Effects of rhG-CSF on neutrophil functions and bone marrow parameters in diabetic rats

Neutrophils have an important role in the host defense. The elevated serum glucose levels of diabetics affect traditional host defenses such as neutrophil counts and functions. The causes of these impairments are not clear. We aimed to investigate changes of peripheral neutrophil counts and functions and their relation with bone marrow cells in diabetic rats. Thirty-two rats were divided into four equal groups. Group 1 were controls and Groups 2 and 4 were made diabetic by a single intraperitoneal injection of streptozotocin. Granulocyte colony stimulating factor (G-CSF) was injected subcutaneously into Groups 3 and 4. White blood cell count, neutrophil counts and function and bone marrow cell count were determined. Peripheral blood cell counts, neutrophil phagocytosis index were decreased but neutrophil adhesivity index was not different in the diabetes-induced group. There was a difference in circulating white blood cell counts and neutrophil counts between the rhG-CSF treated and non-treated groups. The phagocytosis index of neutrophil in diabetic rats was significantly diminished by rhG-CSF treatment. A hyperplasia of early cells of the myeloid series in G-CSF treated groups was observed when compared with those of nontreated groups (p<0.001). A significant decrease was noted in the number of mature marrow segmented cells diabetic groups (p<0.001). Finally, G-CSF has been shown to cause neutrophilia by acting as a releasing factor for mature marrow neutrophils in diabetic rats. These results suggest that G-CSF may be used to improve nonspecific immunity in diabetic patients.     

Regulation of neutrophil homeostasis

PURPOSE OF REVIEW: Neutrophils are an essential component of the innate immune response and a major contributor to inflammation. Consequently, neutrophil number in the blood is tightly regulated. Herein, we review recent studies that have greatly advanced our understanding of the mechanisms controlling neutrophil homeostasis. RECENT FINDINGS: Accumulating evidence shows that stromal derived factor-1 (CXCL12) through interaction with its major receptor CXCR4 provides a key retention signal for neutrophils in the bone marrow. Granulocyte colony-stimulating factor induces neutrophil release from the bone marrow, in major part, by disrupting stromal derived factor-1/CXCR4 signaling. Granulocyte colony-stimulating factor expression is regulated by a novel feedback loop that senses neutrophil emigration into tissues. Specifically, engulfment of apoptotic neutrophils by tissue phagocytes initiates a cytokine cascade that includes interleukin-23, interleukin-17, and ultimately granulocyte colony-stimulating factor. SUMMARY: Granulocyte colony-stimulating factor plays a central role in the dynamic regulation of neutrophil production and release from the bone marrow in response to environmental stresses. Recent studies have begun to elucidate both the pathways linking neutrophil clearance to granulocyte colony-stimulating factor expression and the mechanisms by which the factor induces neutrophil release from the bone marrow. These studies may lead to novel strategies to modulate neutrophil responses in host defense and inflammation.     

Morphologic and functional heterogeneity of chronic neutropenia of childhood with normal neutrophil colony formation in vitro

In order to obtain further knowledge of chronic neutropenia of childhood, we studied nine neutropenic infants six to ten months of age by in vitro techniques, including bone marrow culture, electron microscopy, and chemotaxis assay. Eight of the nine patients had a benign clinical course and the bone marrow aspirates showed a reduced number of segmented neutrophils. The ninth patient had a moderately severe course and the bone marrow showed maturation arrest at the promyelocyte stage. Bone marrow cultures demonstrated that the in vitro neutrophil colony formation and production of colony-stimulating activity were normal in all of the eight patients studied. Neutrophils from one of the nine patients had ultrastructural abnormalities such as a decrease in number of primary and secondary granules and the presence of myelin figures in primary granules. Neutrophil chemotaxis was defective in three of the nine patients. All of the six patients in whom the neutrophil colony formation in agar, the ultrastructure of neutrophils, and neutrophil chemotaxis were normal recovered from the neutropenia between 11 and 30 months of age. These in vitro parameters appear to be useful for evaluating chronic neutropenia of childhood.     

Migration across the sinusoidal endothelium regulates neutrophil mobilization in response to ELR + CXC chemokines

An increase in circulating neutrophils is a characteristic feature of many inflammatory reactions and is a result of the rapid mobilization of neutrophils from the bone marrow, driven by inflammatory mediators, including the ELR + CXC chemokines. In this paper, using a combination of light and electron microscopy and an in situ perfusion system of the rat femoral bone marrow, we examined this mobilization process in detail. We show that mobilization of neutrophils stimulated by the CXC chemokine, rat MIP-2, involves neutrophil migration from the haematopoietic compartment of the bone marrow across the bone marrow sinusoidal endothelium via a transcellular route. The critical role of the bone marrow sinusoidal endothelium in regulating neutrophil mobilization was demonstrated by artificially disrupting the bone marrow endothelial barrier by treatment with cytochalasin D, which results in the non-selective release of leucocytes from the bone marrow. In contrast, inhibiting the activity of p38 mitogen-activated protein kinase, inhibited both MIP-2 stimulated chemotaxis of bone marrow neutrophils in vitro and neutrophil mobilization in situ while, a broad spectrum matrix metalloproteinase inhibitor, BB94, had no effect on neutrophil mobilization. These results support the hypothesis that neutrophil migration drives their mobilization and highlights the function of the sinusoidal endothelium in regulating this process.     

Transplantation of HLA-haploidentical T-cell-depleted marrow for leukemia: autologous marrow recovery with specific immune sensitization to donor antigens

Autologous marrow recovery without engraftment of donor marrow was observed after bone marrow transplantation (BMT) for two patients with acute lymphoblastic leukemia. Each had received marrow from a haploidentical mixed lymphocyte culture (MLC) reactive donor after pretransplant conditioning with total body irradiation and high-dose cyclophosphamide. To minimize graft-vs-host disease, the marrow was depleted of T cells in vitro by treatment with a monoclonal anti-T-cell antibody and complement. Two weeks after each transplant, reactive lymphocytes were noted transiently in the blood of each patient. Analysis of karyotype, HLA type, and in vitro MLC responsiveness proved the lymphocytes to be of host, not donor, origin. MLC studies showed rapid proliferative responses specifically to stimulating cells from the BMT donor, indicating in vivo sensitization to donor antigens. Return of hematopoietic function was markedly delayed, but it eventually normalized after several months, without evidence of chimerism. These studies confirm that some immune and hematopoietic stem cells of host origin survive the high-dose chemoradiotherapy used as transplant conditioning. Because these immune cells are specifically reactive to donor alloantigens, more potent suppression of host immunity may be needed to prevent nonengraftment of T-cell-depleted, HLA-mismatched bone marrow.     

Function of peripheral blood and bone-marrow monocytes in preleukemic patients: normal phagocytosis and intracellular killing of Candida albicans

Studies were performed to evaluate the function of peripheral blood and bone marrow monocytes from 15 patients with preleukemia and 16 healthy controls. The patients were grouped according to the criteria of the FAB collaborative group. No abnormality in phagocytosis and killing of Candida albicans by peripheral blood and bone marrow monocytes was found in patients compared to normal controls. Normal opsonization by autologous serum was found. No differences were found in this respect between the three groups of patients.     

STAT3 governs distinct pathways in emergency granulopoiesis and mature neutrophils

Granulocyte colony-stimulating factor (G-CSF) is essential for the host response to bacterial infection by controlling neutrophil production in the bone marrow. The G-CSF receptor (G-CSFR) activates the Jak/STAT pathway, although little is understood about how these signals regulate basal and stress-induced granulopoiesis. We examined STAT3 function in granulocytes using a bone marrow conditional knockout mouse model. Our results show that STAT3 has a crucial role in emergency granulopoiesis and mature neutrophil function. STAT3-deficient mice have an aberrant response to G-CSF in vivo, characterized by failure to accumulate immature granulocytes and an increased ratio of mature to immature neutrophils in the bone marrow, peripheral blood, and spleen. Acute neutrophil mobilization is impaired in STAT3-deficient mice as judged by their failure to up-regulate circulating neutrophils following short-term G-CSF exposure. STAT3 also controls neutrophil chemotactic responses to natural ligands for CXCR2 and regulates the magnitude of chemoattractant-induced actin polymerization. These functions of STAT3 are independent of its principal target gene Socs3, which encodes a crucial feedback inhibitor of cytokine signaling. Our results demonstrate the existence of distinct STAT3 target pathways in neutrophils required for granulopoiesis and innate immunity.     

Neutrophil kinetics in chronic neutropenia.

Quantitative studies of bone marrow neutrophil pool sizes and production rates and of blood neutrophil kinetics were performed in 16 patients with chronic neutropenia without splenomegaly. Marrow netrophil cellularity was determined from a ferrokinetic estimate of marrow normoblasts and from neutrophil-erythroid ratios determined from marrow sections. Postmitotic pool turnover was derived from the postmitotic pool size and transit time, the latter determined from 3H-thymidine neutrophil emergence time. Blood neutrophil kinetics were studied with 32P-diisopropylfluoophosphate-labeled autologous neutrophils. Mitotic pool size was basal or below basal in 12 of the 16 patients. The turnover of the post-mitotic neutrophils was subbasal in 6, basal in 7, and above basal in 3 patients. Blood neutrophil turnover was within the normal range in 8 patients and decreased in 8. The degree of ineffective granulocytopoiesis was assessed by comparing the relative size of the mitotic pool, postmitotic pool turnover, and blood turnover. On this basis, 13 of the 16 patients showed appreciable degrees of ineffective granulocytopoiesis. Ineffective neutrophil production occurred both early and late in neutrophil development. These studies indicate that most patients with chronic neutropenia without splenomegaly lack a proliferative marrow response to the neutropenia and suggest that ineffective granulocytopoiesis is a common feature of this disorder.     

Neutrophil function and pyogenic infections in bone marrow transplant recipients.

In a consecutive entry trial, the incidence and time course of decreased neutrophil function was assessed in 20 patients treated with allogeneic bone marrow transplantation (BMT). The aim of the study was to assess the prognostic value of low neutrophil function for late pyogenic infections. Chemotaxis, superoxide production, and phagocytic-bactericidal activity were studied before and 2, 6, 9, and 12 months after BMT. Skin window migration was quantitatively assessed 2 months after BMT. Infectious complications were recorded prospectively with preset criteria during 1 year. Six of the 20 leukemic patients had defective neutrophil function before BMT. Two months after BMT all 10 patients with greater than stage II graft-versus-host disease (GVHD), and 6 of 10 patients with less than or equal to stage II GVHD had at least one decreased function. At this time, patients with subsequent pyogenic infections had lower chemotaxis (P less than .05), phagocytic-bactericidal activity (P less than .005), and superoxide production (P less than .025) than those without. Defective skin window migration and combined defects were predictive for late pyogenic infections. At 9 months all tests were normal in seven patients surviving without GVHD. In contrast, at 9 months three of three patients, and at 1 year two of three with chronic GVHD had still decreased neutrophil function. In conclusion, neutrophil function is frequently impaired during the first months after BMT. Combined neutrophil defects predispose to pyogenic infections and indicate the patient at risk.     

Antibody incubation of human marrow graft for prevention graft versus host disease

Summary An in vitro incubation of incompatible donor bone marrow by xenogenic anti-T-cell globulin (ATG) suppressed an otherwise lethal GvH reaction in animal models. An application of this principle to clinical bone marrow transplantation was successfully tried in three patients with acute lymphoblastic leukemia. Preparation of the specific anti-human T-cell globulin (ATCG-H) was carried out by absorption of anti-human thymocyte globulin with liver-kidney homogenate, chronic lymphocytic leukemia cells of B-cell type, and erythrocytes. Subsequent testing revealed that the serum still reacted with human T-cells but no longer reduced the number of colony-forming units in culture (CFU-C).All three bone marrow recipients were treated by chemotherapeutic conditioning and total body irradiation followed by grafting of in vitro treated bone marrow from HLA-identical siblings. The transplantation of the bone marrow was well tolerated and no major side effects were encountered. No patient so far (24, 7, 6 months) has shown any signs of GvHD. The in vitro pretransplantation treatment of bone marrow with anti T-globulin may be a new approach to the prevention for GvHD in man.Supported by Deutsche Krebshilfe, Bonn and Deutsche Forschungsgemeinschaft, SFB 37, B 7, E 9     

Recombinant human granulocyte colony-stimulating factor therapy for patients with neutropenia and/or neutrophil dysfunction secondary to glycogen storage disease type 1b

The purpose of this study was to evaluate the efficacy and toxicity of recombinant human granulocyte colony-stimulating factor (rhG-CSF) therapy in patients with neutropenia and/or neutrophil dysfunction secondary to glycogen storage disease (GSD) type 1b. Thirteen patients with neutropenia and/or neutrophil dysfunction secondary to GSD type 1b were treated with rhG-CSF. The effects of therapy on neutrophil numbers and in vitro neutrophil function and on bone marrow cellularity and morphology were studied. The clinical status of the patients and the occurrence of adverse events associated with rhG-CSF use were monitored. Use of rhG-CSF therapy was associated with a significant increase in circulating neutrophil numbers (P <. 01) and an improvement in neutrophil function as assessed in vitro. In addition, rhG-CSF therapy produced a significant increase in marrow cellularity and an increase in myeloid:erythroid (M:E) ratio, indicating stimulation of granulopoeisis. No adverse effects on marrow function were noted; in particular, no myelodysplasia or marrow exhaustion was seen. Use of rhG-CSF therapy was associated with objective and subjective improvements in infection-related morbidity. The therapy was well tolerated, although all patients developed splenomegaly, and 5 patients developed mild hypersplenism that did not require any specific treatment. rhG-CSF therapy is efficacious in the management of neutropenia and neutrophil dysfunction associated with GSD type 1b. Patients on this therapy need to be monitored for hypersplenism. Continued follow-up will be necessary to confirm long-term safety; however, no significant short-term toxicity was noted.     

Discontinuation of immunosuppression for prevention of kidney graft rejection after receiving a bone marrow transplant from the same HLA identical sibling donor

Induction of tolerance in solid organ transplant recipients has been a long sought goal so that patients will not need lifelong immunosuppression. In this case report we review a patient who received a kidney transplant from an HLA matched related sibling and developed acute leukemia as a consequence of her immunosuppression. The patient was then treated with an allogeneic bone marrow transplant from her kidney donor. After the bone marrow transplant, all immunosuppression therapy for graft rejection and graft versus host disease was stopped. Six months after the bone marrow transplant, the patient's kidney function had no deterioration as a consequence of stopping immunosuppression. This illustrated that a combined solid organ/bone marrow transplant can help to induce tolerance. In fact, the tolerance to the bone marrow transplant for prevention of graft versus host disease may have been accomplished by the prior kidney transplant.     

Neutrophil marrow in chronic benign idiopathic neutropenia

Quantitative studies of neutrophil marrow were carried out in 10 patients with chronic neutropenia (60 to 1,970 cells/μl) with no other abnormalities and no serious infections. Neutrophil marrow cellularity was determined from neutrophil-normoblast ratios in marrow sections and ferrokinetic estimation of normoblasts. The results were interpreted in the light of 95 per cent confidence limits previously observed in 13 normal volunteer subjects. Three distinct neutrophil marrow profiles were determined by the numbers of promyelocytes and myelocytes and of segmented marrow cells. Tentative kinetic interpretation was based on the expectation that the physiologic marrow response to removal of neutrophils from circulation would produce an increase in promyelocytes and myelocytes due to influx and proliferation, and a decrease in marrow segmented cells due to accelerated release. In two patients increased segmented marrow cells were consistent with an abnormality of release. Decreased numbers of promyelocytes and myelocytes in three patients was consistent with decreased proliferation. In five patients basal numbers of promyelocytes and myelocytes suggested abnormal proliferation or abnormal regulation of myelopoiesis. The number of metamyelocytes and band forms relative to promyelocytes and myelocytes was normal in all 10 patients: none had evidence of cell loss during postmitotic maturation. The term “chronic benign idiopathic neutropenia” appears to embrace more than one mechanism for neutropenia. All 10 patients had evidence of abnormal neutrophil marrow function.     

Thymus and T cells are not essential for rat leucopoiesis

It is generally held that T lymphocytes take part in the regulation of haemopoiesis. We have examined whether, in vivo, the influence of thymus of functional T cells is dispensable for the steady-state or accelerated formation of granulocytes (and in some experiments macrophages), utilizing a rat model. Untreated normal and athymic 'nude' rats had similar blood and marrow granulocyte counts and marrow proliferative activity. Bone marrow regeneration after two cytotoxic insults to the two kinds of rats gave no clues to an important regulatory role for thymic factors or T cells. Nor were such clues obtained in experiments where bone marrow cells from normal rats were cultured in vivo in diffusion chambers (DC), in either normal rats or rats undergoing a graft-versus-host (GvH) reaction (with supranormal serum levels of cytokines). On the other hand, when marrow cells from athymic rats were similarly cultured in DC, small but significant differences in leucopoiesis occurred between the three kinds of DC hosts: Marrow cells lacking functional T cells generated fewer proliferative granulocytes and had a lower proliferative activity when cultured in athymic than in normal hosts, whereas the proliferative granulocytes were most numerous in chambers carried by GvH rats. No differences were found for macrophages and eosinophilic granulocytes. The results indicate that thymic hormones or T cells or both can stimulate granulopoiesis. They apparently play no indispensable role in short-term leucopoiesis, however, since their influence was weak in our experimental models. Consequently, marked effects seen in vitro in simplified cellular systems may lose some importance in the more complex in vivo setting.     

Granulopoiesis in the alcoholic and cirrhotic patient. Exploration of neutropenia in cirrhosis using bone marrow cultures and a hydrocortisone test

Twenty per cent of 60 patients with cirrhosis (55 alcoholic patients) had neutropenia (defined as neutrophil polymorphonuclear (NP) count under 2,000/mm3). The authors tried to define the mechanism of this neutropenia and compared it with the acute bone marrow cytotoxicity of alcohol. They found that bone marrow neutrophil colony growth was normal in 7 cases of cirrhosis compared to 7 controls (with and without cirrhotic serum incubation). On the other hand, the granulocyte response to hydrocortisone test was less marked than in controls. This result suggests involvement of the late maturation process of NP and delayed release from bone marrow.     

成人Still's病的血液学特点

目的总结成人Still’s病(AOSD)的血液学特点。方法回顾性对比分析15例AOSD和败血症患者外周血常规检查主要参数,并就AOSD骨髓细胞形态学描述归类。结果AOSD组外周血白细胞数、中性粒细胞比值和中性粒细胞绝对值、血小板计数分别为18.3±9.5×109/L,82.6%±10.4%,15.9±9.3×109/L,306.4±115.1×109/L,均较败血症组明显升高。但仅中性粒细胞绝对值和血小板计数具有显著和极显著的统计学差异。AOSD组骨髓象表现为粒系统显著增生,大部分粒细胞胞浆存在中毒性颗粒。结论AOSD患者血液学特点类似于感染性疾病,也有其独自的特征。结合临床分析有助于AOSD诊断和鉴别诊断。     

Restriction fragment length polymorphisms as markers of engraftment in allogeneic marrow transplantation

We have used DNA hybridization techniques employing restriction fragment length polymorphisms (RFLPs) to quantitate the level of donor cell engraftment in bone marrow transplantation recipients. The genetic origin of the bone marrow cells and various peripheral blood populations was analyzed in 14 patients. We found at least one informative polymorphism for each donor-recipient pair. Additional markers of engraftment included cytogenetic analysis, HLA typing, and red cell typing. By DNA analysis, four patients had complete engraftment, five had partial engraftment, and five had no evidence of donor cell engraftment. In three cases, DNA analysis permitted detection of minor populations (5% to 10%) of donor or host cells. Eight of fourteen patients were evaluable for chimerism posttransplant by cytogenetic analysis. In five cases, cytogenetic results were completely concordant with DNA analyses. In two cases of apparent autologous recovery, as assessed using RFLPs, a small number of cells of donor karyotype was seen. In one other case, a small number of cells of host karyotype was not detected by RFLP studies. HLA typing in three partially engrafted patients was purely either of donor or host type. Red cell typing was discordant with DNA and/or cytogenetic results in four of eight cases. We conclude that DNA analysis at a limited number of informative genetic loci is useful for quantitating the degree of engraftment in multiple populations of nondividing cells following allogeneic bone marrow transplantation.     

Mechanisms of resistance of Aspergillus fumigatus Conidia to killing by neutrophils in vitro

Despite the critical role for neutrophils in host defenses against invasive aspergillosis, previous studies have established that neutrophils are unable to kill resting conidia of Aspergillus fumigatus. The mechanisms of resistance of the conidia were therefore investigated. Electron microscopy studies showed the fusion of phagosomes containing A. fumigatus conidia with lysosomes of the neutrophil. Resting conidia of A. fumigatus were then compared with those that had been preincubated in broth until swollen, but not germinated, as well as with blastospores of Candida albicans (two fungal forms that are killed by neutrophils) and zymosan particles. Despite comparable susceptibility to phagocytosis, resting conidia of A. fumigatus stimulated production of significantly less superoxide anion, hydrogen peroxide, and hypochlorous acid and induced less myeloperoxidase-dependent iodination by neutrophils than did the preincubated conidia of A. fumigatus, blastospores of C. albicans, or zymosan particles. In addition, resting conidia of A. fumigatus were relatively resistant to cell-free killing by oxidants presumed to be generated by neutrophils. Thus, resistance of resting conidia of A. fumigatus to neutrophil fungicidal mechanisms appears to be secondary to both failure of the conidia to stimulate an optimal respiratory burst as well as resistance of the conidia to neutrophil oxidants. However, the reversal of this resistance by preincubation of the conidia suggests that neutrophils still may form an important host defense against the conidia of A. fumigatus.     

Function of Neutrophils in Preleukaemia

The function of blood neutrophil granulocytes was studied in vitro in 17 patients with preleukaemia. 3 patients had a cellular defect of chemotaxis. 2 of them had monosomy-7 in bone marrow karyotype, in 1 associated with the deletion of the long arm of a chromosome 20. The third patient had trisomy-8. In the patient with trisomy-8, the high percentage of band neutrophils was possibly associated with the chemotactic defect. In another patient with trisomy-8 chemotaxis was normal. There was a statistically significant tendency to reduced phagocytosis and impaired ability to kill Staphylococcus aureus. 1 patient with a chemotactic defect and monosomy-7 suffered from repeated infections. The other 2 patients with defective chemotaxis had several febrile episodes most probably of infectious origin, and 1 of them died in sepsis. All of these 3 patients had cutaneous abscesses. It is concluded that defects in neutrophil granulocyte function are not uncommon in preleukaemia and may result in reduced resistance to infection.     

Apparent Folate Deficiency in Iron-Deficiency Anaemia

S ummary . Examination of 50 patients with iron-deficient hypochromic anaemia showed evidence suggesting a high incidence of folate depletion. The peripheral blood films of 44 patients (88%) showed more than 3% of neutrophils with five nuclear lobes, 35 patients (70%) had a high mean neutrophil lobe count, 20 patients (40%) had hypersegmented neutrophils and 19 patients (38%) had giant metamyelocytes in the bone marrow. The serum folate was below 3 ng/ml in 12 patients and 3–6 ng/ml in 18. Red-cell folate was subnormal in 15%, and 45% had a positive Figlu test.
Correlation between various tests for folate deficiency was not found, apart from a correlation between red-cell folate levels and morphological changes in the neutrophils. The haemoglobin rise following intravenous iron therapy was smaller when the serum folate level was low. There was probably a similar relationship to the red-cell folate level, but the numbers tested were small. The presence of neutrophil multilobing in the peripheral blood film and giant metamyelocytes in the bone marrow did not influence the response to iron therapy, neither did an abnormal Figlu excretion.
Following intravenous iron therapy, both neutrophil multilobing and marrow giant metamyelocytes were significantly reduced in number. This therapy did not significantly alter Figlu excretion measured 6 weeks after treatment, but both serum and red-cell folate levels fell. Intravenous iron therapy did not produce any significant changes in the serum vitamin B₁₂ levels. The part that iron deficiency itself may play in causing apparent folate depletion is discussed.