Granulocyte- colony-stimulating factor induces cytokine secretion by human polymorphonuclear leukocytes.

A Lindemann, … , R Mertelsmann, F Herrmann

J Clin Invest. 1989;83(4):1308-1312. https://doi.org/10.1172/JCI114016.

Research Article

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known as an inducer of proliferation and functional of myeloid cells. This study was carried out to characterize the effects of GM-CSF on polymorphonuclear leukocytes (PMN) more extensively. Using Northern blot analysis, we show that PMN are able to accumulate mRNAs for different cytokines, including tumor factor-alpha (TNF-alpha); G-CSF, and M-CSF, all of which are involved in and hematopoiesis. Biological assays and immunoassays demonstrate that PMN translate these mRNAs, except TNF-alpha, into secretory . However, the expression of these cytokines is dependent on stimulation by exogenous signals, preferentially provided by the -derived GM-CSF. Stimulation of hematopoiesis and amplification of defense mechanisms after T cell activation thus might involve not only but also PMN, a cell type previously believed to be biosynthetically inactive.

Find the latest version: https://jci.me/114016/pdf Granulocyte-Macrophage Colony-stimulating Factor Induces Cytokine Secretion by Human Polymorphonuclear Leukocytes Albrecht Lindemann, Detlev Riedel, Wolfgang Oster, H. W. Loems Ziegler-Heitbrock,* Roland Mertelsmann, and Friedhelm Herrmann Department ofHematology, University ofMainz, D-65 Mainz, Federal Republic of Germany; and the *Institutefor , University ofMunich, D-8 Muenchen, Federal Republic ofGermany

Abstract also affects a multitude of functions of mature , monocytes, and some mesenchymal cells. PMN respond to Granulocyte-macrophage colony-stimulating factor (GM- GM-CSF by decreased migratory activity (4), enhanced phago- CSF) is known as an inducer of proliferation and functional cytosis, -dependent cellular cytoxicity (5), and super- activation of myeloid cells. This study was carried out to char- oxide anion production in response to chemoattractant FMLP acterize the effects of GM-CSF on polymorphonuclear leuko- (6). In monocytes, GM-CSF also modulates tumoricidal activ- cytes (PMN) more extensively. Using Northern blot analysis, ity (7) and induces mRNA accumulation and synthesis we show that PMN are able to accumulate mRNAs for differ- of M-CSF (8) and G-CSF (9). ent cytokines, including -a (TNF-a); PMN share a number of common properties with mono- G-CSF, and M-CSF, all of which are involved in inflammation cytes, including their phagocytic activity, similar membrane and hematopoiesis. Biological assays and immunoassays dem- receptors, and a common progenitor cell. PMN have also been onstrate that PMN translate these mRNAs, except TNF-a, implicated in the generation and secretion of regulatory medi- into secretory proteins. However, the expression of these cyto- ators like plasminogen activator (10), and an IL 1 inhibitor kines is dependent on stimulation by exogenous signals, prefer- (1 1), as well as IL 1-a and IL 1-,3, as shown recently by our entially provided by the T cell-derived lymphokine GM-CSF. group (12). Given the relatedness of PMN and monocytes, we Stimulation of hematopoiesis and amplification of defense asked whether PMN similar to monocytes would respond to mechanisms after T cell activation thus might involve not only GM-CSF by synthesis of a broader spectrum of cytokines like monocytes but also PMN, a cell type previously believed to be M-CSF, G-CSF, and TNF-a. The data reported herein, dem- biosynthetically inactive. onstrate inducible cytokine gene expression by PMN at the RNA and protein level. These findings may provide the basis Introduction for understanding some mechanisms ofT cell/PMN collabora- tion. Granulocyte-macrophage colony-stimulating factor (GM- CSF)' is a T -derived, glycoprotein of 22 kD, that Methods was first identified by its capacity to induce hematopoietic Cell purification. PMN were isolated from heparinized peripheral progenitor cells to proliferate and differentiate to granulocytes blood from normal consenting healthy volunteer donors by Ficoll-Hy- and (1). Cloning of the GM-CSF gene, located paque gradient centrifugation (Pharmacia Laboratories, Uppsala, on chromosome 5q2 l-32 (2) by Wong et al. (3) enabled large- Sweden; density 1.077 g/dl). PMN were recovered from cells present in scale production of this lymphokine and facilitated further the pellet by further sedimentation through dextran sulfate (0.4% vol/ investigation of its functional repertoire in in vitro and in vivo vol in HBSS; Gibco Laboratories, Grand Island, NY) as described (13). settings. Residual red blood cells were eliminated by hypotonic lysis. PMN Several recent studies have shown that GM-CSF is more preparations were shown to be > 99% pure by morphology, cytochem- istry, and reactivity with PMN-specific Ab 1D3 (kindly than a hematopoietic growth-inducing molecule, because it provided by Dr. J. Griffin, Dana-Farber Institute, Boston, MA). Monocytes from consenting healthy donors' buffy coats were separated on Ficoll- Hypaque gradients and purified by repeated adherence steps after re- Address reprint requests to Dr. F. Herrmann, Abt. fuir Haematologie, moval of T cells by rosetting with 2-aminoethyl-isothiouronium bro- Universitaetsklinikum, Johannes Gutenberg-Universitaet, Langen- mide hydrobromide-treated sheep RBC (5% vol/vol solution). Individ- beckstrasse 1, D-6500 Mainz, FRG. ual cell fractions assessed by morphology (Wright-Giemsa and Receivedfor publication 22 July 1988 and in revisedform 23 No- a-naphthyl-acetate esterase [ANAE] staining) and immunofluores- vember 1988. cence analysis, using MAb to the T 1 I (T cells), Mo2 (monocytes), B I (B cells) (14), and lD3 (granulocytes) revealed preparations of 1. Abbreviations used in this paper: ANAE, a-naphthyl acetate ester- cells with > 98% purity. ase; ANLL, acute nonlymphocytic leukemia; CAE, naphtol AS-D- Culture conditions. PMN and monocytes purified as described chloracetate esterase; CFU-GM, granulocyte-macrophage colony- above, were cultured at 0.5-5 X 106 cells/ml in standard culture me- forming units; CSF, colony-stimulating factor; G-CSF, granulocyte- dium (RPMI 1640 medium supplemented with 2 mM glutamine, 100 CSF; GM-CSF, granulocyte-macrophage-CSF; h, human; M-CSF, ng/ml streptomycin, 100 U/ml penicillin, 1% sodium pyruvate, and macrophage-CSF; PMN-CM, PMNL conditioned medium; r, recom- 5% low endotoxin FCS) in 24-well flat-bottom plates (Corning Medi-. binant; TNF-a, tumor necrosis factor-a. cal, Coming Glass Works, Medfield, MA) in the presence or absence of various concentrations of yeast-expressed recombinant human GM- J. Clin. Invest. CSF (kindly provided by Dr. F. R. Seiler, Behringwerke, Marburg, © The American Society for Clinical Investigation, Inc. FRG; specific activity is 4 X 107 U/mg of protein; tests for endotoxin 0021-9738/89/04/1308/05 $2.00 using the revealed < 10 pg/mg of Volume 83, April 1989, 1308-1312 protein). After various periods of time, cell-free supernatants of PMN

1308 Lindemann, Riedel, Oster, Ziegler-Heitbrock, Mertelsmann, and Herrmann cultures (PNM-conditioned medium; PMN-CM) were collected and medium of GM-CSF-induced PMNL was detected using a stored at -700C until assayed. For RNA analysis, PMN and mono- murine CFFU-GM assay. Murine CFU-GM are known to re- cytes were incubated at 1-2.5 X 106/ml in standard culture medium spond to hG-CSF and hM-CSF, although unresponsive to under identical conditions. hGM-CSF, thus providing a suitable means to specifically de- Murine CFU-GM colony assay. Murine CFU-GM were assayed in tect G- and M-CSF activity. Supernatants from PMN cultured a double-layer agar culture system as described previously (15). Briefly, in the absence of GM-CSF for 48-72 h did not induce 0.5-ml underlayers were composed of 0.5% agar (Agar Noble; Difco any Laboratories, Inc., Detroit, MI) in Iscove's modified Dulbecco's mini- detectable growth of day 7 murine CFU-GM derived colonies. mum essential medium supplemented with L-glutamine, penicillin/ However, conditioned medium of GM-CSF activated PMNL streptomycin, 20% FCS, and 5 X l0-5 M 2-mercapto-ethanol. Culture induced considerable growth of both granulocyte and macro- supernatants to be assayed for CSF biological activity were added to phage lineage colonies (Table I). Similarly, total cellular RNA the underlayers at 10% vol/vol final concentrations. Control cultures of GM-CSF-induced PMN was found to strongly hybridize to received medium alone, 250 ng/ml recombinant human (rh) G-CSF M-CSF and G-CSF specific cDNA probes in a similar way as (kindly provided by Dr. L. Souza, Amgen Corp., Thousand Oaks, CA) activated monocytes (Fig. 1 and 2). Maximum expression of or 1,000 U/ml rhM-CSF (specific activity is 2 X 101 U/mg of protein; G-CSF and M-CSF mRNA was found after 12-24 h of culture. kindly provided by Dr. P. Ralph, Cetus Corp., Emeryville, CA). 0.5-ml Unstimulated PMN did not reveal any detectable signal. As overlayers were composed of 0.3% agar in the same medium and contained 5 X 104/ml murine (BDF. 1 mice) marrow cells. CFU- seen in Fig. 1 hybridization of mRNA of LPS- and IFN-y- GM were enumerated at day 7 and lineages of colonies were detected stimulated monocytes and GM-CSF-stimulated PMN re- by ctyochemical in situ staining of whole agar cultures (16). Naphthol- vealed multiple transcripts when probed with a M-CSF-spe- AS-D-chloro-acetate esterase (CAE) staining detects granulocyte colo- cific cDNA probe. This is well in line with recent observations nies; ANAE staining detects macrophage colonies. of RNA obtained from the PMA stimulated pancreatic carci- TNF cytotoxic assay. Biological activity of TNF was assayed with noma line MIA PaCa exhibiting six different M-CSF messages, the WEHI 164/actinomycin D (AcD) system essentially as described due to alternate splicing of exon six and alternatively used (17). In brief, WEHI 164 target cells sensitized with 1 Atg/ml AcD 3'-untranslated sequences (24). Similarly lectin-stimulated (Calbiochem, Frankfurt, FRG) were labeled for 1 h with 100 IACi of PBMC were shown to express several M-CSF transcripts (25). 5'Cr (NaCr5'04, Amersham Buchler, Braunschweig, FRG) washed of stimulation may, however, favor the in Different conditions three times fresh HBSS (2.5% FCS) and seeded in standard culture expression of the most abundant 4.2-kb message, as likewise medium at 5 X 104/ml. The cytotoxic assay was performed in sterile U-bottomed microtiter plates (Costar Corp., Cambridge, MA) by in- the assay procedure (stringency of hybridization, time of ex- cubation of Il100 of target cells in standard culture medium supple- posure) influences detection of less abundant messages. To mented with GM-CSF induced PMN-CM followed by centrifugation exclude any contamination of monocytes in the PMN prepa- (at 50 g for 2 min) and incubation for 6 h at 37°C in a humidified rations, the filters were reprobed with a v-fms-specific cDNA, atmosphere of 5% C02, in air. Half of the SN was then harvested and revealing a specific signal in the RNA-containing counted in a gamma counter. Percent specific release was calculated lanes only. from (experimental release-spontaneous release)/(maximum release Analysis of TNF synthesis by GM-CSF-induced PMN. To -spontaneous release) X 100. Maximum release was determined by detect TNF biologic activity in GM-CSF PMN-CM, the WEHI counting 50 jil of the labeled WEHI 164 cells. Sensitivity of the assay 164 cytotoxic assay was performed. These experiments showed was 1 ng/ml. no measurable TNF activity in the supernatant of GM-CSF- TNF-RIA. A TNF-specific RIA was kindly provided by Dr. G. Lambelin, IRE-Medgenix, Fleurus, Belgium. Sensitivity was shown to stimulated and -unstimulated PMN. Therefore a highly sensi- be 0.1 ng/ml TNF at 10% tracer binding inhibition. In selected experi- ments, cell lysates of GM-CSF-induced PMN were generated by re- peated freezing/thawing cycles and were assayed for TNF activity as Table . Effect ofrhGM-CSF on Secretion described above. ofG- and M-CSF by PMN Northern blot analysis. Total cellular RNA was isolated using the guanidinium/cesium chloride method (18). 10 ,g of glyoxylated RNA, Murine CFU-GM/5 X 104 marrow cells obtained from 1-1.5 X and 5-10 X 107 monocytes 10' granulocytes, Monocyte (ANAE+) Granulocyte (CAE+) were fractioned on a 1.2% agarose gel and blotted onto synthetic membranes (Schleicher & Schuell, Dassel, FRG). IL 1 mRNA was Source of CSF* Exp. I Exp. 2 Exp. 3 Exp. 1 Exp. 2 Exp. 3 detected by endlabeled oligonucleotide probes (I19) (N 19-48 for IL 1-a, and N757-786 for IL 1-13 [20]) as described (21). Specific cDNA probes Medium 5±3$ 11±2 4±1 4±2 7±2 3±1 were obtained for M-CSF by Dr. P. Ralph, Cetus Corp., for G-CSF by PMN-CM 5±3 9±1 3±1 4±2 8±2 4±1 Dr. L. Souza, Amgen Corp., for TNF-a by Dr. G. Adolph, Bender, GM-CSF PMN-CM 57±6 59±9 47±3 48±3 72±3 49±3 Vienna, Austria, and for v-fms by Dr. C. J. Sherr, Department of GM-CSF 4±2 9±1 ND§ 2±0 7±3 ND Tumor Cell Jude Research Biology, St. Children's Hospital, Memphis, G-CSF 11±2 14±2 ND 79±3 78±4 ND TN. and 5% dextran sulfate Radiolabeling hybridization including M-CSF 116±8 89±2 ND 3±2 6±2 ND (500,000 mol wt; Sigma Chemical Co., St. Louis, MO) were performed as described (22). All filters were reprobed with an a-actin cDNA (kindly provided by Dr. R. J. Schwarz, Department of Cell Biology, * Purified PMN were cultured at 2.5 X 106/ml for 48-72 h in the Baylor College of Medicine, Houston, TX) that also hybridizes with presence or absence of 250 ng/ml GM-CSF. Cell-free culture super- nonmuscle 13 actin, expressing 70% sequence homology to a actin (23). natants were used as potential stimuli for murine CFU-GM at final Blots were developed on Kodak X-Omat films with intensifying concentrations of 20% vol/vol: PMN-CM, in the presence of GM- screens. CSF (GM-CSF PMN-CM); rh CSFs at concentrations of 250 ng/ml GM-CSF, 250 ng/ml G-CSF, and 1,000 U/ml M-CSF served as con- Results trols. GM-CSF induces M-CSF and G-CSF secretion by PMN. t Colony numbers are means of triplicate CFU-GM assays (±SD). G-CSF and M-CSF biological activity present in conditioned § Not done. Cytokine Secretion by Polymorphonuclear Leukocytes 1309 Mo PMN Mo PMN

28S- ii h M-CSF

18S ;_~ 4G-CSF

4 c-fms

* Ad 4Actin f a *Act in

ni i 0 6 12 24 48 i ni 0 6 12 24 48 Figure 1. Levels of M-CSF mRNA during treatment of PMNL with Figure 2. Induction of G-CSF mRNA in activated human PMN. GM-CSF (250 ng/ml) for 0, 6, 12, 24, and 48 h. Normal human Total cellular RNA was extracted from PMN cultured with GM-CSF PMN were purified and cultured as described in Methods. Total cel- (250 ng/ml) for 0, 6, 12, 24, and 48 h respectively, was size frac- lular RNA was analyzed by Northern blotting using M-CSF-, a- tioned on an agarose gel, transferred to nylon membranes, and hy- actin-, and v-fms-specific cDNA probes. The transcript sizes were bridized thrice with the indicated probes, respectively. Transcript 4.2-2.8 (M-CSF), 2.0 (actin), and 4.2 kb (c-fms). RNA of unstimu- sizes were 1.8 (G-CSF), 2.0 (actin), and 4.2 kb (c-fms). RNA of rest- lated PMN extracted after various culture intervals up to 32 h did ing PMN cultured for 0-32 h did not hybridize with a G-CSF-spe- not hybridize with a M-CSF-specific probe. Control monocytes were cific probe. Control monocytes (i, induced; ni, not induced) were cultured for 12 h in the presence (i, induced) or absence (ni, not in- cultured as described in the legend to Fig. 1. duced) of 10 ng/ml LPS + 25 ng/ml IFN-y.

has been identified as a major activation factor for both cell types. As shown in this study, GM-CSF not only recruits fac- tive TNF-RIA, capable of detecting 0.1-0.5 ng/ml was ap- tor-mediated regulatory functions of monocytes, but also those plied, but did not reveal any TNF protein (data not shown). To of PMN by inducing mRNA accumulation and protein secre- consider the possibility that GM-CSF-induced PMN suffered tion of various cytokines. from defective TNF-a release, PMN cell lysates generated by In our experiments, the purity of analyzed PMNL samples repeated freezing/thawing cycles were analyzed for TNF in was > 99% with a contamination of < 0.1% monocytes, as both test systems as described. Under these conditions, PMN demonstrated by microscopy of ANAE- and CAE-stained ali- failed to exhibit TNF-a synthesis as well. Induction of TNF-a quots. To further exclude the possibility that cytokine release specific mRNA was, however, seen in the GM-CSF stimulated attributed to PMNL is being executed by contaminating PMNL with peak levels after 12 h of culture (Fig. 3). monocytes, PMNL derived mRNA was reprobed with a v-fms-specific cDNA also detecting c-fms (27), without dem- Discussion onstrating a specific hybridization signal, whereas monocyte RNA hybridized to the same probe. It has been previously PMN are relatively short-lived end-stage cells, considered to shown that PBMC expression of c-fms, coding for the M-CSF have little, if any, biosynthetic capacity. Several recent studies, (28), is restricted to monocyte lineage cells (29). As however, clearly demonstrate that PMN can be induced in shown by cell quantities needed to obtain a comparable vitro to synthesize mRNA and subsequently produce various amount of RNA, the specific capacity per cell is - 5-10 times proteins such as c-fos (26), plasminogen activator (10), IL 1-a less in PMN than in monocytes. This, however, does not argue and IL 1-13 (12). PMN are also shown to share a number of against the possible physiological role of cytokine production common features with monocytes, including their common by PMN in vivo, because of their quantitative predominance cell lineage and their migratory and phagocytic potential. in the peripheral blood and particularly at most sites of inflam- PMN and monocytes have similar receptors and surface anti- mation. gens and may play a similar role in the nonspecific host de- Major sources of M-CSF and G-CSF so far identified are fense. In this regard the T cell-derived lymphokine GM-CSF macrophages, endothelial cells, and . GM-CSF-in-

1310 Lindemann, Riedel, Oster, Ziegler-Heitbrock, Mertelsmann, and Herrmann MO PMNi vided (35). It thus is reasonable to assume, that GM-CSF might be involved in the control of TNF synthesis in a similar way, although the ultimate functional effect is dependent on the presence of synergistically operating agents. Studies are under way to define the nature of these molecules. The capac- ity of PMN to synthesize mediators such as IL 1 (12), and -TNF probably TNF-a, supplements their functional role in inflam- matory reactions, and may additionally be relevant for tumor- icidal effects (TNF-a) as well as immune responses via inter- action with T cells (IL 1) (36, 37). Recent reports of hematopoietic secretion by granulocyte lineage acute nonlymphocytic leukemia (38, 39) relate to our results and suggest that myeloid lineage cells bear the capacity to synthesize various cytokines. A state of dysreg- ulation of the progenitor cell and external stimuli of the ma- ow * c-fms ture PMN may trigger this potential. According to preliminary to results, aside from GM-CSF, TNF-a and LPS were found to induce IL 1 and TNF-a mRNA in PMNL, whereas G-CSF was found to be inactive in this regard. However, none of the stim- ulatory molecules was as active as GM-CSF in inducing a brought spectrum of cytokines in PMNL, suggesting a central role of this T cell-derived lymphokine in the recruitment of *. .. * 4Actin nonspecific effector cells. More importantly, PMN were shown to mediate regulatory functions by demonstrating bio- synthetic activity, thus being responsible for some in vivo functions previously ascribed only to monocytes. ni i 0 4 8 12 24 Figure 3. Expression of TNF-a-specific mRNA by GM-CSF-induced Acknowledgments PMNL. Total cellular RNA from PMNL, cultured (2.5 X 106/ml) in the absence or presence of GM-CSF (250 ng/ml) for 0, 4, 8, 12, and This work was supported by the Deutsche Forschungsgemeinschaft 24 h (analogous lane designation) was subjected to Northern blot grant He 1380-2-1 (to F. Herrmann). analysis, hybridized with a TNF-a-specific cDNA and rehybridized with a actin and v-fms cDNAs, detecting transcripts of 1.8 (TNF-a), References 2.0 (actin), and 4.2 kb (c-fms). RNA of noninduced PMN cultured for 0-32 h failed to hybridize with a TNF-a-specific probe. Control monocytes (i, induced; ni, not induced) were cultured as described in 1. Metcalf, D. 1985. The Granulocyte-Macrophage Colony-stimu- the legend to Fig. 1. lating Factors. Science (Wash. DC). 229:16-22. 2. Huebner, K., M. Isobe, C. M. Croce, D. W. Golde, S. E. Kauf- man, and J. C. Gasson. 1985. 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1312 Lindemann, Riedel, Oster, Ziegler-Heitbrock, Mertelsmann, and Herrmann