Specific binding of radioiodinated granulocyte-macrophage colony-stimulating factor to hemopoietic cells

The hemopoietic growth factor granulocyte-macrophage colony-stimulating factor, GM-CSF, specifically controls the production of granulocytes and macrophages. This report describes the binding of biologically-active 125I-labeled murine GM-CSF to a range of hemopoietic cells. Specific binding was restricted to murine cells and neither rat nor human bone marrow cells appeared to have surface receptors for 125I-labeled GM-CSF. 125I-Labeled GM-CSF only appeared to bind specifically to cells in the myelomonocytic lineage. The binding of 125I-labeled GM-CSF to both bone marrow cells and WEHI-3B(D+) was rapid (50% maximum binding was attained within 5 min at both 20 degrees C and 37 degrees C). Unlabeled GM-CSF was the only polypeptide hormone which completely inhibited the binding of 125I-labeled GM-CSF to bone marrow cells, however, multi-CSF (also called IL-3) and G-CSF partially reduced the binding of 125I-labeled GM-CSF to bone marrow cells. Interestingly, the binding of 125I-labeled GM-CSF to a myelomonocytic cell line, WEHI-3B(D+), was inhibited by unlabeled GM-CSF but not by multi-CSF or G-CSF. Scatchard analysis of the binding of 125I-labeled GM-CSF to WEHI-3B(D+) cells, bone marrow cells and peritoneal neutrophils indicated that there were two classes of binding sites: one of high affinity (Kd1 = 20 pM) and one of low affinity (Kd2 = 0.8-1.2 nM). Multi-CSF only inhibited the binding of 125I-labeled GM-CSF to the high affinity receptor on bone marrow cells: this inhibition appeared to be a result of down regulation or modification of the GM-CSF receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin 1 stimulates human endothelial cells to produce granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor

Endothelial cells are a potent source of hematopoietic growth factors when stimulated by soluble products of monocytes. Interleukin 1 (IL 1) is released by activated monocytes and is a mediator of the inflammatory response. We determined whether purified recombinant human IL 1 could stimulate cultured human umbilical vein endothelial cells to release hematopoietic growth factors. As little as 1 U/ml of IL 1 stimulated growth factor production by the endothelial cells, and increasing amounts of IL 1 enhanced growth factor production in a dose-dependent manner. Growth factor production increased within 2 to 4 hr and remained elevated for more than 48 hr. To investigate the molecular basis for these findings, oligonucleotide probes for granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and multi-CSF were hybridized to poly(A)-containing RNA prepared from unstimulated and IL 1-stimulated endothelial cells. Significant levels of GM-CSF and G-CSF, but not M-CSF or multi-CSF, mRNA were detected in the IL 1-stimulated endothelial cells. Biological assays performed on the IL 1-stimulated endothelial cell-conditioned medium confirmed the presence of both GM- and G-CSF. These results demonstrate that human recombinant IL 1 can stimulate endothelial cells to release GM-CSF and G-CSF, and provide a mechanism by which IL 1 could modulate both granulocyte production and function during the course of an inflammatory response.     

Biologic properties of homogeneous interleukin 3. I. Demonstration of WEHI-3 growth factor activity, mast cell growth factor activity, p cell-stimulating factor activity, colony-stimulating factor activity, and histamine-producing cell-stimulating factor activity

Interleukin 3 (IL 3) was initially defined as a factor in conditioned media from concanavalin A-stimulated lymphocytes (Con A CM) that induces the enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH) in cultures of nu/nu splenic lymphocytes. To determine the spectrum of additional "biologic" activities, IL 3 was purified to homogeneity and its properties were assessed. The protein preparation was judged to be homogeneous IL 3 by the following criteria: 1) elution of a peak of IL 3 with a constant specific activity in the last step of purification, 2) presence of a single protein by SDS-PAGE analysis, 3) receptor-binding activity against IL 3-dependent cell lines, 4) a specific activity of congruent to 0.2 ng/ml required for 50% of maximal biologic activity, and 5) the presence of a single amino terminal sequence. With the use of this preparation of IL 3, the dose-response curves for 20 alpha SDH induction were identical or similar to the dose-response curves for the activities of 1) WEHI-3 growth factor, 2) mast cell growth factor, 3) P cell-stimulating factor, and 4) histamine-producing cell-stimulating factor. In addition, homogeneous IL 3 had colony-stimulating factor activity, although only approximately 2% of the total CSF activity found in Con A CM was associated with IL 3. The major peak of CSF activity could be resolved from IL 3 by DEAE column chromatography and lacked many of the biologic activities associated with IL 3.     

Heparin-binding sites in granulocyte-macrophage colony-stimulating factor. Localization and regulation by histidine ionization

The biological activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) is modulated by the sulfated glycosaminoglycans (GAGs) heparan sulfate and heparin. However, the molecular mechanisms involved in such interactions are still not completely understood. We have proposed previously that helix C, one of the four alpha-helices of human GM-CSF (hGM-CSF), contains a GAG-binding site in which positively charged residues are spatially positioned for interaction with the sulfate moieties of the GAGs (Wettreich, A., Sebollela, A., Carvalho, M. A., Azevedo, S. P., Borojevic, R., Ferreira, S. T., and Coelho-Sampaio, T. (1999) J. Biol. Chem. 274, 31468-31475). Protonation of two histidine residues (His83 and His87) in helix C of hGM-CSF appears to act as a pH-dependent molecular switch to control the interaction with GAGs. Based on these findings, we have now generated a triple mutant form of murine GM-CSF (mGM-CSF) in which three noncharged residues in helix C of the murine factor (Tyr83, Gln85, and Tyr87) were replaced by the corresponding basic residues present in hGM-CSF (His83, Lys85, and His87). Binding assays on heparin-Sepharose showed that, at acidic pH, the triple mutant mGM-CSF binds to immobilized heparin with significantly higher affinity than wild type (WT) mGM-CSF and that neither protein binds to the column at neutral pH. The fact that even WT mGM-CSF binds to heparin at acidic pH indicates the existence of a distinct, lower affinity heparin-binding site in the protein. Chemical modification of the single histidine residue (His15) located in helix A of WT mGM-CSF with diethyl pyrocarbonate totally abolished binding to immobilized heparin. Moreover, replacement of His15 for an alanine residue significantly reduced the affinity of mGM-CSF for heparin at pH 5.0 and completely blocked heparin binding to a synthetic peptide corresponding to helix A of GM-CSF. These results indicate a major role of histidine residues in the regulation of the binding of GM-CSF to GAGs, supporting the notion that an acidic microenvironment is required for GM-CSF-dependent regulation of target cells. In addition, our results provide insight into the molecular basis of the strict species specificity of the biological activity of GM-CSF.     

Interactions among granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, and IFN-gamma lead to enhanced proliferation of murine macrophage progenitor cells

This report examines the actions of IFN-gamma on monocytopoiesis in murine liquid and semisolid bone marrow cultures. The proliferative response of bone marrow cells to macrophage CSF and granulocyte-macrophage CSF was assayed by measuring [3H]TdR uptake in a range of mouse strains. No interstrain difference in kinetics was observed for CSF-1 action, but GM-CSF acted significantly more rapidly on C57B1/6, Swiss, and to a lesser extent A/J mice than on BALB/c or CBA. IFN-gamma inhibited [3H]TdR incorporation elicited by CSF-1, and to a much lesser extent, GM-CSF. When the two CSF were added together, the effects were not additive; in fact, the response was the same as that seen with GM-CSF alone. When IFN-gamma was also added, the response was restored to the level seen with CSF-1 alone. In essence, the inhibitory actions of GM-CSF and IFN-gamma were mutually exclusive. The mechanism of these actions was investigated using colony assays. As expected, CSF-1 caused the formation of pure macrophage colonies, whereas GM-CSF stimulated production of macrophage, granulocyte, and mixed granulocyte macrophage colonies. When the two CSF were added in combination, the total colony count was greater than with either alone, but less than additive. The number of pure macrophage colonies was reduced to the number seen with GM-CSF alone. IFN-gamma reduced the number of colonies in the presence of CSF-1, but slightly increased the number with GM-CSF. In the presence of both CSF, IFN-gamma increased the colony count by around 25 to 40%, so that the numbers were greater than the combined total of CSF-1 plus GM-CSF added separately. Similar results were obtained in all mouse strains tested. The results suggest that the thymidine uptake data reflect changes in the number of progenitor cells responding rather than changes in cell cycle time. The results are discussed in terms of the possibility that coadministration of GM-CSF and CSF-1 could ameliorate the myelosuppressive actions of IFN-gamma in vivo, leading to more effective use of this agent as a biologic response modifier.     

Synergistic effects of cyclic AMP and Ca2+ ionophore A23187 on de novo synthesis of histidine decarboxylase in mastocytoma P-815 cells.

In the preceding paper (Kawai, H. et al. (1992) Biochim. Biophys. Acta 1133, 172-178), we reported that in mastocytoma P-815 cells dexamethasone and 12-O-tetradecanoylphorbol-13-acetate (TPA) synergistically enhanced the de novo synthesis of L-histidine decarboxylase (HDC). Here we found that Ca2+ acted synergistically with cAMP in the induction of HDC mRNA and HDC activity in mastocytoma P-815 cells, and that the mechanism underlying the enzyme induction by Ca2+ plus cAMP was distinguishable from that by dexamethasone plus TPA. Ca2+ ionophore A23187, itself having no significant activity, markedly enhanced the induction of HDC activity by N6,O2'-dibutyryl cAMP (db cAMP) or cAMP-inducible prostaglandins such as PGE1, PGE2 and PGI2 in the presence of the phosphodiesterase inhibitor, Ro201724. However, A23187 had little effect on increases in HDC activity induced by other known stimulants, such as TPA, dexamethasone and sodium butyrate. These results suggest that A23187 has a specific effect on the induction of HDC activity due to an increased level of cAMP. The finding that both A23187 and cAMP enhanced HDC activity suggests that both Ca2+/calmodulin and cyclic nucleotide dependent protein kinase play essential roles in the process of enhancement of HDC activity. To examine this possibility, we studied the effects of W-7, an inhibitor of calmodulin, removal of extracellular Ca2+, and H-8, an inhibitor of cAMP-dependent protein kinase, on the enhancing activity of A23187 plus db cAMP. The enhancement of HDC activity by A23187 plus db cAMP was inhibited by W-7, removal of extracellular Ca2+, and H-8. The increase in HDC activity was due to the de novo synthesis of the enzyme, since it was suppressed by the addition of cycloheximide or actinomycin D, and was well correlated with the marked accumulation of a 2.7 kilobase HDC mRNA. Furthermore, the mechanism underlying the induction of HDC by db cAMP plus A23187 is distinguishable from that in the case of dexamethasone plus TPA, since preexposure to dexamethasone plus TPA for 12 h, for a plateau level to be reached, did not affect the subsequent increase in HDC activity due to db cAMP plus A23187.     

CXCR3 expression on CD34(+) hemopoietic progenitors induced by granulocyte-macrophage colony-stimulating factor: II. Signaling pathways involved

CXCR3, known to have four ligands (IFN-gamma inducible protein 10 (gamma IP-10), monokine induced by IFN-gamma (Mig), I-TAC, and 6Ckine), is predominantly expressed on memory/activated T lymphocytes. We recently reported that GM-CSF induces CXCR3 expression on CD34(+) hemopoietic progenitors, in which gamma IP-10 and Mig induce chemotaxis and adhesion. Here we further report that stimulation with GM-CSF causes phosphorylation of Syk protein kinase, but neither Casitas B-lineage lymphoma (Cbl) nor Cbl-b in CD34(+) hemopoietic progenitors can be blocked by anti-CD116 mAb. Specific Syk blocking generated by PNA antisense completely inhibits GM-CSF-induced CXCR3 expression in CD34(+) progenitors at both mRNA and protein as well as at functional levels (chemotaxis and adhesion). Cbl and Cbl-b blocking have no such effects. Thus, GM-CSF binds to its receptor CD116, and consequently activates Syk phosphorylation, which leads to induce CXCR3 expression. gamma IP-10 and Mig can induce Syk, Cbl, and Cbl-b phosphorylation in CD34(+) progenitors by means of CXCR3. gamma IP-10 or Mig has induced neither chemotaxis nor adhesion in GM-CSF-stimulated Cbl-b-blocked CD34(+) hemopoietic progenitors, whereas SDF-1alpha induces both chemotaxis and adhesion in these cells. Interestingly, gamma IP-10 and Mig can induce chemotaxis and adhesion in GM-CSF-stimulated Syk- or Cbl-blocked CD34(+) hemopoietic progenitors. Thus, Cbl-b, but not Syk and Cbl phosphorylation, is essential for gamma IP-10- and Mig-induced chemotaxis and adhesion in CD34(+) hemopoietic progenitors. This study provides a useful insight into novel signaling transduction pathways of the functions of CXCR3/gamma IP-10 and Mig, which may be especially important in the cytokine/chemokine environment for mobilization, homing, and recruitment during proliferation, differentiation, and maturation of hemopoietic progenitor cells.     

Glycosaminoglycans bind granulocyte-macrophage colony-stimulating factor and modulate its mitogenic activity and signaling in human osteoblastic cells

We recently demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) is an autocrine growth factor for human osteoblastic (hOB) cells. Since GM-CSF is a member of the heparin-binding factor family, we examined the interactions between GM-CSF and glycosaminoglycans (GAGs) present in the osteoblast microenvironment. Using a bioassay in which the mitogenic activity of recombinant human (rh) GM-CSF was measured after incubation in the presence of an hOB cell layer or extracellular matrix (ECM) produced by these cells, we showed that rhGM-CSF binds to GAG components present in the ECM and that the bound rhGM-CSF retains its ability to stimulate hOB cell proliferation. Heparan sulfate compounds on the hOB cell surface were also found to sequester GM-CSF. Moreover, treatment with sodium chlorate, an inhibitor of GAG sulfation, suppressed the mitogenic activity of rhGM-CSF on hOB cells. This inhibitory effect was rescued by a low dose of heparin. Heparin was also found to promote the effect of rhGM-CSF on hOB cell proliferation, allowing nonmitogenic high doses of rhGM-CSF to stimulate hOB cell growth. Western blot analysis showed that undersulfation of cellular GAGs by chlorate inhibited the increased tyrosine phosphorylation of proteins involved in GM-CSF signaling in cloned immortalized hOB cells. The data demonstrate that GM-CSF binds to proteoglycans on the hOB cell surface and in ECM produced by these cells and that the bound GM-CSF is biologically active. Furthermore, this study shows that cellular proteoglycans play an essential role in GM-CSF signaling and biological activity in hOBs. J. Cell. Physiol. 177:187–195, 1998. © 1998 Wiley-Liss, Inc.     

Interleukin 3, granulocyte-macrophage colony-stimulating factor, and transfected erythropoietin receptors mediate tyrosine phosphorylation of a common cytosolic protein (pp100) in FDC-ER cells.

Receptors for the hematopoietic growth factors erythropoietin, interleukin 3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) are members of a structurally related receptor superfamily. Interestingly, while none of these receptors encode tyrosine kinase activities, induced tyrosine phosphorylation has been observed in various responsive cells stimulated with each factor. Toward defining possible common transduction pathways which are activated by these three cytokines, we have studied induced protein phosphorylation in murine myeloid FDC-P1 cells stably transfected with an erythropoietin receptor cDNA (FDC-ER cells). FDC-ER cells proliferate in response to erythropoietin (Quelle, D. E., and Wojchowski, D. M. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4801-4805), and presently are shown to rapidly phosphorylate a M(r) 100,000 cytosolic protein (pp100) at tyrosine residues in response to this factor. Phosphorylation of pp100 also is induced in FDC-P1 and FDC-ER cells in response to IL-3 or GM-CSF. Importantly, quantitative analyses showed identical concentration dependencies for factor-induced pp100 phosphorylation and induced cell proliferation. Moreover, a selective loss of proliferative responsiveness to GM-CSF in FDC-ER cells was associated with a reduced capacity of GM-CSF to induce pp100 phosphorylation. Finally, limited differences in tryptic phosphopeptide maps of pp100 as isolated following exposure to erythropoietin, IL-3, or GM-CSF were observed, suggesting that these factors also may preferentially induce phosphorylation of pp100 at distinct sites. These findings are consistent with a role for pp100 as a common cytosolic transducer in the apparently convergent pathways of erythropoietin-, IL-3-, and GM-CSF-induced proliferation of myeloid progenitor cells.     

The effect of inhibition of leukotriene synthesis on the activity of interleukin-8 and granulocyte-macrophage colony-stimulating factor

The cytokines interleukin-8 (IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced the extracellular release of arachidonate metabolites from ionophore-stimulated neutrophils by 145 +/- 10% (mean +/- S.E.M., n = 13) and 182 +/- 11% (n = 16), respectively. To determine whether enhanced leukotriene production mediates the effects of these cytokines on neutrophil activity, two different specific arachidonate 5-lipoxygenase (5-LO) inhibitors, piriprost and MK-886, were used to inhibit leukotriene synthesis. Neither inhibitor affected the upregulation of CD11b beta(2)-integrin expression or priming of superoxide generation stimulated by IL-8 and GM-CSF. It is concluded that leukotrienes do not mediate either the direct or priming effects of these cytokines and that these classes of anti-inflammatory drugs are therefore unlikely to inhibit the effects of IL-8 and GM-CSF on neutrophil activation.     

Recombinant murine steel factor stimulates in vitro production of granulocyte-macrophage progenitor cells.

The ability of murine Steel factor to promote the in vitro production of granulocyte-macrophage progenitor cells (CFU-GM) was examined in short-term liquid cultures. Bone marrow from C57BL/6J or Sl/Sld mice was placed in culture for seven days with either Steel factor alone or in the presence of IL-3. CFU-GM responsive to GM-CSF, IL-3, and CSF-1 were measured in the input population and again after 3 or 7 days in culture. Steel factor alone increased the number of all CFU-GM types as early as 3 days after culture initiation, with further increases at day 7. This effect was potentiated by the addition of IL-3. Production of CFU-GM by C57BL/6J or Sl/Sld marrow was comparable except for enhanced production of CSF-1 responsive progenitors by Sl/Sld marrow. A recombinant Sld protein was also shown to be equivalent to the wild-type protein in its capacity to promote CFU-GM production from normal bone marrow.     

The human granulocyte-macrophage colony-stimulating factor receptor is capable of initiating signal transduction in NIH3T3 cells.

The ability of the receptor for the hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) to function in non-hematopoietic cells is unknown. NIH3T3 fibroblasts were transfected with cDNAs encoding the alpha and beta subunit of the human GM-CSF receptor and a series of stable transformants were isolated that bound GM-CSF with either low (KD = 860 - > 1000 pM) or high affinity (KD = 20-80 pM). Low affinity receptors were not functional. However, the reconstituted high affinity receptors were found to be capable of activating a number of signal transduction pathways, including tyrosine kinase activity, phosphorylation of Raf-1, and the transient induction of c-fos and c-myc mRNAs. The activation of protein tyrosine phosphorylation by GM-CSF in NIH3T3 cells was rapid (< 1 min) and transient (peaking at 5-20 min) and resulted in the phosphorylation of proteins of estimated molecular weights of 42, 44, 52/53 and 58-60 kDa. Some of these proteins co-migrated with proteins from myeloid cells that were phosphorylated on tyrosine residues in response to GM-CSF. In particular, p42 and p44 were identified as mitogen-activated protein kinases (MAP kinases), and the phosphorylation on tyrosine residues of p42 and p44 MAP kinases occurred at the same time as the phosphorylation of Raf-1. However, despite evidence for activation of many mitogenic signal transduction molecules, GM-CSF did not induce significant proliferation of transfected NIH3T3 cells. These results suggest that murine fibroblasts contain signal transducing molecules that can effectively interact with the human GM-CSF receptor, and that are sufficient to activate at least some of the same signal transduction pathways this receptor activates in myeloid cells, including activation of one or more tyrosine kinase(s). However, the level of activation of signal transduction is either below a threshold of necessary activity or at least one mitogenic signal necessary for proliferation is missing.     

Interaction of serum and colony-stimulating factor for survival of a factor-dependent hemopoietic progenitor cell line

The growth in vitro of the murine myeloid cell line FDC-P1 depends on the presence of serum and a murine hemopoietic growth factor (either granulocyte/macrophage colony-stimulating factor (GM-CSF) or multipotential colony-stimulating factor (multi-CSF, IL3]. To determine the differential roles of serum and colony-stimulating factor (CSF) during the growth of FDC-P1 cultures, we investigated the kinetics of proliferation and death after withdrawal of serum or CSF, using flow cytometry to quantitate the numbers of vital and dead cells. After withdrawal of CSF, the cells died without entering a quiescent state. The life span of cultures lacking CSF increased with increasing concentrations of serum (greater than 50 h at 30% serum), and the cells kept dividing until they died. During the period of population death caused by the absence of CSF, the re-addition of CSF immediately prevented further cells from dying. After the withdrawal of serum in the presence of CSF, the cells continued to live and proliferate for weeks, but required high cell densities (much greater than 10(5)/ml), which suggests that the cells produced an active substance that can substitute for serum. Serum as well as serum-free conditioned medium from dense cultures made the survival and growth of FDC-P1 cultures independent of cell density. Without sufficient quantities of this activity, all cells of the population died within an interval that was much shorter than one cell cycle, which indicates that the factor acts throughout most of the cell cycle. The results suggest that both the CSF and the serum factor act together to permit cell survival, rather than to drive proliferation.     

The biology of granulocyte-macrophage colony-stimulating factor: effects on hematopoietic and nonhematopoietic cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of a family of glycoprotein cytokines that have potent effects in stimulating the proliferation, maturation, and function of hematopoietic cells. Deriving its name from its ability to stimulate the formation of macroscopic colonies containing neutrophils, eosinophils, macrophages, or mixtures of these cell types, GM-CSF stimulates the proliferation and maturation of myeloid progenitors, as well as functionally activating mature neutrophils, eosinophils, and macrophages. As most of the effects observed using GM-CSF in vitro have been shown to occur in vivo either in animal models or in human subjects, it is important to consider that GM-CSF may also exert some biological effects on nonhematopoietic cells. In response to immunologic stimuli, immunologic surveillance cells and cells of the microenvironment are capable of producing GM-CSF. In vitro experiments indicate that GM-CSF production is tightly regulated. In that regard, GM-CSF is not present in measurable quantities in normal serum, but little is known about the in vivo process of GM-CSF production and regulation. The biologic capabilities of GM-CSF have triggered its widespread clinical use in situations where hematopoiesis is compromised. GM-CSF can act as a potent growth factor in vivo, increasing the number and enhancing the function of hematopoietic progenitors and mature cells. However, the precise in vivo effect that GM-CSF may have on normal and neoplastic cells of nonhematopoietic origin remains undefined. The full range of GM-CSF bioactivity is mediated following binding to its receptor. The presence of specific receptors for GM-CSF has been demonstrated in all responsive cells of hematopoietic lineage, as well as in nonhematopoietic cells, both responsive and unresponsive. In conclusion, a large body of work from a number of laboratories has defined the biology of GM-CSF. Currently available reagents and technology will provide additional insights into the biology of this molecule, thereby expanding our present definition and allowing us to explore the mechanisms regulating hematopoiesis.     

Structure-function studies of human granulocyte-macrophage colony-stimulating factor. Identification of residues required for activity

Human granulocyte-macrophage colony stimulating factor (hGM CSF), a protein containing 127 amino acids, was chemically synthesized by using automated stepwise solid-phase methods. The unpurified synthetic hGM-CSF had the same range of actions on hemopoietic cells as the purified recombinant protein. The structural requirements for the activities of synthetic hGM-CSF were examined by the design and synthesis of fragments and analogs. The synthetic fragment, hGM-CSF (54-127), containing all four of the cysteine residues found in the intact protein, lacked detectable activity. Assays of fragments shortened at the N terminus showed that the residues 1-13 were not required for activity, but that the integrity of residues 14-25, particularly residues 16, 17, and 18, was critical for biologic activity. The 14-25 region is predicted to form the first alpha-helix in hGM-CSF. Synthetic peptides within the N-terminal 53 residue region lacked detectable activity. The synthetic analog hGM-CSF (1-121), which lacks the C-terminal 6 residues, had similar activity to hGM-CSF (1-127) indicating that residues 122-127 are not required for activity. An analog, [Ala88] hGM-CSF (14-96), which lacks the hydrophobic C-terminal region and 2 cysteine residues, had low but readily detectable activity suggesting that residues 14-96 are sufficient for detectable synthetic hGM-CSF activity, although the presence of residues 97-121 are required for full activity. No dissociation of the multiple biological activities of hGM-CSF was detected.     

Secretion and partial degradation of granulocyte-macrophage colony-stimulating factor (GM-CSF) of mouse L-P3 cells

Secretion of a granulocyte-macrophage colony-stimulating factor (GM-CSF) was accomplished by L-P3 cells in culture with a serum-free medium. Cell proliferation per se was not requisite for the production of GM-CSF; the cells continued secreting GM-CSF even after their growth had been suspended. The amount of GM-CSF accumulated in the conditioned medium was reasonably accounted by the daily rate of production, and the addition of a proteinase inhibitor such as leupeptin and pepstatin did not result in greater accumulation of GM-CSF in the culture. It is thus postulated that there is no significant proteolytic inactivation of the secreted GM-CSF in the culture. However, when partially purified GM-CSF preparation was chromatographed on a gel-filtration column in the presence of 0.1% Triton X-100, a derivative of the GM-CSF was yielded which had been diminished in the molecular weight and altered in the isoelectric point. On the other hand, when leupeptin was included in the solution during production and isolation of the factor, the yielded GM-CSF did not manifest such a detergent-induced transformation and maintained its isoelectric point at pH 3.5. It is thus assumed that, in the presence of the detergent, GM-CSF suffers deterioration by an endogenous proteinase and releases a sialoglycopeptide fragment without loosing its colony-stimulating activity.