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.     

Harvesting of granulocytes for granulocyte transfusion by means of continuous flow centrifugation or filtration leukapheresis]

More than 10(10) viable granulocytes are necessary for a therapeutical effective granulocyte transfusion. This number of cells can be harvested from normal donors by two techniques basing on different principles: continuous flow centrifugation (CFC) and filtration leucapheresis (FL). Our studies demonstrated that, under certain special conditions, the separation potentials of both methods are comparable yielding 2.5 to 3.0 X 10(10) granulocytes within 4 hrs. Granulocyte collection rate was optimal if donors were treated with dexamethasone during 16 hrs prior to the state of the procedure. However, the costs of CFC exceed those of FL by a factor of about two. The increased occurrence of side effects attributed to the transfusion of FL-granulocytes can be reduced to the level of CFC-granulocytes by repetitive filtration-elution leucapheresis minimizing cell damage. The studies define the efficiency spectrum of CFC which in addition to granulocyte separation includes collection of thrombocytes, cells for immunotherapy, and plasmapheresis.     

Biphasic changes in leukocytes induced by strenuous exercise

Seven healthy male volunteers participated in short- (STR, 1.7 km), middle- (MTR, 4.8 km) and long- (LTR, 10.5 km) term runs at a speed close to their maximum. A prompt mobilization of white cells, and lymphocytes in particular, appeared following the exercise. The initial increase in the number of lymphocytes was succeeded by a significant decrease [(P less than 0.03) lymphopenial], which on average was 32%-39% of the pre-exercise values in all groups. A close correlation was found between the initial increase in plasma cortisol concentration after exercise and the subsequent lymphopenia. A modest enhancement in the number of granulocytes immediately after the exercise was accompanied by a comprehensive increase in polymorphonuclear (PMN) elastase concentration accounting for 78.6%, SEM 16.3%, 140.7%, SEM 31.8% and 241.3%, SEM 48.1% in the STR, MTR and LTR groups. No correlation was found between granulocyte number and the plasma PMN elastase concentration. A delayed granulocytosis was noted in all subjects, reaching a peak between 2 and 4 h after the exercise. The magnitude of the granulocytosis varied among subjects and peak values of the number of circulating granulocytes were found to be 5.7 x 10(9) cells.l-1, SEM 0.5, 6.7 x 10(9) cells.l-1, SEM 0.6 and 8.8 x 10(9) cells.l-1, SEM 0.5 in STR, MTR and LTR respectively, whereas the mean baseline value was 3.6 x 10(9) cells.l-1, SEM 0.4. The neutrophilic granulocytosis was not accompanied by a corresponding enhancement in PMN elastase concentration. The plasma cortisol concentration reached a peak 30 min after exercise and declined below the control level in 4 h. Neither the initial increase, nor the subsequent decrease in plasma cortisol concentration were found to be essential for the magnitude of the delayed leukocytosis.     

Inhibition of human cytotoxic T lymphocyte activity in vitro by autologous peripheral blood granulocytes

Peripheral blood granulocytes from normal healthy donors were found to reproducibly inhibit the cytolytic effector function of specifically sensitized cytotoxic T lymphocytes in vitro when co-incubated with these effector cells and target cells in 8 hr 51Cr release assays. Inhibition required intact granulocytes, was proportional to the number of granulocytes present, and was independent of granulocyte adherence, phagocytic function, and viability. Equivalent numbers of enriched normal or leukemic peripheral T lymphocytes did not cause inhibition of 51Cr release, and preincubation of granulocytes with effectors did not significantly alter viability or cytotoxic function. Because granulocytes can inhibit natural killer cell function in vitro, these data indicate that granulocytes can regulate diverse antigen-specific and spontaneous cytotoxic functions in vitro, suggesting that circulating granulocytes may have the potential for in vivo regulation of these cytotoxic effectors.     

Mitogenic activity in platelet-poor plasma from rats with persistent liver nodules or liver cancer

Platelet-poor plasma (PPP) from F-344 rats with chemically-induced preneoplastic liver nodules or hepatocellular carcinoma stimulated S-phase DNA synthesis in monolayer cultures of normal rat hepatocytes. Similar mitogenic activity was detected in PPP 6 hrs to 1 week after partial hepatectomy (PH) or after necrotizing doses of CCl4 or diethylnitrosamine (DENA). Very little activity was found in PPP4 from control rats. The mitogenic activity in PPP from animals with nodules was non-dialyzable (greater than 14 kd) and bound to a heparin-sepharose affinity column. None of the mitogenic PPPs competed with [125I] epidermal growth factor (EGF) for binding sites on A431 cells or normal rat hepatocytes. These studies indicate that persistent proliferation of preneoplastic and neoplastic hepatocytes is associated with increased circulating levels of mitogenic hepatocyte growth factor.     

Interleukin-1-induced granulocytopenia and pulmonary leukostasis in rabbits

Pulmonary leukostasis is a postulated prerequisite lesion for acute lung injury. Interleukin-1 (IL-1) mediates components of the acute-phase response, stimulates granulocyte metabolism and secretion, and augments endothelial adhesiveness. We studied the effects of murine IL-1 infusion on circulating granulocytes, their sequestration within the pulmonary microvasculature, lung water, and bronchoalveolar lavage fluid (BALF) protein concentration in rabbits at 3 and 24 h after infusion. IL-1 administration induced significant (P less than 0.01) granulocytopenia compared with saline-injected controls and at 3 h induced significant increases in both mean alveolar septal wall granulocytes per high power field (HPF) (P less than 0.001) and mean myeloperoxidase (MPO) activity per gram lung tissue (P less than 0.001). At 24 h, IL-1 induced a marked granulocytosis and again significantly increased both mean alveolar septal wall granulocytes per HPF (P less than 0.001) and lung MPO (P less than 0.01). Increased lung water or BALF protein concentration could not be demonstrated in animals killed at either 3 or 24 h after IL-1 administration. Therefore, IL-1 can induce an early profound granulocytopenia followed by later granulocytosis, as well as sustained pulmonary leukostasis in the absence of detectable pulmonary edema formation or an alveolar-capillary leak.     

Assumed identity of granulocyte-pyrogen (GP) and leukocytosis inducing factor (LIF).

Pyrogen obtained from granulocytes induces granulocytosis in normal, endotoxin-tolerant and spinal animals alike, whereas heat treatment destroys the granulocytosis inducing effect. It is concluded that GP and LIF are identical, or nearly identical, factors.     

Neutrophil migration in canines: In vivo comparison of granulocyte function following 24-hour storage at 6 or 20 °C of leukocyte concentrates or of granulocytes purified by counterflow centrifugation-elutriation

The use of granulocyte-rich concentrates from leukapheresis purified by counterflow centrifugation—elutriation to obtain pure granulocytes for transfusion studies in cyclo-phosphamide-induced neutropenic animal models is reported. Our data for granulocyterich leukapheresis concentrates indicate that room temperature (20 °C) appears to be preferred to 6 °C for short-term granulocyte storage. The data also indicate that although the granulocytes isolated by counterflow centrifugation—elutration may retain in vitro functions of chemotaxis, phagocytosis, and bactericidal activity, the in vivo function of migration into skin chambers for isolated granulocytes is seriously impaired after storage for 18 to 24 hr at both 6 and 20 °C. This loss of in vivo function of stored granulocytes occurs in isolated granulocytes obtained by both counterflow centrifugation-elutriation and dextran sedimentation, and it is not observed in the leukocyte concentrates held at 20 °C. The results of these studies are fourfold. First, freshly isolated granulocytes display no apparent loss of either in vivo or in vitro function. Second, granulocytes isolated by counterflow centrifugation-elutriation or dextran sedimentation and stored at 6 or 20 °C are severely impaired in terms of their in vivo chemotactic function but display no loss of in vitro efficacy. Third, 20 °C storage of granulocyte-rich leukapheresis concentrates for 18 to 24 hr is superior to 6 °C storage. Fourth, in vitro analysis may be limited in its ability to indicate in vivo function as a measure of success in granulocyte preservation studies.     

Liquid and cryopreservation effects on in vitro function of dog granulocyte concentrates prepared for transfusion

The effects of liquid and Cryopreservation on in vitro function of dog granulocyte concentrates prepared by continuous flow centrifugation leukapheresis and counterflow centrifugation elutriation are presented. These homogeneous granulocyte concentrates (97 ± 2% granulocytes, 99.4 ± 0.3% viable) were cryopreserved in 5% DMSO and 5% HES dissolved in 2 g% BSA, 20% autologous citrated plasma in a modified a minimal essential medium. The granulocyte recovery was 87.6 ± 2.4% relative to the number of intact and viable granulocytes in the washed suspension of cells. In vitro functions of chemotaxis, phagocytosis, bactericidal activity, and selected enzymes were not affected by 12–24 hr storage at 4–6 °C. Frozen, thawed, and washed granulocytes showed a significant decrease (P < 0.01) in chemotactic recognition and response but not chemokinetic response, although it was depressed. Phagocytosis of latex beads and associated burst of O₂ consumption also decreased significantly (P < 0.05) to approximately 50% of the original prefreezing value. However, the killing of live Escherichia coli was not depressed to the extent expected and suggested by loss of O₂ consumption and selected enzyme activity. The cryopreservation of viable homogeneous granulocyte concentrates in sufficient quantity for transfusion in the neutropenic and/or septicemic dog model is demonstrated in these results.     

The healthy donor profile of immunoregulatory soluble mediators is altered by stem cell mobilization and apheresis

Background

Peripheral blood stem cells from healthy donors mobilized by granulocyte colony-stimulating factor (G-CSF) and thereafter harvested by leukapheresis are commonly used for allogeneic stem cell transplantation.

Use of a modified rotor and enlarged separation chamber for isolation of human granulocytes by counterflow centrifugation-elutriation

Human granulocytes were isolated from leukapheresis blood by counterflow centrifugation-elutriation using the Beckman J21B centrifuge and JE6 rotor. Attempts to increase the absolute number of human granulocytes recovered by a single- or double-chamber procedure revealed the physical limitations of the chamber capacity in granulocyte isolation. To improve the absolute number of granulocytes from leukapheresis blood, an enlarged separation chamber and accompanying rotor were fabricated. The enlarged chamger has three times the capacity of the standard 4.5-ml Beckman chamber. Aside from increased yield of granulocytes, the 13.2-ml chamber affords a 25% saving in isolation time compared to the Beckman chamber for isolation of a comparable number of cells. In vitro analysis of the isolated granulocytes for viability, latex bead ingestion, and chemotactic response indicate no apparent loss of granulocyte function as a result of the isolation procedure.     

Detection of anti-CD32 alloantibody in donor plasma implicated in development of transfusion-related acute lung injury

Transfusion-related acute lung injury (TRALI) occasionally causes serious symptoms that may be fatal to recipients. Polymorphonuclear neutrophils (PMNs) and alloantibodies specific to PMN cell surface antigens are suspected to cause TRALI. The aim of this study is to establish a sensitive and stable procedure of detecting alloantibodies not only in donor blood, but also in recipient's plasma. We have introduced a new method of detecting alloantibodies based on double-determinant enzyme-linked immunosorbent assay (DD-ELISA) and a monoclonal antibody-immobilized granulocyte antigen (MAIGA) test (arbitrarily designated as modified DD-ELISA). We verified the specificity of alloantibodies against PMN cell surface antigens in plasma samples from three normal healthy donors of blood that induced respiratory distress in recipients after a blood transfusion. Anti-CD32 (Fc gamma RIII) alloantibodies were detected in all the plasma samples using two different clones of the monoclonal anti-CD32 antibody. The specificities of these plasma samples could not be identified by the granulocyte immunofluorescence test (GIFT) using typed test cells. Except for the anti-CD32 alloantibodies, one plasma sample was proved to have the anti-HNA-1a alloantibodies. In another plasma sample, the anti-HNA-2a alloantibodies were detected. By modified DD-ELISA, we could clearly specify the presence of alloantibodies in the three plasma samples. Our results also suggest that the anti-CD32 alloantibodies can be generated in vivo and may play some roles in the development of TRALI.     

Role of circulating granulocytes in sheep lung injury produced by phorbol myristate acetate

Phorbol myristate acetate (PMA) and endotoxin cause pulmonary granulocyte sequestration and alteration in lung fluid and solute exchange in awake sheep that are felt to be analogous to the adult respiratory distress syndrome in humans. The basic hypothesis that PMA causes lung injury by activating circulating granulocytes has never been tested. The effects of infused PMA on lung mechanics and the cellular constituents of lung lymph have also not been reported. We therefore characterized the effects of intravenous PMA, 5 micrograms/kg, on lung mechanics, pulmonary hemodynamics, lung fluid and solute exchange, pulmonary gas exchange, blood and lymph leukocyte counts, and plasma and lymph cyclooxygenase products of arachidonate metabolism in 10 awake sheep with normal granulocyte counts and after granulocyte depletion with hydroxyurea. PMA significantly altered lung mechanics from base line in both nongranulocyte depleted and granulocyte-depleted sheep. Dynamic compliance decreased by over 50% and resistance to airflow across the lungs increased over threefold acutely following PMA infusion in both sets of experiments. Changes in lung mechanics, pulmonary hemodynamics, lung fluid and solute exchange, pulmonary gas exchange, and plasma and lymph arachidonate metabolites were not significantly affected by greater than 99% depletion of circulating granulocytes. We conclude that the lung injury caused by PMA in chronically instrumented awake sheep probably is not a result of activation of circulating granulocytes.     

A mathematical model of canine granulocytopoiesis

Summary The granulocyte cell renewal system of the dog is represented by a mathematical model consisting of the following compartments: The pool of pluripotential stem cells, the committed stem cell pool, divided into a blood and a bone marrow compartment, the proliferation pool, the maturation pool, the reserve pool and the blood pool of functional granulocytes. This chain of compartments is described by a system of non-linear differential equations. Cell losses anyplace in the system provoke increased production in all pools containing cells capable to divide. A reduced number of granulocytes in the blood pool stimulates production of a granulocyte releasing factor which mobilizes a rising number of cells to transit from the marrow reserve into the blood pool.The model was simulated on a digital computer. It was found to be capable to reproduce the steady state conditions and it also fits the data of two distinct experimental perturbations of the system both equally well. These perturbations are a loss of proliferating cells as it occurs after the administration of cytostatic drugs and losses of functional cells as they are induced by leukapheresis experiments of differing leukapheresis rates.This study was supported by the Deutsche Forschungsgemeinschaft (SFB 112)     

Platelet-vessel wall interactions: effects of platelets and plasma on the antiaggregatory activity and 6 keto-PGF1 alpha production in isolated perfused aortas

An experimental model for the study of platelet-vessel wall interactions has been developed, based on perfusion of rat platelet rich plasma (PRP) through isolated rat aortas. In the perfused PRP, platelet aggregation was inhibited and levels of 6 Keto PGF1 alpha and cAMP were elevated over the values found in non perfused PRP. When PPP or buffer were perfused through the isolated artery, elevations of 6 Keto PGF1 alpha levels in the perfusate were smaller (in perfused PPP) or of shorter duration (in both perfused PPP and buffer). The presence of platelets in the perfusion fluid thus enhanced the formation of Prostacyclin by the arterial wall. Levels of 6 Keto PGF1 alpha in PRP obtained from aspirin-treated animals and in PRP from normal animals, both perfused through normal aortas, were the same, and also levels of the above metabolite in normal PRP perfused through aortas of aspirin-treated animals did not differ from those found in non perfused PRP. It is concluded, from these data, that PRP does not stimulate PGI2 formation in perfused aortas by providing cyclic endoperoxides. The experimental model developed allows the study of interactions between normal platelets and aortas from experimentally treated animals or viceversa.     

Cytochemical and functional characterization of blood and inflammatory cells from the lizard Ameiva ameiva

The fine structure and differential cell count of blood and coelomic exudate leukocytes were studied with the aim to identify granulocytes from Ameiva ameiva, a lizard distributed in the tropical regions of the Americas. Blood leukocytes were separated with a Percoll cushion and coelomic exudate cells were obtained 24 h after intracoelomic thioglycollate injection. In the blood, erythrocytes, monocytes, thrombocytes, lymphocytes, plasma cells and four types of granulocytes were identified based on their morphology and cytochemistry. Types I and III granulocytes had round intracytoplasmic granules with the same basic morphology; however, type III granulocyte had a bilobued nucleus and higher amounts of heterochromatin suggesting an advance stage of maturation. Type II granulocytes had fusiformic granules and more mitochondria. Type IV granulocytes were classified as the basophil mammalian counterpart based on their morphology and relative number. Macrophages and granulocytes type III were found in the normal coelomic cavity. However, after the thioglycollate injection the number of type III granulocyte increased. Granulocytes found in the coelomic cavity were related to type III blood granulocyte based on the morphology and cytochemical localization of alkaline phosphatase and basic proteins in their intracytoplasmic granules. Differential blood leukocyte counts showed a predominance of type III granulocyte followed by lymphocyte, type I granulocyte, type II granulocyte, monocyte and type IV granulocyte. Taken together, these results indicate that types I and III granulocytes correspond to the mammalian neutrophils/heterophils and type II to the eosinophil granulocytes.     

Stability of granulocytosis-promoting activity of post-pheresis plasma on freezing and thawing.

Pretreatment of rats with fresh post-pheresis plasma (PPP) obtained by filtration leukopheresis results in a significant increase in the yield of granulocytes. The practical application of this method in human beings requires a period of storage of the PPP without substantial loss of its activity. These studies demonstrate the maintenance of granulocytosis-promoting activity of PPP for at least 2 weeks after storage at ?150 °C.     

Platelet-vessel wall interactions: Effects of platelets and plasma on the antiaggregatory activity and 6 keto-PGF1α production in isolated perfused aortas

An experimental model for the study of platelet-vessel wall interactions has been developed, based on perfusion of rat platelet-rich plasma (PRP) through isolated rat aortas. In the perfused PRP, platelet aggregation was inhibited and levels of 6 Keto PGF_1α and cAMP were elevated over the values found in non perfused PRP. When PPP or buffer were perfused through the isolated artery, elevations of 6 Keto PGF_1α levels in the perfusate were smaller (in perfused PPP) or of shorter duration (in both perfused PPP and buffer). The presence of platelets in the perfusion fluids thus enhanced the formation of Prostacyclin by the arterial wall. Levels of 6 Keto PGF_1α in PRP obtained from aspirin-treated animals and in PRP from normal animals, both perfused through normal aortas, were the same, and also levels of the above metabolite in normal PRP perfused through aortas of aspirin-treated animals did not differ from those found in non perfused PRP. It is concluded, from these data, that PRP does not stimulate PGI₂ formation in perfused aortas by providing cyclic endoperoxides. The experimental model developed allows the study of interactions between normal platelets and aortas from experimentally treated animals or viceversa.     

Effects of intravenous IL-8 administration in nonhuman primates.

IL-8, a cytokine known for its potent and specific neutrophil activation and chemoattractant properties, has been recently detected in the circulation during septic shock, endotoxemia, and after IL-1 alpha administration. Because of its observed in vitro actions, it has been hypothesized that IL-8 may contribute to the dynamics of circulating granulocytes and to the pathologic sequelae seen in sepsis. Here, human rIL-8 is administered to healthy nonhuman primates as a single i.v. injection or as a continuous 8-h i.v. infusion. We demonstrate that both methods of i.v. administration result in a rapid but transient, severe granulocytopenia, followed by a granulocytosis that persists as long as IL-8 levels are detectable in the circulation. There were no hemodynamic changes after IL-8 administration, and animals remained clinically stable during the 24-h observation period. No detectable circulating TNF-alpha, IL-1 beta, or IL-6 response was induced by either IL-8 administration regimen. Histopathologic examination revealed mild to moderate neutrophilic margination in lung, liver, and spleen, of greater severity in baboons receiving the 8-h infusion. There was no associated neutrophilic infiltration or tissue injury. Thus, IL-8 modulates circulating granulocyte dynamics and likely directs their actions, but when administered i.v. to healthy animals, either as a bolus dose or as a continuous infusion for up to 8 h, does not induce the hemodynamic and metabolic aberrations or the acute organ damage seen during sepsis.     

The single cell origin of normal granulocyte colonies in vitro

It has been shown by re-cloning of colonies formed in vitro from rat bone marrow cells, that normal granulocyte colonies can originate from single cells. No mixed macrophage (M) and granulocyte (G) colonies were obtained after re-cloning either M or G colonies. The results indicate, that clones of normal granulocytes and macrophages can be obtained in vitro, and that the mixed primary M and G colonies formed after seeding hematopoietic cells from animals presumably originate from a mixture of M and G cells.