Interdependence of the radioprotective effects of human recombinant interleukin 1 alpha, tumor necrosis factor alpha, granulocyte colony-stimulating factor, and murine recombinant granulocyte-macrophage colony-stimulating factor

Interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), granulocyte-colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) are molecularly distinct cytokines acting on separate receptors. The release of these cytokines can be concomitantly induced by the same signal and from the same cellular source, suggesting that they may cooperate. Administered alone, human recombinant (hr)IL-1 alpha and hrTNF alpha protect lethally irradiated mice from death, whereas murine recombinant GM-CSF and hrG-CSF do not confer similar protection. On a dose basis, IL-1 alpha is a more efficient radioprotector than TNF alpha. At optimal doses, IL-1 alpha is a more radioprotective cytokine than TNF alpha in C57BL/6 and B6D2F1 mice and less effective than TNF alpha in C3H/HeN mice, suggesting that the relative effectiveness of TNF alpha and IL-1 alpha depends on the genetic makeup of the host. Administration of the two cytokines in combination results in additive radioprotection in all three strains. This suggests that the two cytokines act through different radioprotective pathways and argues against their apparent redundancy. Suboptimal, nonradioprotective doses of IL-1 alpha also synergize with GM-CSF or G-CSF to confer optimal radioprotection, suggesting that such an interaction may be necessary for radioprotection of hemopoietic progenitor cells.     

Specific release of granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha, and IFN-gamma by human tumor-infiltrating lymphocytes after autologous tumor stimulation

Tumor-infiltrating lymphocytes (TIL) have been cultured from a variety of human tumors, and some melanoma TIL have demonstrated specific, MHC-restricted recognition of autologous tumor in short term lysis assays. The current study investigates cytokine release by TIL as an indicator of specific tumor recognition. We have identified two of four melanoma and one of seven breast carcinoma TIL cultures that specifically release granulocyte-macrophage-CSF, TNF-alpha, and IFN-gamma after autologous tumor stimulation. The other cultures either do not secrete cytokine or secrete cytokine in a nonspecific fashion. The amount of specific cytokine released is directly related to the number of TIL and stimulating tumor cells. Studies of TIL, from two melanoma patients, separated into CD4+ and CD8+ populations revealed that CD8+ cells were responsible for virtually all of the specific cytokine secretion, although both populations released cytokines when activated by immobilized anti-CD3 antibody. Specific cytokine release by CD8+ TIL was inhibited by anti-MHC class I mAb. Specific cytokine release was also detected from a CD4+ breast cancer TIL culture, and this was inhibited by anti-MHC class II mAb. The clinical significance of this specific mode of immune antitumor reactivity is currently under investigation.     

Characterization of the human granulocyte-macrophage colony-stimulating factor receptor

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine derived from activated T cells, endothelial cells, fibroblasts, and macrophages. It stimulates myeloid and erythroid progenitors to form colonies in semisolid medium in vitro, as well as enhancing multiple differentiated functions of mature neutrophils, macrophages, and eosinophils. We have examined the binding of human GM-CSF to a variety of responsive human cells and cell lines. The most mature myelomonocytic cells, specifically human neutrophils, macrophages, and eosinophils, express the highest numbers of a single class of high affinity receptors (Kd approximately 37 pM, 293-1000 sites/cell). HL-60 and KG-1 cells exhibit an increase in specific binding at high concentrations of GM-CSF; computer analysis of the data is nonetheless consistent with a single class of high affinity binding sites with a Kd approximately 43 pM and 20-450 sites/cell. Dimethyl sulfoxide induces a 3-10-fold increase in high affinity receptors expressed in HL-60 cells, coincident with terminal neutrophilic differentiation. Finally, binding of 125I-GM-CSF to fresh peripheral blood cells from six patients with chronic myelogenous leukemia was analyzed. In three of six cases, binding was similar to the nonsaturable binding observed with HL-60 and KG-1 cells. GM-CSF binding was low, or in some cases, undetectable on myeloblasts obtained from eight patients with acute myelogenous leukemia. The observed affinities of the receptor for GM-CSF are consistent with all known biological activities. Affinity labeling of both normal neutrophils and dimethyl sulfoxide-induced HL-60 cells with unglycosylated 125I-GM-CSF yielded a band of 98 kDa, implying a molecular weight of approximately 84,000 for the human GM-CSF receptor.     

Selective synthesis and secretion of a 23 kD protein by neutrophils following stimulation with granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha

We tested a wide range of pro-inflammatory cytokines for their capacity to activate protein synthesis in neutrophils as analyzed b y [35S] methionine metabolic labelling experiments. Of all the cytokines tested, only GM-CSF and TNF alpha stimulated significant synthesis and secretion of a 23 kD protein which resolved into two bands on two dimensional gels. Under non-reducing conditions on one dimensional gels, its migration pattern remained the same indicating that the two bands most likely represent isoforms of the same protein. Immunoisolation studies using antibodies directed against size-relevant molecules did not lead to the identification of this molecule. The fact that this 23 kD molecule is induced in a highly specific and selective manner by GM-CSF and TNF alpha indicates that it may play a key role in some of the responses of neutrophils to these two cytokines. Therefore, full characterization of this 23 kD protein could provide important new knowledge on the mechanisms by which these two cytokines exert their biological effects on neutrophils.     

Frequent expression of receptors for granulocyte-macrophage colony-stimulating factor on human nonhematopoietic tumor cell lines

Receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) were identified on 9 of 35 (26%) human nonhematopoietic tumor cell lines including non-small cell lung cancer, stomach cancer, colon cancer, and osteosarcoma cells. GM-CSF receptors distributed on these human tumor cells were low affinity types with an equilibrium dissociation constant of 1.5-10.0 nM. Cross-linking studies revealed that the molecular weights of the low affinity GM-CSF receptors were 65-85 kilodaltons. The high affinity receptors identified on hematopoietic cells were not detected on human nonhematopoietic tumor cells which we studied, and we could detect no effects of GM-CSF on cell growth of these tumor cells.     

Immune interferon enhances functional properties of human granulocytes: role of Fc receptors and effect of lymphotoxin, tumor necrosis factor, and granulocyte-macrophage colony-stimulating factor

We report here a comparative study of the effects of several cytokines known to affect myeloid cell differentiation on functional properties of human mature granulocytes. We show that recombinant interferon-gamma (rIFN-gamma), recombinant granulocyte/macrophage-colony stimulating factor (rGM-CSF), recombinant tumor necrosis factor (rTNF), and lymphotoxin (LT) purified to homogeneity are potent stimulators of polymorphonuclear cells (PMN) activity. All cytokines enhance antibody-dependent cell-mediated cytotoxicity (Ab-CMC) mediated by human PMN; however, rGM-CSF, rTNF, and LT have an immediate and short-lived effect on the PMN, whereas the activation by rIFN-gamma requires several hours of induction but can be observed up to 24 to 48 hr of culture. Only the effect of rIFN-gamma is in part dependent on induction of a high-affinity FcR for monomeric IgG on PMN, as suggested by two-color sorting analysis, and on mechanisms that result in prolonged survival of PMN in a functionally active state to mediate oxidative burst, phagocytosis, and bactericidal activity. Greater enhancement of Ab-CMC is obtained by using rIFN-gamma in combination with the other cytokines. Our data indicate that cytokines previously defined on the basis of their cytotoxic effects mediate a wide spectrum of activities on mature myeloid cells and provide evidence for their possible role in vivo, alone or in combination with rIFN-gamma, in modulating functional activities of cells responsible for non-adaptive systems of defense.     

Two neutralizing monoclonal antibodies against human granulocyte-macrophage colony-stimulating factor recognize the receptor binding domain of the molecule.

Using a series of mutant and chimeric human-mouse granulocyte-macrophage-CSF molecules the binding epitopes of two neutralizing mAb antibodies to human GM-CSF have been mapped. Both intact antibody and Fab fragments neutralize the biologic activity of human GM-CSF. The epitope of one of the antibodies contains residues widely separated in the primary structure of the growth factor that suggests that these two regions are adjacent in the tertiary structure of the molecule. In addition, evidence is presented that both mAb neutralize the activity of this cytokine by blocking the receptor binding domain of human GM-CSF.     

Effect of tumor necrosis factor and granulocyte/macrophage colony-stimulating factor on neutrophil degranulation

Both TNF and and granulocyte/macrophage CSF (GM-CSF) can activate neutrophils. The aim of this work was to determine the effect of these cytokines on neutrophil degranulation. The secretion of lactoferrin of secondary granules and myeloperoxidase (MPO) of primary granules from single adherent human neutrophils was assayed by use of a reverse hemolytic plaque assay. Both rTNF and rGM-CSF caused secretion of lactoferrin in a dose-dependent manner. Both agents also caused secretion of MPO, but only in the presence of cytochalasin B. Preincubation with pertussis toxin inhibited rGM-CSF-induced secretion of both lactoferrin and MPO. rTNF-induced MPO secretion was also blocked by pertussis toxin, whereas lactoferrin secretion was only slightly affected. Neither rTNF nor rGM-CSF caused any detectable changes in the concentration of cytoplasmic free Ca2+ in fura-2-loaded cells. However, when neutrophils were loaded with increasing concentrations of quin-2 to buffer any local, not detectable, changes in the concentration of cytoplasmic Ca2+, both rTNF- and rGM-CSF-induced secretion of lactoferrin and MPO were almost totally abolished at a relatively low quin-2 concentration. These results suggest a role of a regulatory G-protein and minute local changes in the concentration of cytoplasmic Ca2+ in TNF- and GM-CSF-induced neutrophil degranulation.     

Characterization of the cell surface receptor for granulocyte-macrophage colony-stimulating factor

125I-labeled recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to characterize receptors specific for this lymphokine on the surface of cells of both myelomonocytic and T-cell origin. GM-CSF binding to these cells was specific and saturable. Equilibrium binding studies revealed that on all cell types examined, GM-CSF bound to a single class of high affinity receptor (1000-5000 receptors/cell) with a Ka of 10(8)-10(9) M-1. More extensive characterization with P388D1 cells showed that binding of GM-CSF was rapid at 37 degrees C with a slow subsequent dissociation rate. Among a panel of lymphokines and growth hormones, only unlabeled natural or recombinant GM-CSF were able to compete for the binding of 125I-GM-CSF to these cells. Affinity cross-linking experiments with the homobifunctional cross-linking reagents disuccinimidyl suberate, disuccinimidyl tartrate, and dithiobis(succinimidyl propionate) resulted in the identification of a receptor protein with a Mr of 130,000 on five out of the seven cell types examined. This protein was extremely sensitive to proteolysis and in the absence of protease inhibitors was degraded to a form with an approximate Mr of 70,000. A receptor protein of Mr 180,000, in addition to the Mr 70,000 protein, was found on bone marrow cells and on P815 cells. The potential tissue-specific molecular heterogeneity associated with the GM-CSF receptor may help to explain some of the diverse biological effects associated with this growth and differentiation factor.     

Molecular determinants of the granulocyte-macrophage colony-stimulating factor receptor complex assembly

The granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) is composed of two chains that belong to the superfamily of cytokine receptors typified by the growth hormone receptor. A common structural element found in cytokine receptors is a module of two fibronectin-like domains, each characterized by seven beta-strands denoted A-G and A'-G', respectively. The alpha-chain (GMRalpha) confers low affinity GM-CSF binding (K(d) = 1-5 nM), whereas the beta-chain (beta(c)) does not bind GM-CSF by itself but confers high affinity binding when associated with alpha (K(d) = 40-100 pM). In the present study, we define the molecular determinants required for ligand recognition and for stabilization of the complex through a convergence of several approaches, including the construction of chimeric receptors, the molecular dynamics of our three-dimensional model of the GM.GMR complex, and site-directed mutagenesis. The functional importance of individual residues was then investigated through ligand binding studies at equilibrium and through determination of the kinetic constants of the GM.GMR complex. Critical to this tripartite complex is the establishment of four noncovalent bonds, three that determine the nature of the ligand recognition process involving residues Arg(280) and Tyr(226) of the alpha-chain and residue Tyr(365) of the beta-chain, since mutations of either one of these residues resulted in a significant decrease in the association rate. Finally, residue Tyr(365) of beta(c) serves a dual function in that it cooperates with another residue of beta(c), Tyr(421) to stabilize the complex since mutation of Tyr(365) and Tyr(421) result in a drastic increase in the dissociation rate (Koff). Interestingly, these four residues are located at the B'-C' and F'-G' loops of GMRalpha and of beta(c), thus establishing a functional symmetry within an apparently asymmetrical heterodimeric structure.     

Modulation of colony-stimulating factor-1 receptors on macrophages by tumor necrosis factor

The effect of murine rTNF-alpha on the binding of human 125I-rCSF-1 to murine thioglycolate-elicited peritoneal exudate macrophages (PEM) was investigated. At 4 degrees C, 125I-CSF-1 binding to PEM was inhibited by preincubation with human rCSF-1, but not by other cytokines. When PEM were incubated with various cytokines at 37 degrees C, murine rTNF-alpha caused greater than 90% decrease in 125I-CSF-1 binding. This decrease was time, temperature and TNF dose dependent, and was not affected by preincubation with cycloheximide. The reduction in CSF-1-binding activity was reversed by prolonged incubation at 37 degrees C even in the presence of TNF. However, PEM preincubated with TNF subsequently washing free of residual TNF resulted in a rapid recovery of CSF-1 binding. This recovery of CSF-1-binding activity required protein synthesis. Binding studies suggested that the decrease in 125I-CSF-1 binding was most likely caused by a reduction in the number of CSF-1 receptors. In addition, preincubation with TNF at 37 degrees C inhibited 125I-CSF-1 binding on mononuclear phagocytes, including the macrophage cell line J774, bone marrow-derived macrophages, and nonelicited macrophages from three different strains of mice. In contrast, 125I-murine rTNF-alpha binding to PEM was not inhibited by preincubation with CSF-1 at 4 degrees C or 37 degrees C. These data suggest that TNF may play a role in the modulation of receptor expression on blood cells, and may point to a role for this pleiotropic cytokine in the regulation of hemopoiesis.     

Identification of an antigenic domain near the C terminus of human granulocyte-macrophage colony-stimulating factor and its spatial localization

The goal of this study was to map an epitope on the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) at its C terminus, a region whose integrity is fundamental in maintaining the normal function of this molecule. Residues including the fourth alpha-helix (D, 103-116) were analyzed for their role in the interaction with antibodies (Abs) raised against the protein. Five peptides homologous to different segments of the C terminus of hGM-CSF were synthesized. Peptide-(102-121) included the same residues of the alpha-helix D and the next five amino acids toward the C terminus; peptide-[E108A]-(102-121) introduced the mutation E108A in order to verify the role of acidic residues; peptide-[C96A](93-110) encompassed the beta-sheet 2 and half of the alpha-helix D; peptide-[C121A]-(110-127) included the second half of the alpha-helix D and the C terminus of hGMCSF; peptide-(13-31)-Gly-Pro-Gly-(103-116) included both the alpha-helices A and D connected by the tripeptide Gly-Pro-Gly, which allows the original antiparallel orientation of the two alpha-helices to be maintained. Both anti-protein and anti-peptide-(102-121) antibodies, capable of neutralizing the stimulatory activity of hGMCSF in the bone marrow colony-forming assays, recognized a specific epitope in the C terminus of hGM-CSF. Molecular modeling estimated the surface accessibility of hGM-CSF and the stability of the synthetic peptides in aqueous solution. Altogether, our results showed that the immunogenic region includes part of the alpha-helix D and the residues 116-120, which are external to this helix and particularly exposed on the protein surface, confirming the feasible participation of this region in antibody binding.