Kinetics of bone marrow eosinophilopoiesis and associated cytokines after allergen inhalation

Allergen inhalation is associated with increased eosinophil/basophil progenitors in bone marrow 24 hours after allergen inhalation. This study examined the kinetics of eosinophilopoiesis in dual (n = 14), compared with isolated early, responders (n = 12). Dual responders, in contrast to isolated early responders, develop significant sputum and blood eosinophilia and prolonged airway hyperresponsiveness. Bone marrow aspirates were taken before and 5, 12, 24, and 48 hours after allergen inhalation. In dual responders, increases in interleukin (IL)-3-responsive progenitors were detected as early as 5 hours after allergen inhalation, and IL-5-responsive progenitors were detected at 12 and 24 hours. No changes were detected in isolated early responders. Bone marrow IL-5 protein levels increased at 12 and 24 hours in dual responders only and these increases correlated with increases in IL-5-responsive progenitors. In addition, bone marrow IFN-gamma levels increased in dual responders at 48 hours. These data demonstrate that, in dual responders, there is allergen-induced activation of an eosinophilopoietic process that is rapid and sustained, and a relationship between increased bone marrow IL-5 levels and increased eosinophil production. We propose that after allergen inhalation, time-dependent changes in cytokine levels in the bone marrow control differentiation of eosinophil/basophil progenitors.     

Allergen-induced increases in bone marrow T lymphocytes and interleukin-5 expression in subjects with asthma

Inhaled allergen challenge of subjects with atopic asthmatic increases bone marrow eosinophil progenitor cells. Interleukin-5 (IL-5) specifically induces growth and maturation of eosinophils. This study examined the effect of allergen challenge on the number of bone marrow total and CD3+ cells expressing IL-5 protein and IL-5 mRNA in subjects with asthma who developed either allergen-induced isolated early responses, or early and late asthmatic responses (dual responders). At 24 hours after allergen challenge, dual responders had significantly greater blood and airway eosinophilia compared with early responders. There were significant increases in the percentage of bone marrow CD3+ cells (p < 0.005) in both groups. However, there were significant differences in the increases in bone marrow IL-5 mRNA+ (p < 0.005), CD3+ (p < 0.005), and IL-5 mRNA+ CD3+ (p < 0.005) cells between the dual and early responder groups. These results suggest that, in subjects with atopic asthma, inhaled allergen causes trafficking of T lymphocytes to the bone marrow, and that in subjects who develop late responses and greater blood and airway eosinophilia after inhalation of allergen, there is a significant increase in the ability of bone marrow cells, particularly T lymphocytes, to produce IL-5.     

The bone marrow aspirate of healthy subjects

Bone marrow aspirates were obtained from the right or left posterior superior iliac spine of 50 healthy volunteers, 30 men and 20 women. Reference ranges were derived for each cell type and for the myeloid : erythroid (M : E) ratio. The M : E ratio and the percentage of neutrophils were significantly higher in the women and the erythroid component significantly lower. All 28 evaluable men and 11/17 evaluable women had storage iron present in more than trace amounts. The percentage of erythroblasts with detectable iron granules varied very widely, from 3% to 69% in those with more than a trace of storage iron. Minor dyserythropoietic features were present in a high percentage of subjects and 19/50 subjects had one or two dysplastic megakaryocytes. Granulocytic dysplasia was not detected.     

过敏性哮喘与骨髓反应

过敏性哮喘是以嗜酸细胞在气道浸润为主的慢性炎症 ,激活的嗜酸细胞可分泌多种前炎性蛋白和脂质介质 ,在白三烯 (ＬＴＢ4 )和血小板激活因子 (ＰＡＦ)刺激下 ,它还可释放基质蛋白酶、嗜酸细胞阳离子蛋白、嗜酸细胞神经毒素等引起气道上皮破坏、平滑肌痉挛 ,粘液分泌增多[1 ] ,形成慢性气道炎症和气道高反应性 ,介导迟发性哮喘反应 (ＬＡＲ) [2 ] ;在哮喘患者外周血和气道中嗜酸细胞及其祖细胞明显增多 ,最近发现 ,由于抗原刺激气道后引起炎性介质释放或气道中炎细胞趋化至骨髓 ,引起骨髓中炎细胞的祖细胞 (主要是嗜酸 /嗜碱细胞祖细胞 )扩增…     

The effect of bone marrow transplantation on the osteoblastic differentiation of human bone marrow stromal cells.

Osteoporosis is a serious and relatively common complication of transplantation procedures. However, little is known about the exact mechanism or severity of osteoporosis complicated by bone marrow transplantation (BMT). We conducted both ex vivo and clinical studies to identify the mechanism and extent of bone loss after BMT. In a prospective clinical study, we intended to identify the changes in bone turnover markers and bone mineral density (BMD) after BMT. During a 1-yr follow-up, BMD was measured before BMT and 1 yr after BMT in 67 patients undergoing BMT. Biochemical markers of bone formation and resorption were measured in all patients at short-term intervals during the yearlong follow-up. In ex vivo study, we cultured human bone marrow cells of normal controls and BMT recipients in osteogenic medium and compared their osteogenic potential. Using a DNA fingerprinting method, we also investigated the origin of bone marrow stromal cells that were harvested 3-4 wk after BMT. In a clinical study of 67 patients undergoing BMT, the mean serum carboxy-terminal cross-linked telopeptide of type I collagen increased progressively until 4 wk after BMT. Thereafter, it began to decrease and reached basal values after 1 yr. Serum osteocalcin decreased progressively until 3 wk after BMT and reached basal values after 3 months. One year after BMT, lumbar spine BMD had decreased by 3.3% (P < 0.05), and total proximal femoral BMD had decreased by 8.9% (P < 0.001). For the ex vivo study, bone marrow was obtained from healthy donors (n = 7) and transplant recipients (n = 7). Then, mononuclear cells including marrow stromal cells were isolated and cultured to osteoblastic lineage. Alkaline phosphatase activities of each group were measured by the time course of secondary culture, and the mineralizing potentials were compared between the two groups. Cells cultured in our system showed characteristics of osteoblast-like cells differentiated from marrow stromal cells. They were initially in a fibroblastic-like spindle shape and became cuboidal with the formation of nodules that were later confluent. The cells were stained to both alkaline phosphatase histochemistry and Von Kossa histochemistry, demonstrating that these cells were of osteoblastic lineage differentiating from marrow stromal cells. The mean time required for the near-confluence in the primary culture was 15 and 22.9 d in healthy donors and transplant recipients, respectively (P = 0.003). Alkaline phosphatase activity was significantly lower in the bone marrow recipients than in the healthy donors at d 7 and 10 of the secondary cultures. The period at which peak activity of alkaline phosphatase was reached was also delayed in the osteoblasts derived from BMT recipient bone marrow compared with those of healthy donors. Using Von Kossa histochemistry, much more mineralization was observed in osteoblasts of healthy donors than those of BMT recipients. After BMT, although the peripheral mononuclear cells in the recipients were of donor origin, the bone marrow stromal cells were of recipient origin according to the PCR analysis using YNZ 22 mini-satellite probe. In conclusion, the differentiation of bone marrow stromal cells into osteoblasts was impaired after BMT, and this might contribute to post-BMT bone loss.     

The effects of Mycobacterium vaccae on allergen-induced airway responses in atopic asthma.

T-helper (Th)2 cytokines play a central role in asthma. Therefore, a double-blind randomised study was conducted to investigate whether heat-killed Mycobacterium vaccae (SRL172), a potent downregulator of Th2 cytokines, can reduce allergen-induced airway responses in patients with atopic asthma. A total 24 male asthmatics participated in this study. A bronchial allergen challenge was performed along with early (EAR) and late asthmatic responses (LAR) 2 weeks before and 3 weeks after a single intradermal injection of SRL172 or placebo. Before and after treatment, serum immunoglobulin (Ig)E levels and in vitro production of interleukin (IL)-5 by peripheral blood lymphocytes were studied. Neither treatment affected the EAR. SRL172 caused a mean 34% reduction of the area under the curve of the forced expiratory volume in one second (FEV1) changes during the LAR, which failed to reach conventional statistical significance when compared with placebo. SRL172 also caused a mean 25% decrease in the maximum fall in FEV1 during LAR, but this was not significantly different from placebo. SRL172 caused a reduction in serum IgE and IL-5 synthesis in vitro 3 weeks post-treatment (p = 0.07). This study shows a trend toward significance for the effects of heat-killed Mycobacterium vaccae (SRL172) on allergen-induced airway responses. Further clinical trials, involving multiple dosing, are needed.     

Expression of interleukin-5 by human bone marrow microvascular endothelial cells: implications for the regulation of eosinophilopoiesis in vivo

We have shown that bone marrow microvascular endothelial cells (BMEC) support growth and differentiation of haemopoietic progenitors in vitro by elaboration of haemopoietic cytokines. Since generation of eosinophils can be observed in these coculture experiments, and BMEC do not produce interleukin (IL)-3, we evaluated BMEC for expression of IL-5, a specific growth factor for the eosinophilic lineage. Using RT-PCR, IL-5 mRNA was expressed by BMEC after stimulation (12 h) with lipopolysaccharide (LPS), IL-1, IL-2 and phorbol myristate acetate (PMA), but not by resting BMEC, after stimulation with TNF-α or interferon (IFN)-γ. Moreover, IFN-γ suppressed expression of IL-5 in response to LPS and IL-2. The identity of the PCR products was confirmed by restriction enzyme digestion, which resulted in fragments of the predicted size. T lymphocytes were not present in the endothelial cultures as demonstrated by absence of CD2 mRNA. Using a sensitive (1 pg/ml) ELISA assay, IL-5 was detected after 48 h incubation of BMEC with IL-2 (4.1 pg/10⁶ cells) or with a combination of LPS and IL-2 (4.8 pg). However, the number of eosinophils generated after 4 weeks coculture of CD34⁺ haemopoietic cells with BMEC was not increased by addition of IL-2. RT-PCR revealed that BMEC in coculture with haemopoietic cells expressed IL-5 even without addition of exogenous cytokines or stimulating agents. In conclusion, expression of IL-5 by BMEC can be stimulated by cytokines (IL-1, IL-2), LPS, PMA, and coculture with proliferating haemopoietic cells. Thus, BMEC may support proliferation and differentiation of eosinophils in the bone marrow. IFN-γ represents a cytokine with an inhibitory effect on IL-5 expression by BMEC. In addition, eosinophilia in response to circulating IL-2 or bacterial products (LPS) in vivo may be partially mediated by BMEC or vascular endothelium.     

The effect of hyperinflation on respiratory muscle strength and efficiency in healthy subjects and patients with asthma

We evaluated respiratory muscle strength and efficiency in 15 patients with asthma. There was a significant reduction in mean Plmax (89.3 +/- 4.7 versus 110.5 +/- 9.4 cm H2O, p less than 0.001) and efficiency (2.41 +/- 0.2 versus 4.2 +/- 0.6%, p less than 0.01). This reduction in strength and efficiency was seen only in the male patients. Following bronchodilator, there was a significant increase in Plmax (from 89.3 +/- 4.7 to 96.2 +/- 5.4 cm H2O, p less than 0.005) and efficiency (from 2.41 +/- 0.2 to 3.22 +/- 0.2%, p less than 0.001). There was no correlation between the change in strength and efficiency and the degree of improvement in FEV1 following bronchodilator. However, there was a significant correlation with the fall in lung volume. To determine whether hyperinflation would result in a reduction in respiratory muscle strength and efficiency, we induced a mean increase in end-expiratory lung volume of 0.66 L by applying continuous negative pressure around the chest in 10 healthy individuals. This was associated with a significant fall in Plmax (from 110.5 +/- 9.4 to 100.5 +/- 8.93 cm H2O, p less than 0.001) and efficiency (from 4.2 +/- 0.6 to 2.6 +/- 0.5%, p less than 0.005). The data suggest that the strength and efficiency of the respiratory muscles are reduced in asthmatic males but not in the females. The strength and efficiency of the respiratory muscles improve significantly following bronchodilator, and this improvement is related to reduction in lung volume.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on bone marrow in vitro; the effect of anoxia and hyperoxia on explanted bone marrow

The effect of various oxygen tensions on explanted bone marrow fragments wasstudied. It was found that gas mixtures containing 1, 3, 5, 10 and 12 per cent oxygenhave an injurious effect on hemic cells. Bone marrow maintained in these gas mixtures showed various degrees of degeneration, which was the more pronounced thelower the oxygen tension. Mitotic activity was also found to be reduced under theinfluence of low oxygen tension.

Bone marrow cultures maintained in a gas mixture containing 15 per cent oxygendid not show appreciable changes and were similar to the controls.

Increased rate of maturation and multiplication occurred in bone marrow cultures maintained in an excess of oxygen, i.e. 50 per cent.

The significance of these findings in the light of observations on the effect ofanoxia in vivo has been discussed, and reported findings on the effect of low oxygentensions on other tissues in vitro have been briefly reviewed.

     

The effect of G-CSF on the composition of human bone marrow

The effects on bone marrow cellularity and morphology of 6 days' treatment with granulocyte colony-stimulating factor (G-CSF) in 35 patients were studied. Examination of trephine biopsies showed a highly significant increase in cellularity (P < 10-13). Assessment of aspirates revealed an increase in the myeloid to erythroid (M : E) ratio (P = 0.00006), the proportion of myeloid cells (P < 10-8), myelocytes (P = 0.00007), metamyelocytes (P = 0.04), band forms (P = 0.0005) and neutrophils (P = 0.02). This study presents a comprehensive analysis of the effects of six days' administration of G-CSF on human bone marrow.     

The effect of challenge method on sensitivity and reactivity to adenosine 5'-monophosphate in subjects with suspected asthma

BACKGROUND: The following two methods of inhalation challenge have been used to determine the airway responsiveness: the tidal-breathing method; and the dosimeter method. The objective of the study was to determine the influence of the challenge method on the response to adenosine 5'-monophosphate (AMP). METHODS: This study measured airway responsiveness to AMP by dosimeter and tidal-breathing methods in 25 subjects with suspected asthma. The two AMP challenges were conducted in random order, on separate days, at the same time of day, at intervals of 1 to 5 days. Concentration-response curves were characterized by the provocative concentration of a substance causing a 20% fall in FEV1 (PC20) and slope. RESULTS: The dosimeter PC20 values were significantly higher than the tidal-breathing PC20 values, with geometric mean (95% confidence interval [CI]) values of 50.35 mg/mL (95% CI, 19.50 to 129.72 mg/mL) and 28.97 mg/mL (95% CI, 11.99 to 69.98 mg/mL; p = 0.02), respectively. The mean difference in the PC20 values obtained with each method was 0.80 doubling concentrations (95% CI, 0.16 to 1.44 doubling concentrations). The mean values for the slope were 17.0%/log mg/mL (95% CI, 12.5 to 21.5 mg/mL) with the tidal breathing method and 13.8%/log mg/mL (95% CI, 9.0 to 18.7 mg/mL; p = 0.03) with the dosimeter. CONCLUSIONS: The tidal-breathing method produces AMP PC20 values that are significantly lower than the dosimeter method and slope values that are significantly higher than the dosimeter method. These data suggest that the results obtained with each method of testing may not be comparable.     

The effect of lithium on growth factor production in long-term bone marrow cultures

We have previously reported that lithium chloride (LiCl) stimulates the production of granulocyte-macrophage colony-forming cells (GM-CFC), pluripotent stem cells (CFU-S), and differentiated granulocytes, macrophages and megakaryocytes in murine Dexter marrow cultures and that this effect appears to be mediated indirectly by a radioresistant adherent marrow cell. In this study we have established that exposure of murine Dexter cultures to LiCl (4 mEq/L) causes an increase of colony-forming cell megakaryocytes (CFU-meg) over 1 to 6 weeks of culture in both supernatant (188% to 611%) and stromal phases (123% to 246%). Moreover, we have shown that lithium treatment of either irradiated (1,100 rad) or unirradiated stromal cells increased production of activities stimulating formation of megakaryocyte, granulocyte, macrophage, and mixed lineage colonies and proliferation of the factor-dependent cell line, FDC-P1. This FDC-P1 stimulatory activity was completely blocked by an antibody to purified recombinant granulocyte-macrophage colony stimulating factor (rGM-CSF). The baseline or lithium-induced--stromal-derived bone marrow colony stimulating activity was partially blocked by the antibody to rGM-CSF and by an antibody to purified colony stimulating factor I (CSF-1); the two antibodies combined resulted in greater than 90% inhibition of the lithium-induced marrow stimulatory activity. In addition, radioimmunoassay (RIA) showed that although CSF-1 was detectable in supernatants of these cultures, exposure to lithium did not increase CSF-1 levels. These data indicate that Dexter stromal cells produce CSF- 1 and GM-CSF and that lithium appears to exert its stimulatory effects on in vitro myelopoiesis by inducing production of GM-CSF.     

The effect of oral diltiazem on airway reactivity to methacholine and exercise in subjects with mild intermittent asthma

The effect of increasing doses of oral diltiazem on airway reactivity to methacholine was evaluated in 10 volunteers with mild asthma. Then the highest tolerated dose was compared with placebo in preventing exercise-induced bronchoconstriction. Methacholine challenges were performed 1 h before and 100 min after placebo or after 30, 60, 90, 120, or 180 mg of oral diltiazem, given in a single-blind, crossover manner on different days within 2 wk. Diltiazem, at doses above 60 mg prolonged the P-R interval of the electrocardiograph but had no significant effect on FVC, FEV1, or FEF25-75. The mean +/- SEM ratio of the dose of methacholine required to produce a 20% decrease in FEV1 (PD20) after diltiazem to the PD20 before diltiazem, i.e., the fold increase in PD20, was not significantly different from placebo at any dose: 0.93 +/- 0.11 after placebo, 1.2 +/- 0.1 after 30 mg, 1.3 +/- 0.3 after 60 mg, 1.2 +/- 0.2 after 90 mg, 1.1 +/- 0.1 after 120 mg, and 1.0 +/- 0.1 after 180 mg. One hundred minutes before a standardized exercise challenge, 120 to 180 mg of oral diltiazem and identically appearing placebo tablets were administered in a randomized, double-blind, crossover design on separate days at least 48 h apart. The mean +/- SEM maximal postexercise decrease in FEV1 was 25.5 +/- 3.3% after placebo and 17.0 +/- 4.8% after diltiazem (p less than 0.01). There was no correlation between change in FEV1 and serum concentrations of diltiazem or its active metabolite desacetyldiltiazem.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of recombinant GM-CSF on the recovery of monkeys transplanted with autologous bone marrow

The regulatory function of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) on granulocyte production in vivo was evaluated in an autologous bone marrow transplantation model using rhesus monkeys. Monkeys were exposed to 9.0 Gy total body irradiation and then transplanted with 5.0 x 10(7) low-density bone marrow cells/kg. Alzet miniosmotic pumps were subcutaneously implanted to deliver rhGM-CSF at a rate of 50,400 U/kg/d. Minipumps, containing either rhGM-CSF or saline, were implanted between zero and five days after transplantation for seven days. Kinetic recoveries of peripheral blood cells after either saline or rhGM-CSF treatment were compared. Treatment with rhGM-CSF accelerated the recovery of neutrophils. Neutrophils in rhGM-CSF-treated animals recovered to 80% (3.4 x 10(3)/mm3) pre-irradiation control levels by day 20, in comparison with only 33% (0.9 x 10(3)/mm3) recovery for saline control monkeys. In addition, the recovery of neutrophils was enhanced over that of the controls, reaching 140% v 70% on day 30. Another prominent feature of rhGM-CSF-treated monkeys was the accelerated recovery of platelets, reaching near 50% normal levels by day 24 in comparison with 20% of normal levels for controls. The infusion of rhGM-CSF was shown to be an effective regulator of early hematopoietic regeneration, leading to the accelerated recovery of both neutrophils and platelets and then providing a consistent sustained increase of neutrophils even in the absence of rhGM-CSF.     

Churg-Strauss syndrome after reduction of inhaled corticosteroid in a patient treated with pranlukast for asthma

Recently, various forms of Churg-Strauss syndrome (CSS) have been reported in association with the use of leukotriene receptor antagonists. A 53-year-old woman with a 5-year history of asthma associated with chronic sinusitis presented mononeuropathy, hypereosinophilia, and positive P-ANCA in October 1999. She had been treated with pranlukast (450 mg/day) and beclomethasone dipropionate (BDP) at a dose of 1,200 microg/day which had gradually been tapered to 800 microg/day over the previous 17 months. She was found to have CSS, and 60 mg/day of prednisolone was administered instead of pranlukast, resulting in an improvement of her symptoms and eosinophilia. Later, we confirmed that serum P-ANCA had been positive before the pranlukast treatment, but CSS vasculitis had not appeared at that time. We speculated that an underlying incomplete form of CSS was being masked in this case and that the reduction of inhaled corticosteroid might have been responsible for the unmasking of CSS.