Effect of Granulocyte-Macrophage Colony- Stimulating Factor on Chemiluminescence of Human Neutrophils

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1 Original Paper International Journal of Cell Cloning (1989) Effect of Granulocyte-Macrophage Colony- Stimulating Factor on Chemiluminescence of Human Neutrophils Shigeru Yamaga"-', Seiichi Okamura', Teruhisa Otsuka", Yoshiyuki Niho" 'The First Department of Internal Medicine and bcancer Center, Faculty of Medicine, Kyushu University, Fukuoka, Japan Key words. GM-CSF LDCL Neutrophils * EDso Abstract. We investigated the capacity of recombinant human granulocyte-macrophage colony-stimulating factor (rhgm-csf) to enhance the function of neutrophils. Neutrophil function was measured in terms of N-formyl-methionyl-leucyl-phenylalanine (MLP)- induced luminol-dependent chemiluminescence (LDCL). LDCL of MLP-stimulated neutrophils was enhanced up to 4.5 fold following preincubation with rhgm-csf. This enhancement depended on the length of preincubation, reaching an optimal level at 120 min. The dose-response relationship for MLP-induced LDCL of neutrophils preincubated with rhgm-csf revealed that half-maximum enhancement was achieved at an approximately 20-fold higher concentration than that of colony-forming units in culture-derived colony formation. These results suggest that differences in dose dependency may be explained by differences in the distribution of receptor(s) for GM-CSF. This may also enable GM-CSF to affect the hematopoietic system, which contains cells at various levels of differentiation, thus mediating the host-defense mechanism. Introduction Human granulocyte-macrophage colony-stimulating factor (hgm-csf), a 22 to 23 kd glycoprotein, is one of the cytokines produced by activated T cells or interleukin 1 (IL-1)-stimulated fibroblasts [I-31. It stimulates hematopoietic precursor cells for the granulocyte-macrophage lineage in bone marrow and was recently found to potentiate superoxide generation by neutrophils [4]. In this study, we investigated the dose effect of hgm-csf on the generation of active oxygen radicals by neutrophils. Such generation was potentiated by hgm- CSF, and the dose for half-maximum effect (ED50) was found to be considerably Correspondence: Dr. Seiichi Okamura, Cancer Center, Faculty of Medicine, Kyushu University, Maidashi, Higashi-ku, Fukuoka 812, Japan. Received June 3, 1988; provisionally accepted July 6, 1988; accepted for publication August 26, Press

2 GM-CSF on LDCL of Neutrophils 51 higher than that required for growth-stimulating activity on colony-forming units in culture (CFU). Materials and Methods Cells Non-adherent bone marrow cells (NA cells) were prepared as previously described [5]. Briefly, bone marrow aspirates from healthy volunteers were diluted 3 times with Dulbecco's phosphate-buffered saline (PBS; Nissui Pharmaceutical Co., Tokyo, Japan), and light-density ( < g/ml) cells were separated by density gradient centrifugation on lymphocyte separation medium (LSM; Organon Teknika Cop, Durham, NC) at x600 g for 30 min and washed 3 times with PBS. The light-density cells were then resuspended in Iscove's modified Dulbecco's medium (IMDM; GIBCO Laboratories, Grand Island, NY) supplemented with 20% (v/v) heat-inactivated fetal calf serum (FCS; Boehringer, Mannheim, FRG) at 1 X lo6 cells/ml. The culture was transferred to plastic Petri dishes (NUNC, Roskilde, Denmark), pretreated with FCS and incubated at 37 C overnight in a fully humidified atmosphere of 5% C02 in air. Cells which had not adhered to the dish were recovered and treated as NA cells. Human neutrophils were obtained from venous blood of healthy volunteers. Briefly, 1 volume of saline containing 6% (w/v) dextran (mean MW, 66,300; Sigma Chemical Co., St. Louis, MO) was added to 3 volumes of heparinized venous blood and left at room temperature for 30 min to induce erythrocyte sedimentation. After dextran sedimentation, neutrophils were separated by density gradient centrifugation at X600 g for 30 min on LSM. The pellet in the bottom was recovered and subjected to hypotonic hemolysis in order to eliminate any contaminating erythrocytes. The pellet was resuspended in 5 ml of ice-cold 0.2% NaCl solution and stirred for 30 sec; 5 ml of 1.6% NaCl solution was then added and centrifuged at ~430 g for 5 min. After washing twice with Ca", Mg++-free Hanks' balanced salt solution (CMF-Hanks), the cells containing neutrophils at a purity of more than 95% were used for the luminol-dependent chemiluminescence (LDCL) assay. Recombinant hgm- CSF (rhgm-csf) The rhgm-csf and mock were kindly provided by Dr. K. Ami of DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA. It was expressed in large quantities in Sacchammyces cerevisiae using a secretion vector containing the promotor and leader sequences of the mating pheromone a-factor, and the activity of human GM-CSF was confirmed by stimulation of granulocyte-macrophage colony formation using human cord blood cells [6]. Measurement of Colony-Stimulating Activity Colony-stimulating activity was assayed using the modified colony formation technique, as previously reported [7]. Briefly, NA cells (5 X 104) were cultured in 35 mm Petri dishes using 1 ml of IMDM supplemented with 20% (vlv) FCS, 0.88% methylcellulose (Shin-Etsu Kagaku Co., Ibkyo, Japan) and various concentrations of rhgm-csf. The rhgm- CSF was serially diluted with IMDM containing 1% FCS. The culture was incubated at 37 C in a fully humidified atmosphere of 5% C02 in air for 14 days. Cell aggregates containing greater than 20 cells following incubation were counted as CFU-derived colonies. Colony-stimulating activity was calculated from the linear portion of the dose-response curve as described by Nicola et al. [7].

3 YamagalOkamura/Otsukaa/Ni ho 52 LDCL Assay 18, 91 Neutrophils (2 X lo7) were incubated at 37 C with CMF-Hanks supplemented with 2% FCS, 0.5% gelatin (Sigma) and appropriate amounts of rhgm-csf for a specific time. Then a proportion of them (1 x lo5) were suspended with 0.2 ml of Hanks' balanced salt solution supplemented with 1 x 10-"M luminol (Sigma) and incubated at 37 C for 1 min. Then 0.1 ml of Hanks' balanced salt solution supplemented with 3 X 10-7M N-formylmethionyl-leucyl-phenylalanine (fmlp; Sigma) was added unless otherwise specified. The LDCL was measured with the use of a lumiphotometer (TD400; Lab0 Science Co., Tokyo, Japan). The peak CL) response of fmlp-stimulated neutrophils occ u d between 2-5 min post-stimulation with a subsequent rapid decline toward the baseline (Fig. 1). The peak CL of the neutrophils was expressed as counts per min (cpm) [8, 91. Results Colony-Stimulating Activity of rhgm-csf The number of CFUderived colonies formed with optimal dilution of rhgm- CSF was f 14.W x lo4 NA cells. The dose-response relation between the number of CFU-derived colonies formed and rhgm-csf dilution is shown in 'hble I. The half-maximum stimulation for CFU formation was achieved at 1,700 times the dilution of the original preparation. As the concentration of rhgm-csf for this condition was defined as 50 U/ml, this original preparation contained 85,000 U/ml of rhgm-csf. Direct Effect of rhgm-csf on LDCL of Neutrophils The LDCL of neutrophils incubated with 5,000 U/ml of rhgm-csf was measured for 60 min. In the absence of fmlp, no stimulation of neutrophils was observed in the presence of rhgm-csf (Fig. 1). LDCL ofjmlp-stimulated Neutrophils after Preincubation with rhgm-csf The effect of the duration of neutrophil preincubation with rhgm-csf on neutrophil fmlp-induced LDCL is shown in Figure 2. No significant effect was noted immediately after the addition of rhgm-csf. LDCL was enhanced as the preincubation time became longer, and the effect was prominent by 120 min. No enhancement of LDCL was observed with mock. Thus, the optimal preincubation time was 120 min, and the subsequent experiments were performed during this period. The peak CL of fmlp-stimulated neutrophils preincubated with mock for 120 min was 3.34 x 104 cpm/l x lo5 cells. The peak CL was significantly enhanced by preincubation even with 50 U/ml of rhgm-csf. This effect was observed to be dose-dependent, and the peak CL reached 14.5 x lo4 cpm/l x lo5 cells when 2,500 U/ml of rhgm-csf was added (Fig. 3). Discussion GM-CSF, one of the known CSFs, is a glycoprotein that stimulates the clonal

4 ~~~ GM-CSF on LDCL of Neutrophils 53 'hble I. Colony-stimulating activity of rhgm-csf on normal human bone marrow cells Ratio of dilution Number of CFU-derived colonies' Ratio of colony Exp. 1 Exp. 2 Exp. 3 formationb 4, f 3.8 2, f 2.0 1,OOo f f f 2.1 'CFU-derived colonies formed per 5 x lo4 NA cells at day 14 bratio in comparison with maximum colony formation in each experiment, mean f SD growth of human hematopoietic precursor cells in vitro [6, 101, as well as in vivo [ll]. It enhances the functional activity of mature neutrophils, for example, granulocyte antibodydependent cell-mediated cytotoxicity [lo], and neutrophil superoxide production, which is dependent on a secondary stimulus such as fmlp [lo, 121. Thus, GM-CSF is thought to contribute to the regulation of neutrophil production and function, which comprises an important part of the primary defense mechanism. In the regional reaction which occurs in the early phase of infection, neutrophils play the most important role [13]. They begin to assemble at the site of inflammation in a few minutes, and the neutrophil reaction reaches its maximum by 10 to 20 h [14]. When neutrophils come into contact with foreign substances or are stimulated by chemoattractants like MLP or Cs fragments, their consumption of oxygen increases explosively, producing the so-called respiratory burst, and superoxide is produced [l5-17]. The respiratory burst also causes the chemiluminescence phenomenon of neutrophils. Allen et al. [9] have proved that the frequency of the chemiluminescence is correlated with hexosemonophosphate shunt activity. Chemiluminescence also reflects the function of neutrophils, including met cellkilling ability and post-phagocytic O2 consumption, and disorders of neutrophil function, such as chronic granulomatous disease [9, 181 or leukocyte myeloperoxidase deficiency [19] can be detected by this assay. This phenomenon is considered to be the result of 0; and its oxidants, and the frequency of chemiluminescence has been proved to be related to 0; production [8,9]. Therefore, the chemiluminescence phenomenon reflects the biological function of neutrophils. In our experiments, the chemiluminescence of MLP-stimulated neutrophils was measured using luminol as a photointensifier, and enhanced function of rhgm-csf-activated neutrophils was measured as LDCL. The present data indicate that rhgm-csf does not directly stimulate neutrophils to produce the respiratory burst, although neutrophils can be primed by exposure to GM-CSF. After GM-CSF priming, increased LDCL was observed upon

5 YamagalOkamura10tsukalNiho 54 Fig. 1. Direct effect of fmlp and rhgm-csf on LDCL of neutrophils. Either rhgm- CSF or fmlp was added to a suspension containing 1 x los neutrophils, and LDCL was recorded every second for 60 min. Addition of fmlp caused stimulation of neutrophils, but rhgm-csf did not. Fig. 2. Effect of duration of preincubation with rhgm-csf on fmlp-induced LDCL of neutrophils. Neutrophils were preincubated for various periods of time with or without rhgm-csf. The concentration of rhgm-csf was 5,OOO Ulml.

6 GM-CSF on LDCL of Neutrophils 55 NLP stimulation. Optimal priming of neutrophils was achieved by 120 min preincubation with rhgm-csf. A preincubation period shorter than 120 min caused partial time-dependent enhancement of LDCL. This suggests that some mechanisms, including enzymatic activity in the cytosol and/or cell membrane, may play a role in the priming of neutrophils exposed to rhgm-csf. This type of neutrophil priming has also been shown for bacterial lipopolysaccharides [20]. Possible contamination by lipopolysaccharides present in commercial culture medium, deionized water, animal sera or albumin, Ficoll solutions, buffers and salts must be eliminated, since it might disturb the results of this type of examination. We therefore used the mock which had originated from the same species of yeast without induction of cdna for rhgm-csf, and this did not enhance the LDCL of neutrophils. Therefore, the effect of preincubation with rhgm-csf on the LDCL of MLP-stimulated neutrophils proved to be due to rhgm-csf itself. In addition, the time-dependency of preincubation with rhgm-csf reached an optimal value at 120 min, longer than that with bacterial lipopolysaccharide [20]. This also suggests that there is some difference in the mode of priming between rhgm-csf and bacterial lipopolysaccharide. The EDSo for the priming effect of rhgm-csf on neutrophils calculated from the dose-response curve was 1,OOO U/ml (Fig. 4, 0), which was significantly (20 times) higher than the effect of rhgm-csf on the clonal growth of < 0.01), defining the half-maximum stimulating activity for CFU as 50 U/ml (Fig. 4, 0). We studied the effect of rhgm-csf on human neutrophil functions using LDCL as a function parameter. Previous investigators using a cytochrome c reduction assay system reported that the dose of rhgm-csf required for enhancement of neutrophil functions is less than or equal to that necessary for CFU-gm colony formation [lo, 21, 221; whereas, we have shown here that the former is greater than the latter. Neutrophil functions measured by LDCL and by cytochrome c reduction show different results [23] because LDCL is dependent on the myeloperoxidase-hydrogen system [19,24], however cytochrome c is related only to 0; generation [14]. CFU are hematopoietic precursor cells of the myeloid lineage, and the continuous presence of GM-CSF is essential for CFU clonal growth [25]. Neutrophils are differentiated mature myeloid cells and are the effector cells that actually play a role in the host-defense mechanism [D]. Both CFU and neutrophils are influenced by rhgm-csf [I, 2,4, 101, but the optimal doses required were significantly different according to our data. In this regard, our findings support the distributional heterogeneity of GM-CSF receptors on myeloid cells at different levels of maturation [26]. Sullivan et al. recently demonstrated that NLP receptors are not detectable on normal human myeloblasts and that they gradually increase in number as the cells mature [27]. In contrast to MLP receptors, GM-CSF receptors might decrease as the cells mature, but GM-CSF can still modify the level of the respira-

7 YamagalOkamuralOtsuklNiho 56 Fig. 3. Dose-response relationship for fmlp-induced LDCL of neutrophils preincubated with rhgm-csf. Neutmphils were preincubated with various concentrations of rhgm- CSF. Preincubation period was 120 min, which had proven to be optimal (Fig. 2). Fig. 4. Difference in the dose-response relationship between two effects of rhgm-csf on myeloid cells: the effect on CFU growth (0) and on enhancement of neutrophil LDCL (0). The data are shown as mean percentages and 1 SD of the maximum effect for 3 individual experiments.

8 GM-CSF on LDCL of Neutrophils 57 tory burst and superoxide production following attachment of chemotactic peptides to the receptors. This phenomenon might be due to transmodulation between receptors of different types. It is known that mitogen-stimulated lymphocytes, or fibroblasts exposed to IL-1 [3] or tumor necrosis factor in vitro [28] can express the GM-CSF gene. It is suggested that the high level of GM-CSF secreted from activated cells infiltrating or located close to the site of inflammation might actually mediate the biological information necessary for enhancing neutrophil function. As neutrophils tend to dmage normal tissue distant from the site of inflammation by oversecretion of superoxides, it may be rational for a higher level of GM-CSF to be required in order to activate neutrophils. The relatively lower level of GM-CSF required for the clonal growth of progenitor cells in bone marrow may also be rational for the recruitment of mature neutrophils. Acknowledgment This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan (Nos , , and ) and from the Fukuoka Anti-Cancer Society. The authors are also indebted to Drs. K. Arai and A. Miyajima (DNAX Research Institute) for providing the rhgm-csf, to Drs. S. Hayushi and E Omori of our Department for helpful discussion, and to Miss l! Haruuono for typing the manuscript. References Wong GG, Witek J, Temple PA, et al. Human GM-CSF: molecular cloning of the complementary DNA and purification of the natural and recombinant proteins. Science 1985 ;228: Lee F, Yokota T, Otsuka T, et al. Isolation of cdna for a human granulocyte-macrophage colony-stimulating factor by hnctional expression in mammalian cells. Proc Natl Acad Sci USA 1985;82: Zucali JR, Dinarello CA, Oblon DJ, Gross MA, Anderson L, Weiner RS. Interleukin 1 stimulates fibroblasts to produce granulocyte-macrophage colony-stimulating activity and prostaglandin E2. J Clin Invest 1986; Weisbart FU-I, Golde DW, Clark SC, Wong GG, Gasson JC. Human granulocytemacrophage colony-stimulating factor is a neutrophil activator. Nature 1985;314: Messner H, Till JE, McCulloch EA. Interacting cell populations affecting granulopoietic colony formation by normal and leukemic human marrow cells. Blood 1973; 42 : Miyajima A, Otsu K, Schurs J, Bond MW, Abrams JS, Arai K. Expression of murine and human granulocyte-macrophage colony-stimulating factors in S. ceraisiae: mutagenesis of the potential glycosylation sites. EMBO J 1986;5: Nicola NA, Metcalf D, Matsumoto M, Johnson GR. Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells: identification as granulocyte colony-stimulating factor. J Biol Chem 1983;258:

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