Glucocorticoid receptor mediates the expansion of splenic late erythroid progenitors during chronic psychological stress.

Stress evokes an integrated neuroendocrine response perturbing the homeostasis of different physiological systems. In contrast to well established physiologica linteractions between neuroendocrine and immune systems during chronic stress, there has been relatively little information on the effects of psychological stress on erythroid cells. Since stress-induced erythropoiesis occurs predominantly in the spleen, in the current study, we investigated the influence of chronic psychological stress on splenic erythroid progenitors and examined a role of glucocorticoid receptor (GR) in observed effect using a mouse model of restraint. The adult male mice were subjected to 2 hours daily restraint stress for 7 or 14 consecutive days and the role of GR in erythropoietic response to stress was assessed by pretreatment of mice with GR antagonist mifepristone 60 min prior to restraint. The results showed that chronic restraint stress induced an increase in spleen weight as well as in the cellularity of red pulp, as compared to controls. Furthermore, 7 and 14 days of restraint stress resulted in markedly increased number of both splenic early (BFU-E) and late (CFU-E) erythroid progenitors. Blockade of GR with mifepristone did not affect the number of BFU-E in stressed mice, but it completely abolished the effect of repeated psychological stress on CFU-E cells. Additionally, plasma corticosterone concentration was enhanced whereas the GR expression was significantly decreased within splenic red pulp after one and two weeks of stress exposure. Obtained findings suggest for the first time an indispensable role for GR in the expansion of CFU-E progenitors in the spleen under conditions of chronic psychological stress.

Effects of IL-17 on erythroid progenitors growth: involvement of MAPKs and GATA transcription factors.

Interleukin-17 is Th17 cell cytokine implicated in regulation of hematopoiesis and inflammation. Besides promoting granulopoiesis, we have previously shown that IL-17 also affects erythropoiesis stimulating the development of early erythroid progenitors, BFU-E, but suppressing, at least partly via p38 MAPK, the growth of late stage erythroid progenitors, CFU-E. The aim of the present study was to investigate the involvement of other MAPKs, JNK and ERK1/2, as well as GATA transcription factors, in IL-17-mediated effects on murine bone marrow erythroid progenitors. Data obtained by use of specific MAPKs inhibitors indicated that MEK1/2-ERK1/2 MAPK signaling mediates IL-17-induced CFU-E inhibition, as well as that JNK and/or MEK1/2-ERK1/2 MAPKs activation underlies IL-17-induced stimulation of BFU-E growth. Furthermore, Western blot analyses demonstrated no effect on early hematopoiesis transcription factor, GATA-2, and enhanced expression level of erythroid-specific factor GATA-1 in murine bone marrow cells after IL-17 stimulation, which in light of previous reports that GATA-1 overexpression inhibits erythroid differentiation, could be related to IL-17-mediated inhibition of CFU-E growth. Although, no contribution for p38, JNK and ERK MAPKs in IL-17-induced GATA-1 expression was shown, data obtained using specific inhibitors pointed to the role of JNK and MEK1/2-ERK1/2 in GATA-1 downregulation. Overall, obtained data gave an insight into the mechanisms by which IL-17 exerts its effects on erythropoiesis, implying the involvement of JNK and ERK MAPKs, as well as GATA-1, in IL-17-regulated growth of erythroid progentors.

Combined effect of IL-17 and blockade of nitric oxide biosynthesis on haematopoiesis in mice.

AIM: The study was undertaken to extend our investigation concerning both the in vivo activity of interleukin (IL)-17 and the specific role of nitric oxide (NO) in IL-17-induced effects in the process of haematopoiesis. METHODS: CBA mice were simultaneously treated with IL-17 and/or nitric oxide synthase (NOS) inhibitor, l-NAME, for 5 days and changes within various haematopoietic cell lineages in bone marrow, spleen and peripheral blood were analysed. RESULTS: Findings showed that administration of both IL-17 and l-NAME stimulated increase in net haematopoiesis in normal mice. IL-17-enhanced myelopoiesis was characterized by stimulation of both femoral and splenic haematopoietic progenitor cells and morphologically recognizable granulocytes. Additionally, IL-17 induced alterations in the frequency of erythroid progenitor cells in both bone marrow and spleen, accompanied with their mobilization to the peripheral blood. As a consequence of these changes in the erythroid cell compartments, significant reticulocytosis was observed, which evidenced that in IL-17-treated mice effective erythropoiesis occurred. Exposure of mice to NOS inhibitor also increased the number of both granulocyte-macrophage and erythroid progenitors in bone marrow and spleens, and these alterations were followed by the mobilization of erythroid progenitors and elevated content of reticulocytes in peripheral blood. The specific role of NO in IL-17-induced haematopoiesis was demonstrated only in the IL-17-reducing effect on bone marrow late stage erythroid progenitors, CFU-E. CONCLUSION: The results demonstrated the involvement of both IL-17 and NO in the regulation of haematopoietic cell activity in various haematopoietic compartments. They further suggest that IL-17 effects are differentially mediated depending on the haematopoietic microenvironments.

The effect of interleukin-17 on hematopoietic cells and cytokine release in mouse spleen.

To evaluate whether the response of hematopoietic cells to interleukin-17 (IL-17) depends on the tissue microenvironment in which hematopoiesis occurs, the influence of recombinant mouse IL-17 on spleen hematopoietic cells and cytokine release was assessed in normal mice in vitro and in vivo. In vitro, IL-17 did not significantly affect the growth of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E and CFU-E) derived colonies. A single injection of IL-17 in vivo exhibited stimulatory effects on hematopoietic cells from both granulocytic and erythroid lineages. The increased number of metamyelocytes 48 h after treatment imply to the IL-17-induced stimulation of granulopoiesis. The number of BFU-E was increased at 24 h, while the number of CFU-E increased 6 h and 24 h after treatment. Since the same treatment in the bone marrow decreased the number of CFU-E, it may be concluded that the local microenvironment plays an important role in IL-17-mediated effects on CFU-E. IL-17 increased the release of IL-6 both in vitro and in vivo, but showed tendency to suppress the constitutive secretion of IL-10 by spleen cells. Our results suggest the complexity of target cell response and interplay of secondary induced cytokines by IL-17 in different hematopoietic organs.

In vivo effects of interleukin-17 on haematopoietic cells and cytokine release in normal mice.

In order to gain more insight into mechanisms operating on the haematopoietic activity of the T-cell-derived cytokine, interleukin-17 (IL-17) and target cells that first respond to its action in vivo, the influence of a single intravenous injection of recombinant mouse IL-17 on bone marrow progenitors, further morphologically recognizable cells and peripheral blood cells was assessed in normal mice up to 72 h after treatment. Simultaneously, the release of IL-6, IL-10, IGF-I, IFN-gamma and NO by bone marrow cells was determined. Results showed that, in bone marrow, IL-17 did not affect granulocyte-macrophage (CFU-GM) progenitors, but induced a persistant increase in the number of morphologically recognizable proliferative granulocytes (PG) up to 48 h after treatment. The number of immature erythroid (BFU-E) progenitors was increased at 48 h, while the number of mature erythroid (CFU-E) progenitors was decreased up to 48 h. In peripheral blood, white blood cells were increased 6 h after treatment, mainly because of the increase in the number of lymphocytes. IL-17 also increased IL-6 release and NO production 6 h after administration. Additional in vitro assessment on bone marrow highly enriched Lin- progenitor cells, demonstrated a slightly enhancing effect of IL-17 on CFU-GM and no influence on BFU-E, suggesting the importance of bone marrow accessory cells and secondary induced cytokines for IL-17 mediated effects on progenitor cells. Taken together, these results demonstrate that in vivo IL-17 affects both granulocytic and erythroid lineages, with more mature haematopoietic progenitors responding first to its action. The opposite effects exerted on PG and CFU-E found at the same time indicate that IL-17, as a component of a regulatory network, is able to intervene in mechanisms that shift haematopoiesis from the erythroid to the granulocytic lineage.

Hematopoiesis during acute Toxoplasma gondii infection in mice.

We studied hematopoiesis in bone marrow and blood of CBA mice following infection with Toxoplasma gondii. Our data showed that acute infection with the virulent RH strain was associated with leucopenia, thrombocytopenia and bone marrow hypoplasia while, in spite of the infection-induced damage of the granulocyte cell lineage, in bone marrow stimulated production of granulocytes was revealed. In peripheral blood, T. gondii infection caused a significant decrease in the total number of white blood cells, reticulocytes and platelets. However, the relative proportion of granulocytes and lymphocytes was changed in favor of granulocytes, as compared to pre-infection levels. The functional activity of granulocytes was also increased. The bone marrow alterations were characterized by a decrease in the total number of nucleated cells due to the reduced numbers in all cell compartments of erythroid and megakaryocytic lineage, as well as in the number of mature granulocytes and lymphocytes. In contrast, femoral granulocytic proliferative compartments, colony forming unite granulocyte-macrophage (CFU-GM) and morphologically recognizable proliferative granulocytes (PG), exhibited stimulated granulopoiesis, while the number of mature monocytes was close to the control value. In summary, we have shown that acute T. gondii infection results in profound alterations of the hematopoietic system that markedly contribute to the clinical onset of the disease and the, ultimately lethal, outcome.

Effect of IL-17 on in vitro hematopoietic progenitor cells growth and cytokine release in normal and post-irradiated murine bone marrow.

The influence of recombinant human IL-17 on granulocyte-macrophage (CFU-GM) and erythroid (BFU-E and CFU-E) progenitors and the release of IL-1alpha/beta, IL-6 and erythropoietin (EPO) was estimated in the bone marrow cells obtained from normal and sublethally irradiated mice. In normal mice IL-17 increased CFU-GM and BFU-E and reduced CFU-E derived colonies numbers and augmented release of IL-6 and EPO. In irradiated mice the effects of IL-17 on hematopoietic progenitors were lineage-dependent, as well as dependent on their stage of differentiation and the time after the irradiation. IL-17 had no major effects on CFU-GM on day 1 and 3, but decreased their number on day 2, while enhanced both BFU-E and CFU-E on day 1 and 2 after irradiation, whereas on day 3 its effect on erythroid progenitors was again as observed in normal mice. After irradiation, IL-17 increased the release of IL-1alpha, IL-6 and EPO. The observed effects suggested the involvement of IL-17 in the regulation of hematopoiesis and indicated that its effects on both hematopoietic progenitors and cytokine release are dependent on the physiological/pathological status of the organism.

Peripheral blood granulocyte activity following contact sensitization of rats with dinitrochlorobenzene.

Contact hypersensitivity (CHS) reaction is a classic example of a cell-mediated reaction. As the afferent phase of the reaction includes inflammation, CHS is a suitable model for investigating non-specific immunity. Some aspects of granulocyte activity in the afferent phase of experimentally induced CHS to dinitrochlorobenzene (DNCB) in two genetically different rat strains, AO and DA were examined in this study. A shift in the ratio of granulocytes to lymphocytes in favour of granulocytes and an increase in granulocyte survival were noted in DA rats. Granulocytes from both strains demonstrated increased levels of NBT reduction and an increase in their adhesion to plastic. Decreased granulocyte adhesion in the presence of monoclonal antibodies to beta2 integrins (anti-CD11b/c and anti-CD18) points to the contribution of these molecules to granulocyte adhesiveness during the sensitization phase of CHS. Stimulation of adhesion in the presence of anti-CD11a antibody, points to a differential modulation of adhesion molecule activity during the afferent phase of CHS. Changes in functional activity of granulocytes demonstrated in this study might contribute to the development of CHS in rats.

Effects of treatment with interleukin-1 receptor antagonist on endogenous interleukin-1 levels in normal and irradiated mice.

The in vivo effects of recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) administration on endogenous IL-1 levels in the circulation and conditioned media (CM) from different immunohematopoietic organ/tissues were studied in CBA mice under steady state and postirradiation conditions. In normal mice, constitutive IL-1 levels were demonstrated in the plasma, CM of peritoneal exudate cells and full-thickness skin explants with low or undetectable levels in CM of splenic and bone marrow cell suspensions. In irradiated mice (2 Gy, X rays) on day 3 post exposure a significant increase of IL-1 levels was seen in the circulation and CM of peritoneal exudate cells, with no significantly different levels in postirradiation bone marrow, spleen and skin. After rhIL-1Ra treatment of the animals (2 x 50 microg/mouse, i.p.), significantly elevated IL-1 levels were observed in the skin and CM of peritoneal exudate cells in normal mice, whereas slightly increased levels were detected in CM of splenic cells. The rhIL-1Ra administration in irradiated mice led to decreased IL-1 concentrations in the circulation, and CM of peritoneal exudate cells and skin. The results pointed out the importance of IL-1 secretion and receptor expression in the maintenance of homeostasis in steady state, as well as during recovery after irradiation. Modulatory effects of IL-1Ra on IL-1 production were dependent on basic endogenous IL-1 concentration.

Donor leukocyte infusion--the effect of mutual reactivity of donor's and recipient's peripheral blood mononuclear cells on hematopoietic progenitor cells growth.

Donor leukocyte infusions are an effective therapy for patients who relapse with leukemia after bone marrow transplantation. We report the case of 14-year-old boy who relapsed 34 months after sibling donor bone marrow transplant for Philadelphia-positive chronic myeloid leukemia. Subsequently, he received three infusions of donor mononuclear cells (DMNC) harvested in steady state hematopoiesis and one G-CSF mobilized-peripheral blood mononuclear cells (PBMC) infusion. Simultaneously, test named as--"Test of Mixed Progenitors" (TMP) was performed for the assessment whether the outcome of donor leukocyte infusion treatment could be predicted. Prior to DMNC infusions, the CFU-GM and BFU-E colony assays were performed for donor's and recipient's PBMC individually, as well as for the mixture of these cells at 1:1 ratio. The cells were plated either directly in the semisolid medium or after 24 h preincubation treatment. Significantly lower values for CFU-GM derived colonies were determined in TMP in comparison to the CFU-GM values obtained for the recipient's cells. The reduced number of CFU-GM was determined both in TMP performed without preincubation treatment, app. 80% and after the 24 h preincubation, app. 55%. The reduced number of BFU-E derived colonies (app. 44%) was observed only related to recipient's cells and after the preincubation treatment of the cells. The patient did not develop GVHD and currently (40 months after the first infusion). He remained well in complete hematological, cytogenetic, molecular and clinical remission, which was the most direct evidence of the GVL effect. The novel in vitro TMP test in which the specific contribution of donor's leukocytes to the growth of recipient's hematopoietic precursor cell growth was determined, correlated with the clinical outcome.

Cytochemical & ultrastructural alteration of cytoplasmic granules of rat peripheral blood neutrophils induced by chronic alcoholism & malnutrition.

The specific influence of malnutrition on the pathophysiologic changes induced by chronic alcoholism is controversial. In an attempt to determine and demarcate the effects of protein malnutrition from those produced by alcoholism and to evaluate the precise effect of alcohol per se on cytochemical and ultrastructural properties of rat polymorphonuclear neutrophil (PMN) granules, we investigated the influence of chronic protein malnutrition or chronic alcoholism alone and in combination, in rats. After a 4 month experimental period various PMN properties, such as cytochemical, morphometrical and ultrastructural, as well as neutrophil functions were studied. It was found that the degree of damage of PMNs induced either by ethanol or protein malnutrition alone was similar whereas their combination led to worsening of all markers of PMN functional ability. Ultrastructural changes of neutrophil granules including reduction, redistribution and atypical accumulation as well as appearance of autophagic vacuoles, confirmed their alteration which was emphasised by the additive pathophysiological interaction of alcoholism and chronic hypoprotein malnutrition.

The cryopreservation protocol optimal for progenitor recovery is not optimal for preservation of marrow repopulating ability.

The efficiency of five different cryopreservation protocols (our original controlled-rate and noncontrolled-rate protocols) was evaluated on the basis of the recovery after thawing of very primitive pluripotent hemopoietic stem cells (MRA(CFU-GM), pluripotent progenitors (CFU-Sd12) and committed granulocyte-monocyte progenitors (CFU-GM) in mouse bone marrow. Although the nucleated cell recovery and viability determined immediately after the thawing and washing of the cells were found to be similar, whether controlled-rate or noncontrolled-rate cryopreservation protocols were used, the recovery of MRA(CFU-GM), CFU-Sd12 and CFU-GM varied depending on the type of protocol and the cryoprotector (DMSO) concentrations used. It was shown that the controlled-rate protocol was more efficient, enabling better MRA(CFU-GM), CFU-Sd12 and CFU-GM recovery from frozen samples. The most efficient was the controlled-rate protocol of cryopreservation designed to compensate for the release of fusion heat, which enabled a better survival of CFU-Sd12 and CFU-GM when combined with a lower (5%) DMSO concentration. On the contrary, a satisfactory survival rate of very primitive stem cells (MRA(CFU-GM)) was achieved only when 10% DMSO was included with a five-step protocol of cryopreservation. These results point to adequately used controlled-rate freezing as essential for a highly efficient cryopreservation of some of the categories of hematopoietic stem and progenitor cells. At the same time, it was obvious that a higher DMSO concentration was necessary for the cryopreservation of very primitive stem cells, but not, however, for more mature progenitor cells (CFU-S, CFU-GM). These results imply the existence of a mechanism that decreases the intracellular concentration of DMSO in primitive MRA cells, which is not the case for less primitive progenitors.

The in vivo effect of liposomes on hematopoiesis.

The influence of liposome structure on hematopoiesis in vivo was assessed in relation to the different contents and origins of phospholipids that make up their membrane structures. Changes within different hematopoietic cells and serum tumor necrosis factor alpha (TNF-alpha) levels were estimated up to 14 days following intravenous administration of liposomes made of either pure egg yolk phosphatidylcholine (LEY) or a soybean phospholipid preparation (LSB) into normal CBA mice. In peripheral blood, only transient changes within white blood cells were observed. In bone marrow, a persistent decline in the number of mature granulocytes, monocytes, and lymphocytes was found. The changes within femoral granulocytic proliferative compartments in various stages of differentiation and a maturation compartment pointed out that, parallel with the depletion of the granulocyte-storage pool, stimulation of de novo production of granulocytic cells occurred. Although both types of tested liposomes induced similar cellular changes, only liposomes made of pure egg yolk phosphatidylcholine induced a transient increase in serum TNF-alpha levels.

Evaluation of cryopreserved murine and human hematopoietic stem and progenitor cells designated for transplantation.

The influence of five different cryopreservation protocols on the quality and/or quantity of frozen cells was investigated on mouse bone marrow cells and human peripheral blood mononuclear cells (MNC). The efficiency of the protocols was evaluated on the basis of the recovery of very primitive pluripotent hematopoietic stem cells (MRA), pluripotent progenitors (CFU-Sd12), committed granulocyte-monocyte progenitors (CFU-GM) of mouse cells after thawing. The recovery of MRA, CFU-Sd12 and CFU-GM varied depending on the type of freezing procedure and cryoprotector (DMSO) concentrations used. It was shown that the controlled-rate protocol was more efficient, enabling better recovery of all categories of progenitor cells in frozen samples. The most efficient was the controlled-rate protocol of the cryopreservation designed to compensate for the release of fusion heat, which enabled the best recovery of CFU-GM (73.0 +/- 8.8%) and CFU-Sd12 (90.0 +/- 15.9%) when combined with 5% DMSO concentration (protocol 4). On the contrary, a better recovery (79.8 +/- 13.5%) of very primitive stem cells (MRA) was achieved only when the higher concentration (10%) DMSO was used in combination with a five-step protocol of cryopreservation (protocol 1). These results pointed out the adequately used controlled-rate freezing to be essential for a highly efficient cryopreservation of some of the categories of hematopoietic stem and progenitor cells. At the same time, it was obvious that a higher DMSO concentration was necessary for the cryopreservation of MRA, but not for more mature progenitor cells (CFU-S, CFU-GM). These results imply the existence of a mechanism that decreases the intracellular concentration of DMSO in MRA cells, which is not the case in less primitive progenitors. For human MNC, the recovery and viability of the cells, as well as the engraftment potential of cryopreserved cells after thawing were investigated. Cryopreservation protocol 1 resulted in better MNC recovery (82.7 +/- 10.4%) than protocol 3 (49.9 +/- 15.1%). The mean recovery of MNCs (collected from patients for autologous transplantation) was 78.5 +/- 7.3% (protocol 1) and 53.1 +/- 26.2% (protocol 3). The obtained favorable recovery of thawed cells and rapid reconstitution of hematopoiesis (on the day 11th following the transplantation) in patients confirmed that the controlled-rate freezing in combination with optimal DMSO concentration was able to obtain sufficient progenitor cryoprotection.

Alpha- and beta-globins of the anemic Belgrade laboratory rat. I. Status of alpha- and beta-globins in bone marrow and spleen.

In this study we have demonstrated that the bone marrow of the anemic Belgrade laboratory (b/b) rat, as the primary site of erythropoiesis, has a decreased globin polypeptide synthesis in total protein cell extracts. Therefore, it is the source of red blood cells containing decreased amounts of globin mRNAs and polypeptides. In spite of the fact that the b/b rat shows a splenomegaly, the spleen is not capable of compensating for the anemic state. Spleen erythroid cells are defective in differentiation and contain a decreased share of globin polypeptides in total protein cell extracts compared to the control. Spleen cells are also characterized by a drastic imbalance of alpha- to beta-globin resulting in the beta-globin chains surplus.

The influence of acute sterile inflammation on erythropoiesis in rats.

Many different cell types, coordinated by proinflammatory mediators, take part in the acute inflammatory reaction, but there is a lack of evidence regarding the role of erythroid cells in such conditions. In this study, changes in bone marrow, splenic, and peripheral blood erythroid cells and in erythropoietin (Epo) blood levels were investigated up to 72 hours after polyvinylpyrrolidone (PVP)-induced sterile inflammation in male Wistar rats (two intraperitoneal injections of 15 mL 3.5% PVP at 18-hour intervals). Transient changes within progenitor erythroid cells were observed in the bone marrow. Significant increases in the number of splenic immature erythroid progenitors (BFU-E) 6 hours and mature erythroid progenitors (CFU-E), erythroblasts, and orthochromatic erythroblasts 48 and 72 hours after the induction of inflammation pointed to stimulated splenic erythropoiesis. This was confirmed by semiquantitative assessment of splenic smears, which demonstrated expansion of erythroid cells at hours 48 and 72. The changes observed in the bone marrow and spleen indicated that during acute inflammation erythropoiesis was stimulated and that the spleens of PVP-treated rats were favorable to erythroid development. The significant increase in the percentage of peripheral blood reticulocytes 48 and 72 hours after PVP-induced inflammation provided evidence that effective erythropoiesis occurred. In spite of the stimulated erythropoiesis, serum levels of Epo remained unchanged, implying that other non-Epo regulatory molecules may be responsible for erythroid cellular changes.

Granulocyte and plasma cytokine activity in acute cadmium intoxication in rats.

Changes in the number and ex vivo function of peripheral blood neutrophils were investigated following intraperitoneal administration of cadmium-chloride in rats. Besides a dose-dependent increase in the number of peripheral blood neutrophils, changes were found in the functional state of isolated polymorphonuclear leukocytes (PMNs). Increased spontaneous adhesion and activation, and TNF activity in a conditioned medium were observed in cultures of granulocytes in comparison to granulocytes from control (saline-treated) animals. Increased levels of plasma activity of inflammatory cytokines, tumor necrosis factor (TNF) and interleukin-6 (IL-6) were noted following cadmium administration. Cytological signs of pulmonary inflammation were revealed histologically and the majority of neutrophils recovered from the lungs by enzyme digestion exhibited a capacity of nitroblue tetrazolium (NBT) reduction. Our data demonstrate that acute cadmium intoxication leads to a systemic inflammatory response characterized by numerical and functional changes in the granulocyte compartment and to increased levels of inflammation-related cytokine activity in the circulation. Correlations between the increased number of peripheral blood neutrophils and IL-6 plasma activity (r=0.776, p<0.00001) and the number of neutrophils recovered from the lung tissue (r=0.893, p<0.00001) suggested that systemic cadmium-induced inflammation might be involved in the pulmonary toxicity of cadmium.

Effect of a single dose of ethanol on granulopoiesis in female rats: relationship to phase of estrous cycle.

OBJECTIVE: The purpose of this study was to investigate the acute effect of ethanol (4g/kg, IP) on granulopoiesis at two phases of the rat estrous cycle, proestrus and diestrus Day 1. METHOD: The following parameters were estimated: in peripheral blood, the ethanol concentration, progesterone and estradiol levels, the total number of WBC and differential count: in the bone marrow, the total number of nucleated cells, the number of granulocyte-macrophage committed stem cells (CFU-GM) and differential count at various time points (5, 3, 6 and 20 hours) after treatment. The experiments were conducted twice and 4 to 7 rats were used per groups for each time point. RESULTS: The results indicated that a single dose of ethanol significantly increased the number of granulocytes and decreased the number of lymphocytes in peripheral blood. These changes were observed earlier at the proestrus compared to the diestrus Day 1, and were consistent with faster ethanol disappearance from blood during the proestrus. Additionally, the ethanol treatment induced a significant increase in progesterone levels at both phases. This effect was prolonged at the diestrus Day 1 and thus was also associated with differences in ethanol metabolism. In the bone marrow, the total number of nucleated cells and morphologically recognizable hematopoietic cells were not affected by ethanol treatment at any of the observed time points. However, at both phases of the estrous cycle ethanol treatment induced an increase in the number of CFU-GM derived colonies 20 hours after administration. CONCLUSIONS: The data obtained suggest active involvement of the granulocytic cell line in response to acute ethanol administration which is modulated by the current hormone status of the treated animals.

The effect of IL-1 receptor antagonist on the proliferation of hematopoietic progenitor cells in regenerating bone marrow.

To evaluate the involvement of IL-1 on bone marrow granulocyte-macrophage (CFU-GM) and erythroid (BFU-E and CFU-E) progenitor cell regeneration during the recovery of hematopoiesis after sublethal irradiation of CBA mice, we examined the effects of IL-1 receptor blockade by recombinant human IL-1 receptor antagonist (rhIL-1ra). The actual number of progenitors and proportion of these cells in S phase of the cell cycle were determined in regenerating bone marrow cells obtained 3 days after 2 Gy irradiation both following the in vivo administration of rhIL-1ra, as well as after the in vitro preincubation with increasing amounts of rhIL-1ra. The results revealed that rhIL-1ra decreased the number and the proportion of CFU-GM in the S phase in regenerating bone marrow. As concerning erythroid progenitors, rhIL-1ra treatment suppressed BFU-E and enhanced CFU-E-derived colony growth, indicating that the biological effects of IL-1 might be different depending on the stage of differentiation. The observed effects pointed to the importance of the basal levels of IL-1, as well as IL-1 receptor expression during the recovery of hematopoiesis.

In vivo effects of interleukin-1 receptor antagonist on hematopoietic bone marrow progenitor cells in normal mice.

The multiple effects of interleukin-1 (IL-1) on hematopoietic cells are mainly documented in disturbed hematopoiesis, but its production and participation during constitutive hematopoiesis are still unproven. To assess the involvement of IL-1 in the regulation of steady-state hematopoiesis in vivo, we have investigated the consequences of IL-1 receptor blockade by recombinant human IL-1 receptor antagonist (rhIL-1Ra) in normal CBA/H mice treated with two i.p. injections of rhIL-1Ra (2 x 50 micrograms/mouse) seventeen and two hours before sacrifice. The cellularity, the number of granulocyte-macrophage (CFU-GM), the number of erythroid (BFU-E) progenitor cells and the percentage of these cells in S phase of the cell cycle, as well as the morphologically recognizable cells in bone marrow were estimated. In peripheral blood, hematocrit, the number and differential count of nucleated cells, the number erythrocytes and the percentage of reticulocytes were determined. IL-1Ra treatment significantly reduced the number of femoral CFU-GM and BFU-E, while all the other analyzed parameters were not different from the level obtained in control, non-treated animals. These findings show that a number of bone marrow IL-1-responsive cells were affected by the IL-1 receptor blockade, indicating that the expression of IL-1 receptors and endogenous IL-1 secretion occur as part of constitutive hematopoiesis.