Physical association between the high-affinity IgG receptor (Fc gamma RI) and the gamma subunit of the high-affinity IgE receptor (Fc epsilon RI gamma).

To investigate the structural basis of transmembrane signaling via the high-affinity IgG receptor (Fc gamma RI), the identity of Fc gamma RI-associated proteins in THP-1 human monocytic cells was examined. Anti-Fc gamma RI monoclonal antibody (mAb) 197 immunoprecipitates from 125I-labeled THP-1 cells solubilized in 1% digitonin buffer were found to contain a protein migrating at 12 kDa on reduction. This protein comigrated with the 12-kDa protein precipitated by the anti-high-affinity IgE receptor gamma chain (Fc epsilon RI gamma) mAb 4D8. Similarly, a 70-kDa band immunoprecipitated by mAb 4D8 comigrated with the 70-kDa protein band corresponding to Fc gamma RI in mAb 197 immunoprecipitates. On two-dimensional nonreducing-reducing gel analysis, the 12-kDa protein present in both mAb 197 and mAb 4D8 immunoprecipitates migrated as a disulfide-linked homodimer. Analysis of 1% Nonidet P-40 eluates of the digitonin immunoprecipitates under reducing conditions demonstrated the presence of a 12-kDa band in the mAb 197 immunoprecipitate that could be reprecipitated with mAb 4D8. Conversely, a 70-kDa band in the mAb 4D8 immunoprecipitate could be reprecipitated with mAb 197. Similar to these findings, both mAb 197 and mAb 4D8 precipitated a 12-kDa disulfide-linked homodimeric protein from digitonin lysates of 125I-labeled human neutrophils after induction of Fc gamma RI expression with interferon gamma but not from unstimulated neutrophils. Northern blot analysis confirmed the presence of Fc epsilon RI gamma mRNA in interferon gamma-induced human neutrophils. We conclude that Fc epsilon RI gamma, a member of a family of proteins implicated in transmembrane signaling via immune recognition receptors, associates with Fc gamma RI in human cells.     

FcR gamma-chain is essential for both surface expression and function of human Fc gamma RI (CD64) in vivo

Most Ig receptors exist as hetero-oligomeric complexes with separate ligand binding (alpha) and signal transducing (beta, gamma, or zeta) subunits. For Fc gamma RIIIa and Fc epsilon RI, association with the FcR gamma-chain is essential for surface expression. However, the human high affinity IgG receptor, hFc gamma RI, was found to be surface- expressed by itself in transient transfection models. We have now analyzed the integrity of hFc gamma RI expression in more detail in stable transfectants. In vitro we noted that, in the absence of FcR gamma-chain, surface expression of hFc gamma RI rapidly declined to background levels, in both IIA1.6 B cells and NIH3T3 fibroblasts. The effect of FcR gamma-chain on hFc gamma RI surface expression in vivo was evaluated by using two newly generated transgenic mouse lines, selectively expressing hFc gamma RI on myeloid cells. These transgenic mice were crossed with FcR gamma-chain-deficient mice. Analysis of blood monocytes and peritoneal macrophages showed that surface expression of hFc gamma RI was reduced by approximately 80%. The remaining approximately 20% of receptors were still capable of binding IgG-opsonized RBC, suggesting FcR gamma-chain not to be critical for hFc gamma RI ligand-binding capacity. Importantly, however, hFc gamma RI signaling capacity was lost in FcR gamma-chain-deficient cells. No phagocytosis could be observed using either ligand sensitized (EA- IgG2a) or CD64-targeted erythrocytes (using a bispecific antibody) in both hFc gamma RI transgenic lines. This documents the FcR gamma-chain to be indispensable for both surface membrane expression and function of human Fc gamma RI in vivo.     

Association of the high-affinity receptor for IgG (Fc gamma RI) with the gamma subunit of the IgE receptor.

How receptors for the Fc portion of IgG antibodies (Fc gamma R) trigger a variety of immune functions by clustering upon engagement of ligand is largely unknown. Of the three distinct classes of human Fc gamma R, only Fc gamma RIIIA has been shown to associate with a potential signal-generating subunit, either the gamma chain or the zeta chain. With these studies we show that Fc gamma RI, the high-affinity receptor for IgG, also is found in association with gamma chain. This association can be demonstrated by using anti-Fc gamma RI antibodies, Fc gamma RI ligand, and anti-gamma-chain antibodies that coadsorb the proteins from detergent lysates of Fc gamma RI-expressing cells. The association of Fc gamma RI and gamma chain can be reconstituted by cotransfection of cDNAs for both proteins into COS cells.     

Endotoxin down-modulates granulocyte colony-stimulating factor receptor (CD114) on human neutrophils

During infection, the development of nonresponsiveness to granulocyte colony-stimulating factor (G-CSF) may be influenced by the down-modulation of G-CSF receptor (G-CSFR) by cytokines. This down-modulation was studied during experimental human endotoxemia. Healthy volunteers received either 2 ng/kg endotoxin (lipopolysaccharide [LPS], n=20) or placebo (n=10) in a randomized, controlled trial. Endotoxin infusion increased the mean fluorescence intensity of the neutrophil activation marker CD11b >300% after 1 h (P<.001 vs. placebo). LPS infusion down-modulated G-CSFR expression in as early as 60 min (-17%; P=.001 vs. placebo). Down-modulation was almost maximal at 90 min and persisted for 6 h (-50% from baseline; P<.0001 vs. placebo). Plasma levels of G-CSF started to increase only after G-CSFR down-modulation had occurred and peaked 37-fold above baseline at 4 h (P<.0001 vs. placebo). In conclusion, LPS down-modulates G-CSFR expression in humans, which may render neutrophils less responsive to the effects of G-CSF and, thereby, compromise host defense mechanisms.     

In vivo stimulation of granulopoiesis by recombinant human granulocyte colony-stimulating factor.

Osmotic pumps containing Escherichia coli-derived recombinant human granulocyte colony-stimulating factor (rhG-CSF) were attached to indwelling jugular vein catheters and implanted subcutaneously into Golden Syrian hamsters. Within 3 days, peripheral granulocyte counts had increased greater than 10-fold with a concomitant 4-fold increase in total leukocytes. Microscopic examination of Wright-Giemsa-stained blood smears from rhG-CSF hamsters showed that only the neutrophil subpopulation of granulocytes had increased. No significant changes in lymphocyte or monocyte counts were observed during the course of continuous rhG-CSF treatment. After subcutaneous injection at rhG-CSF doses of up to 10 micrograms X kg-1 X day-1 only granulocyte counts were affected. However, at higher dose levels, a transient thrombocytopenia was noted. Erythrocyte had lymphocyte/monocyte counts remained unaffected by rhG-CSF over the entire dose range (0.3-300 micrograms X kg-1 X day-1) studied. Total leukocyte counts increased 3-fold within 12 hr after a single s.c. injection of rhG-CSF. This early effect was associated with an increase in the total number of colony-forming cells and the percent of active cycling cells in the marrow. A sustained elevation of peripheral leukocyte and marrow progenitor counts was observed following seven daily s.c. injections of rhG-CSF. The ability of rhG-CSF to increase the production and release of granulocytes from the marrow may underlie the beneficial effect it produced on the restoration of peripheral leukocyte counts in hamsters made leukopenic by treatment with 5-fluorouracil.     

Purified murine granulocyte/macrophage progenitor cells express a high-affinity receptor for recombinant murine granulocyte/macrophage colony-stimulating factor

Purified recombinant murine granulocyte/macrophage colony-stimulating factor (GM-CSF) was labeled with 125I and used to examine the GM-CSF receptor on unfractionated normal murine bone marrow cells, casein-induced peritoneal exudate cells, and highly purified murine granulocyte/macrophage progenitor cells (CFU-GM). CFU-GM were isolated from cyclophosphamide-treated mice by Ficoll-Hypaque density centrifugation followed by counterflow centrifugal elutriation. The resulting population had a cloning efficiency of 62-99% in cultures containing conditioned medium from pokeweed mitogen-stimulated spleen cells and 55-86% in the presence of a plateau concentration of purified recombinant murine GM-CSF. Equilibrium binding studies with 125I-labeled GM-CSF showed that normal bone marrow cells, casein-induced peritoneal exudate cells, and purified CFU-GM had a single class of high-affinity receptor with an approximate Ka of 10(8)-10(9) M-1. CFU-GM expressed an average of 3783 +/- 4 receptors per cell; normal bone marrow cells, 1518 +/- 242 receptors per cell; and peritoneal exudate cells, 2025 +/- 216 receptors per cell. Affinity crosslinking studies demonstrated that 125I-labeled GM-CSF bound specifically to two species of Mr 180,000 and 70,000 on CFU-GM, normal bone marrow cells, and peritoneal exudate cells. The Mr 70,000 species is thought to be a proteolytic fragment of the intact Mr 180,000 receptor. The present studies indicate that the GM-CSF receptor expressed on CFU-GM and mature myeloid cells are structurally similar. In addition, the number of GM-CSF receptors on CFU-GM is twice the average number of receptors on casein-induced mature myeloid cells, suggesting that receptor number may decrease as CFU-GM mature.     

In vivo radioprotective effects of recombinant human granulocyte colony-stimulating factor in lethally irradiated mice.

The purpose of this study was to investigate the in vivo radioprotective effects of recombinant human granulocyte colony stimulating factor (rhG-CSF) in lethally irradiated BALB/c mice. We initially analyzed the effects of increasing doses of rhG-CSF on survival of mice receiving 700 cGy (LD100/30) single dose total body irradiation (TBI). While 1 microgram/kg to 100 micrograms/kg doses of rhG-CSF were not radioprotective, a dose-dependent radioprotection was observed at 200 micrograms/kg to 4,000 micrograms/kg rhG-CSF. We next compared four different rhG-CSF treatment regimens side by side for their radioprotective effects in LD100/30 irradiated mice. One hundred percent of control mice receiving phosphate buffered saline died within 21 days after TBI with a median survival of 14 days. The median survival was prolonged to 20 days and the actuarial 60-day survival rate was increased to 27% when mice received 2,000 micrograms/kg rhG-CSF 24 hours before TBI (P = .0002; Mantel-Peto-Cox). Similarly, the median survival time was prolonged to 24 days and the actuarial 60-day survival rate was increased to 33%, when mice were given 2,000 micrograms/kg rhG-CSF 30 minutes before TBI. Optimal radioprotection was achieved when 2,000 micrograms/kg rhG-CSF was administered in two divided doses of 1,000 micrograms/kg given 24 hours before and 1,000 micrograms/kg given 30 minutes before TBI. This regimen prolonged the median survival time of LD100/30 irradiated mice to more than 60 days and increased the actuarial 60-day survival rate to 62% (P = .0001; Mantel-Peto-Cox). By comparison, no survival advantage was observed when mice received rhG-CSF 24 hours post-TBI. Similar radioprotective effects were observed when mice were irradiated with 650 cGy (LD80/30). The presented findings provide conclusive evidence that rhG-CSF has significant in vivo radioprotective effects for mice receiving LD100/30 or LD80/30 TBI.     

Effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on circulating platelets

Summary The effect on platelets of rhG-CSF was studied in 20 healthy volunteers with the thrombometer, a specially developed device which is described in detail. Additionally, conventional aggregation tests were performed. Low doses of rhG-CSF enhance functional platelet activity, as shown by significant acceleration of the occlusion of the thrombometer channel. Similar results were found in conventional aggregation tests utilizing collagen for induction. At G-CSF concentrations of 0.1 and 1.0 ng/ml the time to response was significantly accelerated and the maximum response was observed in a higher proportion of platelets. However, the second phase of aggregation induced by epinephrine was significantly inhibited by 1.0 ng/ml G-CSF. The activation of platelets may be beneficial in thrombocytopenia, but it may also increase platelet turnover and platelet loss. Further investigation is needed to clarify the mechanism by which G-CSF exerts its effects on platelets.     

Selective targeting of human lymphokine-activated killer cells by CD3 monoclonal antibody against the interferon-inducible high-affinity Fc gamma RI receptor (CD64) on autologous acute myeloid leukemic blast cells

The potential of the CD3 monoclonal antibody (MoAb) OKT3 to selectively target lymphokine-activated killer (LAK) cells and T-cell clones in vitro against autologous tumor cells was studied using material from patients with acute leukemias (19 acute myeloid leukemias [AML], and 3 acute lymphoblastic leukemias [ALL]). Cytotoxicity mediated by patient LAK cells against AML blasts, but not against ALL cells and autologous Epstein-Barr virus-transformed B cells, was enhanced 1.5-fold to 9.3- fold by OKT3 in all AML patients studied. The following findings suggest that the major target molecule on AML cells for OKT3-coated LAK cells is the high-affinity Fc receptor for IgG (Fc gamma RI; CD64): (1) susceptibility to killing by OKT3-coated effector LAK cells segregated with target cell expression of CD64; (2) preincubation of AML blasts with monomeric OKT3 (murine IgG2a), the Fc portion of which is known to have preferential binding affinity to CD64, resulted in lysis by autologous T cells that were not spontaneously cytotoxic; (3) OKT3- dependent increase in lysis of primary and relapsed AML cells by autologous T-cell clones correlated with the amount of target cell expression of CD64; (4) anti-leukemic cytotoxicity of OKT3-coated T cells could partially be inhibited by monomeric human Ig, the natural ligand of CD64; and (5) expression of CD64 (Fc gamma RI) on fresh AML cells could be increased by interferon-gamma (IFN-gamma) and IFN-alpha translating into further enhancement of lysis by autologous OKT3-coated LAK cells. Nonmalignant CD34+ cells sorted from peripheral blood were found to lack expression of CD64 and hence were not affected by OKT3- triggered T-cell targeting, as detected by colony formation assays. In conclusion, the in vitro data presented provide a rationale for the combined clinical use of recombinant interleukin-2, IFN-gamma, and low doses of CD3 MoAb to eliminate AML cells while sparing nonmalignant hematopoietic progenitor cells, for example, in the setting of purging procedures for autologous bone marrow transplantation.     

Granulocyte-macrophage colony-stimulating factor (CSF) and multilineage CSF recruit human monocytes to express granulocyte CSF

We assessed the capacity of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and multilineage (Multi)-CSF to induce release of granulocyte-CSF (G-CSF) by highly purified peripheral blood monocyte (Mo) preparations. Our results reveal that GM-CSF and Multi-CSF, either alone or in a synergistic concert, activate Mo to transcribe G-CSF messenger (m) RNA and release biologically active G- CSF protein into their culture supernatants. G-CSF had no regulatory effect on Mo expression of cytoplasmic G-CSF mRNA levels and G-CSF protein secretion by itself. These differential actions of CSFs provide further insight into self-regulatory mechanisms within the growth factor hierarchy system.     

Homodimeric cross-over structure of the human granulocyte colony-stimulating factor (GCSF) receptor signaling complex

A crystal structure of the signaling complex between human granulocyte colony-stimulating factor (GCSF) and a ligand binding region of GCSF receptor (GCSF-R), has been determined to 2.8 A resolution. The GCSF:GCSF-R complex formed a 2:2 stoichiometry by means of a cross-over interaction between the Ig-like domains of GCSF-R and GCSF. The conformation of the complex is quite different from that between human GCSF and the cytokine receptor homologous domain of mouse GCSF-R, but similar to that of the IL-6/gp130 signaling complex. The Ig-like domain cross-over structure necessary for GCSF-R activation is consistent with previously reported thermodynamic and mutational analyses.     

Use of recombinant human growth hormone (rhGH) plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) for the mobilization and collection of CD34+ cells in poor mobilizers

The activity of recombinant human growth hormone (rhGH) in enhancing CD34(+) cell mobilization elicited by chemotherapy plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) was evaluated in 16 hard-to-mobilize patients, that is, those achieving a peak of circulating CD34+ cells 10/microL or less, or a collection of CD34(+) cells equal to or less than 2 x 10(6)/kg. Patients who had failed a first mobilization attempt with chemotherapy plus rhG-CSF (5 microg/kg/d) were remobilized with chemotherapy plus rhG-CSF and rhGH (100 microg/kg/d). As compared with rhG-CSF, the combined rhGH/rhG-CSF treatment induced significantly higher (P < or =.05) median peak values for CD34(+) cells/microL (7 versus 29), colony-forming cells (CFCs)/mL (2154 versus 28,510), and long-term culture-initiating cells (LTC-ICs)/mL (25 versus 511). Following rhG-CSF and rhGH/rhG-CSF, the median yields of CD34(+) cells per leukapheresis were 1.1 x 10(6)/kg and 2.3 x 10(6)/kg (P < or =.008), respectively; the median total collections of CD34(+) cells were 1.1 x 10(6)/kg and 6 x 10(6)/kg (P < or =.008), respectively. No specific side effect could be ascribed to rhGH, except a transient hyperglycemia occurring in 2 patients. Reinfusion of rhGH/rhG-CSF-mobilized cells following myeloablative therapy resulted in prompt hematopoietic recovery. In conclusion, our data demonstrate that in poor mobilizers addition of rhGH to rhG-CSF allows the patients to efficiently mobilize and collect CD34(+) cells with maintained functional properties.     

The effect of recombinant human granulocyte colony-stimulating factor (rG-CSF) on childhood neutropenias]

The clinical effect of recombinant human granulocyte colony-stimulating factor (rG-CSF), produced by Chinese hamster ovary cells, was studied in 27 patients with childhood neutropenias. The sample consisted of 8 patients with congenital neutropenia (Kostmann type), 9 with neutropenia with miscellaneous causes (5 chronic benign, 2 associated with hypogammaglobulinemia, 1 drug-induced, and 1 hypoplastic type), 3 with cyclic neutropenia, and 7 with severe aplastic anemia. The rG-CSF was given subcutaneously (or in a few cases intravenously) at a dose of 2 micrograms/kg/day for 7 days and 5 micrograms/kg/day for additional 7 to 28 days in cases with poor response. The rG-CSF was effective in 18 of 27 cases (67%). Patients with congenital neutropenia and aplastic anemia responded less frequently and poorly. The mean level of absolute neutrophil counts of 8 congenital neutropenia cases increased from 88/microliters to 2,718/microliters. That of 9 miscellaneous cases changed from 189/microliters to 7,224/microliters at a dose of 2 micrograms/kg/day. In 7 aplastic anemia cases pretreatment level of 220/microliters rose to 851/microliters, usually after increasing the dose up to 5 micrograms/kg/day. The rG-CSF was apparently effective in 3 cases of cyclic neutropenia. In any type of neutropenia, the effect was largely transient; after the discontinuation of rG-CSF, the absolute neutrophil counts tended to decrease to pretreatment levels within 1 to 2 weeks. The G-CSF was well tolerated, and only one case with mild lumbago and another with minimal elevation of transaminases were observed. We conclude that the rG-CSF can be effective for treating various types of childhood neutropenia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF): receptor biology, signal transduction, and neutrophil activation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) are two of the growing number of recognized cytokines involved in the regulation of hematopoiesis. The purification of these factors and the subsequent cloning of the cDNAs which encode these proteins have led to their widespread clinical use in the setting of therapy or disease-induced myelosuppression. Although originally purified on the basis of their colony-stimulating properties, GM-CSF and G-CSF may also play important roles in the regulation of effector cell function. The mechanisms underlying progenitor cell proliferation and effector cell stimulation remain poorly understood. However, the characterization of the GM-CSF and G-CSF receptors and recent work in signal transduction are helping to elucidate these mechanisms. This paper will review the biology of the GM-CSF and G-CSF receptors, the mechanisms of post-receptor signal transduction, and the resultant effects on neutrophil function. In addition, the current and potential clinical uses of these factors will be examined in light of their ability to activate and perhaps enhance the function of neutrophils.     

Human recombinant granulocyte colony-stimulating factor for the treatment of autoimmune neutropenia.

We have recently treated a case of autoimmune neutropenia in a 57-year-old male. Because neutropenia persisted despite the administration of prednisolone for 30 days, daily subcutaneous injection of human recombinant granulocyte colony-stimulating factor (rhG-CSF) at a dosage of 100 micrograms was started. Neutrophil count increased gradually and reached a plateau of 5,000/microliters by day 25 after administration of rhG-CSF. This observation suggests that rhG-CSF is effective for the treatment of autoimmune neutropenia.     

吉姆欣在治疗急性粒细胞缺乏症伴感染中的临床应用

目的　探讨吉姆欣在急性粒细胞缺乏症伴感染患者中与抗生素联合应用的效果。方法　治疗急性粒细胞缺乏症伴感染患者在接受广谱高效抗生素的同时 ,分为加吉姆欣 (每日 150μg～ 30 0μg皮下注射 )的实验组 ,和不加吉姆欣的对照组。结果　接受抗生素及吉姆欣治疗的实验组有效率为 92 .9% ( 52 /56 ) ,而对照组只有58.8% ( 30 /51)。中性粒细胞数恢复正常的时间及发热患者体温恢复正常的时间 ,实验组均较对照组明显缩短( P<0 .0 1)。结论　吉姆欣可缩短化疗致中性粒细胞减少的持续时间 ,促进中性粒细胞生长 ,并提高抗菌治疗的疗效。     

Treatment of idiopathic neutropenia in the elderly with recombinant human granulocyte colony-stimulating factor.

We administered recombinant human granulocyte colony-stimulating factor (rhG-CSF) intravenously for 2 weeks to 2 elderly patients with severe neutropenia. The absolute neutrophil count (ANC) recovered promptly after the initiation of rhG-CSF therapy and reached a peak (greater than 10 x 10(9)/l) on the 13th day. The ANC fell rapidly after rhG-CSF was discontinued, but it remained within the normal range after therapy. There were no side effects during the entire course of treatment. Therefore, rhG-CSF seems to be a most beneficial treatment in elderly patients with severe neutropenia.     

In vivo dose-response effect of recombinant human granulocyte colony-stimulating factor on increase in granulocytes after peripheral blood stem cell autotransplantation

The in vivo dose-response potential of recombinant human granulocyte colony-stimulating factor (rG-CSF) in facilitating reconstitution of granulopoiesis was evaluated in an 11-year-old girl who received autotransplantation of peripheral blood stem cells (PBSC) after marrow-ablative therapy for relapsing acute lymphoblastic leukemia. The recovery of hematopoiesis was slow when a small number of progenitors were collected and reinfused into the patient, but administration of rG-CSF led to significant dose-dependent increase in peripheral blood granulocytes.