0.5%). All of the leukocytes were found to have a substantial marginated pool within the lung, whereas the platelets did not. The exchange between the circulating and marginated pools of leukocytes in the lung was related to blood velocity, with the least retention occurring in lung regions with shortest RBC transit times. We conclude that cell size is a major factor determining the time that cells will be delayed by the pulmonary microvasculature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of two recombinant human granulocyte colony-stimulating factor glycoforms

Two recombinant human granulocyte colony-stimulating factor (rhG-CSF) isoforms were isolated from the medium conditioned by an engineered Chinese hamster ovary (CHO) cell line. The two rhG-CSFs were characterized and were found to differ in the carbohydrate structure attached to Thr-133. The glycoform, referred to as Peak 1, contains the O-linked glycan Neu5Ac(alpha 2-3)Gal(beta 1-3)GalNAc; the Peak 2 glycoform contains the O-linked glycan Neu5Ac(alpha 2-3)Gal(beta 1-3)[Neu5Ac(alpha 2-6)]GalNAc. The two glycoforms displayed a similar biological activity in cultures of a mouse 32D C13 cell line and human bone-marrow myelo-monocytic progenitor cells (CFU-GM). In the latter test both glycoforms displayed a higher activity than nonglycosylated rMet-hG-CSF from Escherichia coli. The pharmacokinetic profile and activity of the two rhG-CSF glycoforms and of a mixture of them (Pool) were investigated in mice treated with a single injection of rhG-CSF at the doses of 125 micrograms and 250 micrograms/kg, given via the intravenous (i.v.) and the subcutaneous (s.c.) route, respectively. The plasma concentration profiles obtained were similar for all three substances and did not show any relevant differences in absorption or elimination. The pharmacokinetic parameters indicate that the three substances have similar area under the curve (AUCs), volumes of distribution, and terminal half-life. Furthermore, our data indicate a high bioavailability of the two different glycoforms of rhG-CSF when given to mice via the s.c. route either singularly or as a mixture. Detectable levels of rhG-CSF persisted for more than 8 h in the i.v. and more than 24 h in the s.c. route of administration. All three substances induced early neutrophilia in mice. All rhG-CSF-treated mice developed a two-four-fold rise in neutrophil counts as early as 4 h after the intravenous and 2 h after the subcutaneous injection. Relatively high levels of neutrophils were maintained for at least 8 and 24 h after i.v. and s.c. administration, respectively.     

Chronopharmacologic Effects of Recombinant Human Granulocyte Colony-Stimulating Factor (rhG-CSF) on Hematopoiesis in Mice

Chronopharmacologic effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on circulating white blood cell and differential counts as well as bone marrow granulocyte-macrophage colony-forming units (CFU-GM) counts were investigated in B6D2F1 mice. The animals were synchronized with an alternation of 12 h of light (L) and 12 h of darkness (D) (LD 12:12) for 3 weeks prior to study, then received a daily subcutaneous injection of rhG-CSF (400 µg/kg/day) for 4 consecutive days at 3, 9, 15 or 21 h ours a fter l ight o nset (HALO). Samples were obtained on the fifth day at the same circadian stage as that of rhG-CSF injection. rhG-CSF significantly increased the 24-h mean of leukocyte, neutrophil, lymphocyte and CFU-GM counts. Maximum increase in leukocyte and neutrophil counts was observed when rhG-CSF was administered in the middle of the dark span, while maximum stimulatory effect on circulating lymphocytes or on CFU-GM counts was obtained with rhG-CSF administration near the middle of the light span. The results indicate that choosing the dosing time of this cytokine may selectively orient its pharmacologic action. Appropriate chronomodulated delivery schemes of rhG-CSF may further reduce hematological toxicity following chemotherapy.     

Chronopharmacologic Effects of Recombinant Human Granulocyte Colony-Stimulating Factor (rhG-CSF) on Hematopoiesis in Mice

Chronopharmacologic effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on circulating white blood cell and differential counts as well as bone marrow granulocyte-macrophage colony-forming units (CFU-GM) counts were investigated in B6D2F1 mice. The animals were synchronized with an alternation of 12 h of light (L) and 12 h of darkness (D) (LD 12:12) for 3 weeks prior to study, then received a daily subcutaneous injection of rhG-CSF (400 µg/kg/day) for 4 consecutive days at 3, 9, 15 or 21 h ours a fter l ight o nset (HALO). Samples were obtained on the fifth day at the same circadian stage as that of rhG-CSF injection. rhG-CSF significantly increased the 24-h mean of leukocyte, neutrophil, lymphocyte and CFU-GM counts. Maximum increase in leukocyte and neutrophil counts was observed when rhG-CSF was administered in the middle of the dark span, while maximum stimulatory effect on circulating lymphocytes or on CFU-GM counts was obtained with rhG-CSF administration near the middle of the light span. The results indicate that choosing the dosing time of this cytokine may selectively orient its pharmacologic action. Appropriate chronomodulated delivery schemes of rhG-CSF may further reduce hematological toxicity following chemotherapy.     

Effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on cytotoxicity of PSK-induced peritoneal polymorphonuclear leukocytes (PMNs)

We investigated whether recombinant human granulocyte colony-stimulating factor (rhG-CSF) en hanced the cytotoxicity of PSK-induced polymorphonuclear leukocytes (PMNs) in the peritoneal cavity. Male C3H/He mice, 8- to 10-week-old, received single subcutaneous (s.c.) or intraperitoneal (i.p.) injection of 2.5 µg/animal of rhG-CSF at different time points before or after an i.p. administration of PSK. In other experiments, mice were s.c. or i.p. treated with the same dosage of rhG-CSF every day for 7 or 14 consecutive days and i.p. injected with 2.5 mg/animal of PSK on the last day. Peritoneal PMNs were harvested 6 hrs after the administration of PSK and purified to more than 95% by Ficoll-Paque for in vitro cytotoxic assay.In vitro cytotoxic assays with⁵¹Cr labeled MM46 mammary carcinoma cells were added with 5–20 µg/ml of Nocardia rubra cell wall skeleton (N-CWS) at the beginning of the assay to augment the cytotoxic activity of PMNs.In vitro addition of rhG-CSF to the assay did not enhance the cytotoxicity of PSK-induced PMNs. However, the cytotoxicity was signifi cantly increased when rhG-CSF was s.c. administered 12 hrs before a PSK injection or 2 or 5 hrs after that. On the other hand, the cytotoxicity was rather weak when mice s.c. or i.p. received consecutive injections of rhG-CSF. This cytotoxicity may be mediated by H₂O₂, since H₂O₂ production of PMNs during the cytotoxic assay appears to correlate with the levels of cytotoxicity under suppressed H₂O₂ generation by catalase or enhanced generation by rhG-CSF. These results suggest that rhG-CSF augments the cytotoxicity of PSK-induced PMNs when administeredin vivo timely.     

Marginated pool of neutrophils in rabbit lungs

The size and location of the marginated pool of neutrophils (PMNs) in rabbit lungs were evaluated, and the rate of exchange of the PMNs with the circulating pool was determined. 99mTc-labeled erythrocytes (99mTc-RBCs) and 125I-labeled macroaggregated albumin (125I-MAA) were used to determine RBC transit times in the pulmonary circulation. Radiolabeled PMNs were studied on their first passage through the lungs. After 10 min of circulation, the lungs were fixed, gamma counted, and prepared for morphometric and autoradiographic studies; 74 +/- 3% of the PMNs was retained in the lungs on the first passage, and 23 +/- 2% was within the pulmonary marginated pool 10 min later. The regional PMN retention and the rate of exchange between the marginated and circulating PMN pools in the lung were directly related to RBC transit time. The radiolabeled PMNs distributed similarly to the unlabeled cells within the microvasculature and had a similar exchange rate between the marginated and circulating pools (1.4 +/- 0.2%/s using labeled cells and 1.5 +/- 0.5%/s using unlabeled cells). The marginated pool was located primarily within alveolar capillaries and contained two to three times as many PMNs as the total circulating pool.