Monocyte-Macrophage Differentiation Induced by Human Upper Airway Epithelial Cells Takayuki Ohtoshi, Carlo Vancheri, Gerard Cox, Jack Gauldie, Jerry Dolovich, Judah A. Denburg, and Manel Jordana Departments of Pathology and Pediatrics and Medicine, McMaster University, Hamilton, Ontario, Canada

We examined the ability of conditioned medium (CM) generated by human upper airway epithelial (Ep) cells from normal (NN) and inflamed, allergic rhinitis (AR) and nasal polyp (NP) tissues to induce monocytic differentiation of hemopoietic progenitors of the HL-60 myeloid leukemia cell line in vitro. In HL-60 cells cultured in RPMI with 10% FBS, there was differentiation to 0.4 ± 0.4% monocytic cells. NN-, AR-, and NP-EpCM induced differentiation to 23 ± 6 %, 42 ± 11%, and 71 ± 10% monocytic cells, respectively. EpCM also induced isolated peripheral blood nonadherent mononuclear cells to express monocyte/macrophage-specific antigens as detected by immunohistochemistry using FMC-32 monoclonal antibodies (anti-CD14). We also examined the cytokine content of these EpCMs and found .that they contained granulocyte/macrophage colony-stimulating factor (GM-CSF): 126 ± 35, 198 ± 22, and 489 ± 118 pglml for NN-, AR-, and NP-EpCM, respectively. These CMs also contained granulocyte-CSF (G-CSF) and interleukin-6 (IL-6); but there were no significant differences between normal and inflamed tissue-derived cell supernatants. No macrophage-CSF (M-CSF) was detected in these EpCMs. Recombinant human GM-CSF, G-CSF, and IL-6, alone and in combinations, at doses similar to or greater than those found in the EpCMs, did not induce comparable monocytic differentiation of HL-60 cells. Preincubation of the EpCM with neutralizing anti-GM-CSF, anti-G-CSF, or anti-IL-6 antibodies did not significantly inhibit the monocytic differentiation induced by the EpCM. Monocyte/macrophage inducing activity in EpCM was heat sensitive, and high-performance liquid chromatography fractionation showed two peaks of activity (34 to 56 kD and 7 to 12 kD), which were synergistic on HL-60 differentiation. These studies illustrate the effector potential of human upper airway epithelial cells, and demonstrate differences between normal cells and those derived from inflamed tissues. They also show the ability of epithelial cell-derived products to induce monocytic differentiation of human hemopoietic progenitors and suggest that this effect is due to a cytokine(s) other than IL-6 and the CSFs.

Nasal polyps and allergic rhinitis are both conditions associated with the presence of a chronic inflammatory infiltrate in the underlying nasal mucosa that is variable but includes macrophages, lymphocytes, neutrophils, metachromatic cells, and eosinophils (1, 2). Among these cells, the

(Received in original form April 25, 1990 and in revised form September 19,1990) Address correspondence to: Manel Jordana, M.D., Department of Pathology, Room 4H17-21, McMaster University, Hamilton, Ontario, L8N 3Z5 Canada. Abbreviations: allergic rhinitis, AR; conditioned medium, CM; human upper airway epithelial cells, Ep; fetal bovine serum, FBS; granulocyte colony-stimulating factor, G-CSF; granulocyte/macrophage colony-stimulating factor, GM-CSF; hormonally defined Fl2 Ham's medium, HD-Fl2; N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid, Hepes; high-performance liquid chromatography, HPLC; interleukin, IL; macrophage colonystimulating factor, M-CSF; neutrophil chemotactic factor, NCF; normal nasal, NN; nasal polyp, NP; nonadherent peripheral blood mononuclear cells, PBMNC; phosphate-buffered saline, PBS; recombinant human, rho Am. J. Respir. Cell Mol. BioI. Vol. 4. pp. 255-263, 1991

monocyte/macrophage is thought to playa pivotal role in the regulation of the inflammatory process because, in addition to its antigen-presenting and phagocytic abilities, this cell is capable of releasing a number of hormone-like peptide messenger molecules (cytokines), In contrast to the abundant knowledge on the effector potential of this cell, the mechanisms by which monocytes/macrophages accumulate at a particular tissue site, especially in the human system, remain unclear. There is increasing evidence indicating that tissue microenvironments are, in fact, inductive compartments in which immune-effector and inflammatory cell functions are modulated. One of the components of this inductive activity involves soluble signals released by the structural cell population. Indeed, structural cells such as fibroblasts and endothelial cells are capable of releasing a variety of cytokines (3-6) that can, in turn, interact with both immature and mature inflammatory cells and thereby modulate the inflammatory process. With regard to upper airway inflammation, we have previously shown that a potential mechanism for inflammatory cell accumulation in tissues is the ingress, and

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differentiation at local sites, of progenitors (7-12). In the current studies, we sought to investigate whether human upper airway epithelial cells were capable of inducing differentiation of hemopoietic cell progenitors toward mature inflammatory cells, particularly to monocytes/macrophages, and whether epithelial cells derived from inflamed tissues elicited this activity in a fashion distinct from normal epithelial cells. Our results show that conditioned media from cultured epithelial cells, particularly those derived from nasal polyps, induce monocytic differentiation of human hemopoietic progenitors. We also show that epithelial cells release interleukin-6 (IL-6), granulocyte/macrophage colony-stimulating factor (GM-CSF), and granulocyte-CSF (G-CSF) in vitro but no detectable amounts of macrophage-CSF (M-CSF). However, the monocyte differentiation activity present in the epithelial cell-conditioned medium (EpCM), which was heat labile, emerged as two separable peaks on highperformance liquid chromatography (HPLC) fractionation. Both factors were active in the HL-60 assay and showed synergistic activity in monocyte differentiation.

Materials and Methods Subjects Nasal polyp tissues were obtained from subjects undergoing polypectomy for nasal obstruction; inferior turbinate tissue mucosae and normal control tissue mucosae were obtained from patients with positive skin tests (allergic rhinitis) or with negative skin tests (nonallergic) at the time of surgery for corrective or cosmetic reasons. Informed consent was obtained for research purposes; these studies conformed to guidelines for human experimentation and were approved by the Chedoke McMaster Hospital's ethics committee. Preparation of Collagen-coated Plates Collagen-coated culture plates were prepared, with minor modifications, according to the method described by Yankaskas and coworkers (13). Briefly, the bottoms of Linbro 24 flat-bottom well culture plates (Flow Laboratories Inc., McLean, VA) were covered with a small amount of collagen (Vitrogen 100; Collagen Corp., Palo Alto, CA) and allowed to dry. Each well was then exposed to 0.5 ml 3.5 % N~OH in distilled water for 25 min. After washing with sterile distilled water, each well was exposed to 0.5 ml of 2.5% glutaraldehyde in phosphate-buffered saline (PBS) for 10 min, and rinsed with distilled water again. Ham's F12 medium (GIBCO, Grand Island, NY) with 1% penicillinstreptomycin (GIBCO) (Ham's F12) was delivered into each well until used. Preparation of Epithelial Cell Monolayers Nasal polyps or inferior turbinate tissues resected from allergic or nonallergic patients were rinsed three times with Ham's F12. They were then incubated in 0.1% protease solution (Sigma Chemical Co., St. Louis, MO) in Ham's F12 at 4° C for 16 h. After incubation, heat-inactivated fetal bovine serum (FBS) (GIBCO) was added to a final concentration of 10% (vol/vol) to neutralize protease activity. The cells were detached from the tissue by gentle agitation, filtered through a 60-p.m Nitex mesh (Collector; E-C Apparatus Corp., St.

Petersburg, FL), and centrifuged at 800 x g for 10 min. The cell pellet was resuspended in hormonally defined Ham's F12 medium (HD-F12), and 5 x lQ4 cells in 2 ml of medium were plated onto collagen-coated wells. The HD-F12 contained 1% penicillin-streptomycin, 5 p.g/ml insulin (GIBCO), 5 p.g/ml transferrin (GIBCO), 25 p.g/mI epidermal growth factor (Collaborative Research Corp., Lexington, MA), 15 p.g/ml endothelial cell growth supplement (Collaborative Research Corp.), 2 x 10- 10 M triiodothyronin (GIBCO), and 10- 7 M hydrocortisone (GIBCO). Cells were cultured in a humidified atmosphere at 3r C and 5 % CO 2 • The medium was changed at day 1 and subsequently every 2 d. Cells derived from nasal polyps or inferior turbinate tissues reached a confluent monolayer between 6 and 10 d. Generation of EpCM When monolayers of epithelial cells reached confluency, media were switched from HD-FI2 to RPMI-1640 medium (GIBCO) supplemented with 10% heat-inactivated FBS, 1% penicillin-streptomycin, and 25 mM Hepes buffer (Boehringer Mannheim Canada Ltd.). After 48-h incubation period, the cell supernatant was collected, centrifuged at 400 x g for 10 min, and sterilized with a Millipore filter (0.22 p.m; Millipore Corp., Bedford, MA), and stored at - 20° C until use.

a

Characterization of Cultured Epithelial Cells Immunohistochemistry. Confluent monolayers of epithelial cells were stained with anti-keratin (1:50 AEl/AE3; Hybritech, San Diego, CA) or with anti-vimentin (1:50 V9; Dako, Santa Barbara, CA), or with FMC-32 (IgG l , kappa; Cedarlane Laboratories, Hornby, Ontario, Canada) monoclonal antibodies using the Zymed streptavidin-biotin immunoperoxidase method (Zymed Histostain-SP Kit; Zymed Laboratories Inc., San Francisco, CA). Monolayers of fibroblasts derived from nasal tissue, obtained using an outgrowth from an explant method previously described (14), were also stained with these antibodies to control for specificity. Negative controls were included throughout by substituting the diluent (5 % swine serum in 'Iris-buffered saline) for the primary antibodies. Transmission electron microscopy. Cultured epithelial cells on collagen-coated tissue culture plates were rinsed with PBS (pH 7.4) and fixed in 2 % cold glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 h. After overnight rinsing in cacodylate buffer, they were then postfixed in 1% osmium tetroxide for 30 min, dehydrated with a series of increasing concentrations of ethanol, and scraped off from the tissue culture plates. These layers of cultured cells were cut into small pieces and embedded in Spurr's resin. Ultrathin sections were doubly stained with uranyl acetate and lead nitrate and examined with a JEM 1000EX electron microscope. Reagents Recombinant human granulocyte/macrophage colonystimulating factor (rhGM-CSF) and rh interleukin-6 (rhIL6) were obtained from Genetics Institute (Cambridge, MA). The specific activities ofrhGM-CSF and rhIL-6 were 1.7 X 107 U/mg and 106 U/mg, respectively. Recombinant human

Ohtoshi, Vancheri, Cox et al.: Monocytic Differentiation by Upper Airway Epithelial Cells

granulocyte colony-stimulating factor (rhG-CSF) was purchased from Amersham (Buckinghamshire, UK); its specific activity was 2 X 108 U/mg. Rat anti-human GM-CSF monoclonal antibody was kindly provided by Dr. John S. Abrams (DNAX Research Institute, Palo Alto, CA). One microgram per microliter of this anti-GM-CSF antibody (IgG) neutralizes 2 ng/ml human GM-CSF in a K61 bioassay. Mouse anti-human G-CSF was kindly donated by Dr. Bruce W. Altrock (AMGEN Corp., Thousand Oaks, CA); the neutralizing activity of this antibody in a CFU-GM bone marrow assay was lOS V/ml. Rabbit anti-human IL-6 antisera were raised against rhIL-6. Neutralizing activity of this antibody was 4,000 U IL-6/ml in a hepatocyte stimulation assay as previously described (15). Experimental Culture of HL-60 Cells Human myeloid leukemia cell line HL-60 cells were originally obtained at passage level 15 from the American Type Culture Collection (Rockville, MD). Cells were grown in RPMI-I640 medium with 10% heat-inactivated FBS, 1% penicillin-streptomycin, and 25 mM Hepes; passaged twice weekly; and maintained at a concentration of 2.5 X I05/ml. Cell viability was tested twice weekly by trypan blue exclusion. In the initial series of experiments, 105 HL-60 cells were cultured in 2 ml EpCM from either nasal polyp (NP-EpCM), allergic rhinitis (AR-EpCM), or normal nasal epithelial cells (NN-EpCM) in 6-well tissue culture plates (Corning, NY) at 37° C in a 5 % CO 2 incubator. Three days later, the cultured HL-60 cells were centrifuged at 400 X g for 10 min and resuspended in either fresh EpCM or RPMI-I640 containing 10% FBS. Afterwards, a minor change was made to simplify the procedure; cells were not centrifuged at day 3 but rather an equal volume of fresh medium or EpCM was just added into each well. No differences in the extent of HL-60 cells differentiation between these two procedures were noted. At day 7, HL-60 cells were harvested and cytocentrifuge smears prepared. The effect of cytokines such as rhGM-CSF, rhG-CSF, and rhIL-6, alone or in combination, on the differentiation of HL-60 cells was examined using the same experimental protocol as described above. In studies involving the use of antiGM-CSF, anti-G-CSF, and anti-IL-6 antibodies, EpCM was preincubated with these antibodies for 2 h at 37° C before being added to the HL-60 cells. Cellular morphology was evaluated by performing differential counts of smears stained with the Diff-Quik modification of the Wright-Giemsa technique (American Scientific Products, McGraw Park, IL). The criteria of monocytic differentiation of HL-60 cells included the following: cells with a diameter ~ 15 JLm and with an irregular, rather lobulated, shape, large dove blue or gray cytoplasm with very fine granules, and either lobulated or indented nucleus with a low chromatin content. Immunohistochemical analysis of EpCM-induced HL-60 cells was performed by a Zymed streptavidin-biotin using immunoperoxidase method with FMC-32 (lgGI, kappa) (Cedarlane Laboratories), a mouse monoclonal antibody directed against human mature monocytes and macrophages, which recognizes CD14 (16).

257

Immunoassay of GM-CSF, G-CSF, and M-CSF Human GM-CSF content in EpCM was detected by means of a specific immunoassay performed by Dr. John S. Abrams. The limit of sensitivity of this assay was 30 pg/ml. Human G-CSF content in EpCM was quantitated in a mono specific sandwich immunoassay by Dr. Bruce W. Altrock, and the limit of specificity of this assay was 0.2 ng/ml, Human M-CSF content in EpCM was quantitated using a radioimmunoassay by Dr. Peter Ralph (Cetus Corp., Emeryville, CA), the limit of sensitivity of this assay was 0.8 ng/ml. Partial Characterization of Monocyte Differentiation Activity in EpCM Heat treatment ofEpCM. EpCM was incubated at 60° C for 1 h and then tested for HL-60 differentiating activity at a concentration of 50% (vol/vol) in the same assay as described above. HPLC of EpCM. Nasal polyp EpCM was concentrated 10fold and tested for the presence of monocyte differentiating activity in the HL-60 system. This material was. then HPLCfractionated using a TSK 250 molecular sieve column, which had been equilibrated with 0.1 M phosphate buffer (pH 6.8) and eluted at a flow rate of 0.75 ml/min. One and one-half milliliter fractions were collected (from 12 to 36 min). The column was calibrated with thyroglobulin (mol wt = 670 kD), y-globulin (mol wt = 158 kD), ovalbumin (mol wt = 44 kD), myoglobin (mol wt = 17 kD), and vitamin B12 (mol wt = 1.35 kD). Eluate fractions were tested directly or in combination for monocyte differentiating activity on HL-60 cells at a concentration of 25 % (vol/vol).

Effect of EpCM on Peripheral Blood Nonadherent Mononuclear Cells (pBMNC) Heparinized venous blood was obtained from normal volunteers. This was mixed with an equal volume of PBS and centrifuged on a Ficoll-Hypaque gradient. The mononuclear cells were washed with PBS, and monocytes were depleted by adherence over 2 h. Nonadherent cells were resuspended in RPMI. In order to examine the effect of EpCM on normal human hemopoietic progenitor cells, 106 nonadherent mononuclear cells were incubated with 2 ml of either RPMI1640 with 10% FBS or 50% EpCM in Linbro 24 flat-bottom well culture plates (Flow Laboratories). At day 7, half volumes of each medium were replaced, and at day 14, cytospin smears of harvested cells were made. Monocyte/macrophage differentiation was evaluated by immunohistochemistry with FMC-32 antibody as described above.

Results Purity of Cultured Epithelial Cells The high degree of purity of the epithelial cells in our culture system was supported by immunohistochemical evaluation of the monolayers with anti-keratin and anti-vimentin antibodies (Figure 1). More than 95% of the cells in the monolayer were keratin-positive; very occasionally, ~mall vimentin-immunoreactive cells were seen. In companson, fibroblasts derived from nasal tissues were strongly positive for vimentin (~ 95 %) and did not react with the anti-keratin

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Figure 1. Immunohistochemical characteristics of human upper airway epithelial cells and fibroblasts. Monolayer of epithelial cells stained with anti-keratin (a) and anti-vimentin (b) antibodies. Monolayer of fibroblasts stained with anti-keratin (c) and antivimentin (d) antibodies.

antibodies. This was further supported by ultrastructural studies (transmission electron microscopy). Figure f illustrates a representative sample of the cells comprising the epithelial cell monolayer; abundant microvilli on the cell surfaces, desmosomes ubiquitously distributed between the cell junctions, and dense tonofilaments in the cytoplasm are all characteristics typical of epithelial cells. EpCM-induced HL-60 Cell Differentiation HL-60 cell cultures in RPMI with 10% FBS contained 0.4 ± 0.4% (n = 17, mean ± SD) monocytic cells at day 7. Under these conditions, no adherent cells were seen. In contrast, a significant number, 8.1 ± 4.8 % (n = 6), of cells cultured in EpCM became attached to the surface of the tissue culture wells at day 7. The differential count of cytocentrifuge smears of cells harvested at day 7 revealed that EpCM induced a marked monocytic differentiation of HL-60 cells. Figure 3 shows that CM from epithelial cells established from a number of nasal polyp tissues (NP-EpCM) (n = 6) induced considerable monocytic differentiation, 71 ± 10% (mean ± SD), and that this is significantly greater compared to CM obtained from epithelial cells established from normal nasal mucosa tissues (NN-EpCM), 23 ± 6% (n = 2) (P < 0.001). A similar finding was observed with CM from nasal mucosa epithelial cell cultures established from patients with allergic rhinitis (AR-EpCM): 42 ± 11% (n = 4) monocytic cells, although the difference compared to NNEpCM did not reach statistical significance. It should be noted that the cell number between NP- and NN-epithelial cells at confluency was not significantly different: 13.0 ± 3.2 x 1()4 well (n = 6) (mean ± SD) and 15.4 ± 2.3 x 1()4/well (n = 5), respectively. Figure 4 illustrates that NPEpCM induced monocytic differentiation of HL-60 cells in a dose-dependent fashion. We also examined a number of CMs generated by epithelial cells that had been stimulated

Figure 2. Morphologic characteristics of human upper airway epithelial cells laid on collagen (C)-coated tissue culture plates. Abundant microvilli (M) can be seen on the cell surfaces. Tonofilaments (T) are densely located in the cytoplasm, and desmosomes (D) are distributed between the junctions of each cell (original magnification: 19,500x).

with rh interleukin-l (rhIL-1Yimd found no enhancement in the extent of the induced monocytic differentiation compared to unstimulated conditioned media (data not shown). The differentiation of HL-60 cells to monocytes induced by EpCM was further confirmed immunohistochemically by using FMC-32 monoclonal antibodies. Whereas only 0.7% of the control (RPMI-IO% FBS) cultured HL-60 cells were positive, 11% and 61% of the cells cultured in 50% and 100% NP-EpCM, respectively, reacted strongly with the antibody (Figure 5). Immunoassay of GM-CSF, G-CSF, and M-CSF in EpCM Table 1 shows the human CSF content of epithelial cell-conditioned media. NP-EpCM contained a substantial amount of GM-CSF (489 ± 118 pg/ml [n = 10] [mean ± SEM]), which was significantly greater than that in NN-EpCM (126 ± 35 pglml [n = 3] [P < 0.05]). AR-EpCM contained GMCSF at an intermediate level (198 ± 22 pglml [n = 8]) but the difference compared to NN-EpCM did not reach statistical significance.

Ohtoshi, Vancheri, Cox et al.: Monocytic Differentiation by Upper Airway Epithelial Cells

259

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Figure 5. Immunohistochemistry of HL-60 cells inducedby nasal polyp epithelial cell-conditioned medium (NP-EpCM) at day 7. At 50 % and 100% NPEpCM-induced HL-60 cells (b and c), 11% and 61% of the cells, respectively, were positive for FMC-32, a mouse monoclonal antibody against CD14. Only 0.7% of the control HL-60 cells were positive at day 7 (a).

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Figure 3. Effect of conditioned medium from different epithelial cell cultures (EpCM)on monocytic differentiation of HL-60 cells. Normal nasal(NN-), allergicrhinitis (AR-), and nasalpolyp (NP-). *** P < 0.001; ** P < 0.01; NS: not significant.

All EpCMs also contained G-CSF with no significant differences related to patient source (normal versus inflamed tissue-derived cells). M-CSF was virtually undetectable in any EpCM. CM from unstimulated human upper airway epithelial cells also contained IL-6 activity, assessed in a hepatocyte stimulation assay (16): 47.3 ± 12.7 U/ml (n = 5). Stimulation of epithelial cells with rhIL-l resulted in a 2- to 4-fold increase in the amount of G-CSF, GM-CSF, and IL-6 present in the conditioned medium (data not shown). Effect of Recombinant Human Cytokines and of Specific Antibodies on HL-60 Cell Differentiation Recombinant human cytokines (rhGM-CSF, rhG-CSF, and rhIL-6), used at concentrations similar to those present in the NP-EpCM, could not reproduce the monocytic differentiating activity elicited by NP-EpCM. rhGM-CSF (500

U/ml) alone or in combination with rhG-CSF (200 U/ml) plus rhIL-6 (50 U/ml) induced a small degree of monocytic differentiation (3.6 ± 0.1% [n = 2, mean ± SD] and 3.4 ± 0.3% [n = 2], respectively) compared with NP-EpCM (n = 2), which induced 45.0 ± 8.2% monocytic differentiation in the concurrent experiment using the same passage level of HL-60 cells and the same reagents (Table 2). Preincubation of the EpCM with an anti-GM-CSF antibody at doses that inhibited neutrophilic differentiation of HL-60 cells as well as colony growth in methylcellulose induced by fibroblast CM (51) slightly reduced the monocytic differentiation of HL-60 cells induced by this EpCM, from 33.2 ± 3.1% to 25.2 ± 2.8% (n = 2, mean ± SD), and a similar effect was seen by the combination of anti-GM-CSF with anti-G-CSF and anti-IL-6 antibodies. Preincubation of the EpCM with either anti-G-CSF or anti-IL-6 antibodies did not at all inhibit the monocytic differentiation induced by the EpCM (Table 3).

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Partial Characterization of EpCM Heat inactivation. Heat-treatmentof EpCM reduced markedly the monocytic differentiation (CD14 expression) of the HL-60 cells from 19.9 ± 1.0% (n = 2) to 5.5 ± 0.3% (n = 2), just slightly higher than that induced by control medium alone (1.4 ± 0.4%, n = 2). Effect ofHPLC fractions on monocytic differentiation ofHL60 cells. Twelve fractions obtained from the HPLC procedure were tested in the HL-60 assay system; only fractions 8 and 11 induced monocytic differentiation to a substantial degree at a concentration of 25% (vol/vol) (3.0 ± 0.5% and 4.3 ± 0.7% [n = 2, mean ± SD]) of CD14-positive cells, respectively. A 1:1 mixture of these two fractions induced as many as 15.5 ± 0.9% (n = 2) CD14-positivecells, indicating a synergistic effect (Table 4). The mol wt of fractions 8 and II were estimated to be 34 to 56 kD and 7 to 12 ill, respectively (Figure 6). Effect ofEpCM on PBMNCThe effect of EpCM on nor-

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 4 1991

TABLE 1

Content of colony-stimulating factors GM-CSF, G-CSF, and M-CSF in epithelial cell-conditioned medium (EpCM) GM-CSF (pg/ml)* NP-EpCM AR-EpCM NN-EpCM

489 198 126

± ± ±

118 22 35

G-CSF (ng/ml)*

(n = lO)t (n = 9)t (n = 3)

1.63 1.18 1.08

± ± ±

0.26 0.32 0.24

M-CSF (ng/ml)*

< 0.8 < 0.8 1.00 ± 0.10

(n = 6) (n = 5) (n = 5)

(n = 3) (n = 3) (n = 2)

* Mean ± SEM. t p < 0.05. Nasal polyp (NP) versus allergic rhinitis (AR) EpCM, and NP versus normal nasal (NN) EpCM.

mal human PBMNC, which contain a small but significant number of hemopoietic progenitors, was analyzed for CD14 acquisition of these cells by immunohistochemistry with FMC-32 antibody. Medium alone failed to induce PBMNC to differentiate to monocytes/macrophages (0.08 ± 0.11%, n = 2) after 2 wk in liquid culture, nor was there any adherent cells on the plastic plate. In contrast, EpCM induced a substantial number of cells to become adherent, and also 2.90 ± 0.Q7 % (n = 2) of suspended cells acquired CDl4 antigen .: CDl4-positive cells induced by EpCM were mostly large cells (cP = 30 p.mor more) with abundant cytoplasm and a round or egg-shaped mononucleus, likely mature macrophages rather than monocytes.

Discussion The monocyte/macrophage is thought to playa central role in the inflammatory response. In addition to its well recognized scavenger and antigen-presenting capabilities, this cell has remarkable effector potential (17, 18). Indeed, monocyte/macrophages are capable of releasing proteolytic enzymes, oxygen radicals, arachidonic acid metabolites, as well as a host of hormone-like polypeptide messenger molecules (cytokines) that can interact with a wide variety of cells and thereby modulate numerous processes. However, relatively little is known, particularly in the human system, about the mechanisms that mediate the accumulation, sur-

TABLE 2

Effect of recombinant cytokines on monocytic differentiation of HL-60 cells Conditions

Monocytes* (%)

Control (Medium Alone) rhGM-CSF 500 U/ml 100 U/ml rhG-CSF rhIL-6 rhGM-CSF

0.3 3.6 0.9

500 U/ml 200 U/ml

1.4 0.6

50 U/ml

0.0

± ± ± ± ± ±

0.1 0.1 0.1

200 U/ml

Conditions

0.0

Control (HL-60 alone)

3.4

± 0.3

4~.0

± 8.2

+ rhIL-6

50 U/ml

NP-EpCMt 100% vol/vol

TABLE 3

Effect of specific antibodies for cytokines on monocytic differentiation of EpCM-induced HL-60 cells

0.1 0.1

500 U/ml

+ rhG-CSF

vival, and activation of these cells at particular tissue sites. Monocytes derive from progenitor cells within the bone marrow, and it is generally thought that these cells migrate into the tissues, where they evolve into macrophages (19). However, there is evidence indicating that part of the kinetic process of monocyte/macrophage tissue accumulation probably occurs at the local level and may well be controlled by locally produced cytokines (20-25). Our group has been interested for some time in inflammatory control mechanisms as they apply to human upper airway inflammation. Our previous observations on bloodborne progenitor cells and epithelial cell scrapings in human atopic subjects (7-12) led us to postulate that microenvironmental influences originating from local structural cells could contribute to the regulation of inflammation at tissue sites (26, 27). One component of such a contribution could involve the release of cytokines by the structural cells themselves. Indeed, fibroblasts are capable of releasing IL-6 (6), GM-CSF, and G-CSF (4, 5) as well as low amounts ofM-CSF (5) and neutrophil chemotactic factor (NCF) (28), originally described from monocytes (29) and more recently referred to as IL-8 (31); similarly, endothelial cells can release GCSF and GM-CSF (3), IL-1 (31), IL-6 (32), and NCF/IL-8 (33). We have recently shown that human upper airway fibroblasts from either normal or inflamed tissues induce neutrophilic but not monocytic differentiation of human hemopoietic progenitors (51). We have focused our attention in the current studies on the epithelial cell. To this end, we have established pure primary cultures of epithelial cells from human upper airway tissues derived from patients with different clinical presentations and studied the ability of CM derived from such cells to induce monocytic differentiation

* Resultsrepresentmean ± SO (n = 2) percentageof monocytic cells of the differential count of HL-60 cells with Diff-Quik stain at day 7. t EpCM derived from nasal polyp tissues,

Monocytes* (%) 0.1

NP-EpCMt 75% vol/vol

33.2

NP-EpCMt + anti-GM-CSF 1:50 NP-EpCMt + anti-G-CSF 1:500 NP-EpCMt + anti-IL-6 1:200 NP-EpCMt + anti-GM-CSF 1:50 1:500 + anti-G-CSF + anti-IL-6 1:200

25.2 34.0 31.0 22.2

± ± ± ± ± ±

0.1 3.1 2.8 2.8 1.6 3.1

* Resultsrepresentmean ± SO (n = 2) percentage of monocyticcells of the differential count of HL-60 cells with Diff-Quik stain at day 7. t EpCM derived from nasal polyp tissues.

Ohtoshi, Vancheri, Cox et al.: Monocytic Differentiation by Upper Airway Epithelial Cells

TABLE 4

261

10'

Effect of HPLC fractions of nasal polyp EpCM on monocytic differentiation of HL-60 cells Monocytes* (%)

Conditions

------------

Control (RPMI alone) EpCM 25% (vol/vol) Fraction 8 25% (vol/vol) Fraction I I 25 % (vol/vol) Fractions 8 + 11 (I: I mixture, vol/vol)

0.9 26.2 3.0 4.3 15.5

± ± ± ± ±

0.2 4.1 0.5 0.7 0.9

* Results represent mean ± SD (n = 2) percentage of CD 14-positive cells of HL-60 cells at day 7. detected by immunohistochemistry with FMC-32 antibody.

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of inflammatory cell progenitors in vitro. We established the purity of these cultures by demonstrating > 95 % of cells stained positively for keratin. Contamination by significant numbers of fibroblasts and macrophages was excluded by the finding of minimal expression of vimentin and no expression of the CDl4 antigen (not shown) by the epithelial cell cultures. To investigate inflammatory cell differentiation, we used human HL-60 cells as targets. These represent a myeloid leukemia cell line that is capable of differentiation into functionally mature inflammatory cells of various lineages depending on the inducing agent (34-36). Thus, this assay system is useful to study biochemical and molecular events associated with differentiation of progenitor cells into functionally mature inflammatory cells. Supernatants from human upper airway epithelial cells induce a marked differentiation of HL-60 cells to monocytes in a dose-dependent manner (Figures 3 and 4). These same supernatants induced monocyte/macrophage differentiation in normal peripheral blood mononuclear cell cultures. Supernatants from epithelial cells derived from nasal polyp tissues induced a significantly greater differentiating effect compared to supernatants derived from control epithelial cells, and supernatants from epithelial cells derived from allergic rhinitis mucosa induced monocytic differentiation at an intermediate level. Since the method of isolation and culture was identical, and the cell number of normal and inflamed tissue-epithelial cells at confluency was similar, this difference would indicate that cells derived from inflamed tissues may be activated in vivo. A number of cytokines have been documented to date as being involved in the regulation of monocyte/macrophage maturation. Whereas M-CSF appears to act primarily on progenitors committed to the monocytic lineage, GM-CSF stimulates the differentiation of both macrophage and granulocyte lineages. Begley and colleagues showed that rhGMCSF induced the expression of granulocyte and macrophage membrane antigens on HL-60 cells, although this effect was not accompanied by morphologic evidence of differentiation (37). G-CSF, which was initially characterized by its ability to stimulate the emergence of neutrophilic colonies in semisolid systems (38), has also been implicated in monocytic differentiation. For example, Souza and associates showed that cells of the murine myelomonocytic leukemia cell line WEHI-3B(D+) could be induced to monocytic differentiation by 100 U/ml rhG-CSF; a similar effect could be induced

o

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3. 2 --=~.l-.-_--'

1

2

3

4

5

6

7 8

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9 10 11 12 Control

FRACTION

Figure 6. High-performance liquid chromatography (HPLC) fractionation of nasal polyp EpCM. The closed bars show the extent of monocytic differentiation of HL-60 cells induced by each fraction at 25% (Vol/vol), as detected by immunohistochemistry with FMC-32 antibodies. Only fractions 8 and 11 induced monocytic differentiation (CDl4 expression) of HL-60 to a substantial degree. The molecular weights of these two fractions were estimated to be 34 to 56 kD and 7 to 12 kD, respectively, according to regression line (r = 0.99446, P < om, and n = 4).

in human leukemia cells exposed to a much larger dose of this cytokine (39). In addition to the colony-stimulating factors, IL-6 has also been implicated in monocyte differentiation (40-42). We examined supernatants from unstimulated epithelial cells for their content of hemopoietic colony-stimulating factors and IL-6. As shown in Thble 1, human upper airway epithelial cells constitutively produce GM-CSF, G-CSF and IL-6 in vitro. We could not detect M-CSF using a specific radioimmunoassay that did detect low amounts of this cytokine in supernatants from human upper airway-derived fibroblasts (51). Thble 1 also shows that supernatants of epithelial cells established from inflamed tissues, particularly nasal polyps, contained significantly greater amounts of GM-CSF. On the basis of these findings, we then examined the effect of these human recombinant cytokines in the HL-60 differentiation assay. None of the cytokine combinations, at similar or greater levels as found in the epithelial cell supernatants, induced monocytic differentiation except for GM-CSF (500 U/ml), which had a small effect (Table 2). We did not examine the ability of rhM-CSF to induce monocytic differentiation of progenitor cells. One reason was that its content in the epithelial cell supernatant, if present, was below the level of detection. In addition, HL-60 cells do not possess receptors for this cytokine nor do they express the mRNA for it (Dr. Peter Ralph, personal communication). It could be argued that IL-6 or CSFs contained in the epithelial cell supernatants could act as co-factors of an as yet unidentified cytokine present in the EpCM or, alternatively, stimulate HL-60 cells to release another cytokine, which then would induce

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 41991

progenitor cells toward the monocytic lineage in an autocrine/paracrine manner. Toaddress these issues, we examined whether neutralizing antibodies, anti-G-CSF, anti-GM-CSF, and anti-Il-6, could abrogate the monocytic differentiation induced by epithelial cell supernatants: none of these antibodies abrogated this effect except for anti-GM-CSF antibodies, which slightly inhibited the monocytic differentiation induced by EpCM (Table 3). There are therefore several lines of evidence arguing against the hypothesis that IL-6 and the CSFs are the cytokines present in the EpCM responsible for monocytic differentiation of HL-60 cells. Firstly, nasal polyp fibroblast CM, which contains, except for M-CSF, similar amounts of IL-6, G-CSF, and GM-CSF as the epithelial cell CM, does not induce monocytic differentiation of HL-60 cells (51). Secondly, EpCM from IL-l-stimulated epithelial cells, which contains greater amounts ofIL-6, GM-CSF, and G-CSF, did not induce an enhanced monocytic differentiation. Thirdly, recombinant human cytokines, alone or in combination, at concentrations similar to or greater than those present in the EpCM, did not induce significant or comparable monocytic differentiation of HL-60 cells. Finally, neutralizing antibodies against these cytokines did not fully inhibit the monocytic differentiation induced by the EpCM. We have not directly examined whether or not our EpCMs contain interleukin-3 (IL-3); this is, however, unlikely as Niemeyer and associates recently examined human fibroblasts as well as endothelial cells and concluded that expression of IL-3 is restricted to human lymphocytes and T-cell tumor lines (43). The monocyte/macrophage differentiating activity is heat labile and emerges from HPLC fractionation as two peaks at 34 to 56 kD and 7 to 12 kD. These two factors act synergistically in monocyte differentiation. On the basis of molecular weight, the heavier may be GM-CSF, but the smaller factor is different from IL-6, G-CSF, GM-CSF and M-CSF. In further studies (data not shown), rhIL-8 alone or in combination with rhGM-CSF could not induce monocyte/macrophage differentiation, nor could anti-IL-8 antisera (Dr. Steven L. Kunkel, Ann Arbor, MI) neutralize the activity in EpCM. Similar findings were made with leukemia inhibitory factor (LIF) (44, 45), a molecule sharing many activities with IL-6, and suitable antibody preparation. (Dr. Heinz Baumann, Roswell Park, Buffalo, NY) (45). The data presented here do not contradict any previous evidence in the literature as differences in the effector cell or the species might account for different observations. Our data indicate that airway epithelial cells produce cytokines capable of influencing the differentiation of monocytes, and that in inflammatory conditions, this function is increased. In this way,structural cells might contribute to the accumulation of monocytes/macrophages in affected tissues. Obviously, epithelial cell-derived factors likely influence other aspects of monocyte-macrophage biology during the inflammatory process. Recombinant GM-CSF has been shown to modulate surface markers (46), Ia antigen expression (47), prostaglandin and cytokine production (48), macrophage differentiation, and antibody-dependent cellular cytotoxicity (49), all of which are relevant to the accumulation and activation of monocytes/macrophages during inflammation. Furthermore, IL-6 can modulate monocyte/

macrophage behavior, providing another pathway for interaction (50). Much remains yet to be elucidated with regard to how the inflammatory response is controlled. The findings presented here lend support to the concept of microenvironmental control of inflammation by structural cells and to the existence of a lineage-specific monocyte/macrophage differentiation factor, probably different than IL-6 and the CSFs, elaborated by these cells. Whether this or other epithelial cell-derived factors is/are relevant to the control of the inflammatory process in vivo remains to be established. Should this be proven, interventions directed at modulating cytokine production by epithelial cells might be helpful in regulating the tissue inflammatory response. Acknowledgments: We are much indebted to Dr. John S. Abrams (DNAX Research Institute), Dr. Bruce W. Altrock (AMGEN Corp.), and Dr. Peter Ralph (Cetus Corp.) for measuring specific cytokines in the EpCMs and for providing specific antibodies for them and to Dr. Steven L. Kunkel and Dr. Heinz Baumann for providing NCF/IL-8 and LIF with appropriate antisera. We are thankful to Dr. Shinichi Kawabori for his expert assistance in the ultrastructural and immunohistochemical studies. The technical assistances ofMr. Ron Stead, Mr. Steve Polyak, Mr. Duncan Chong, and Mrs. Karen Howie are gratefully acknowledged. We are also indebted to Drs. D. Hitch and P. Lapp from the ENT Department who provided us with tissues for study. The useful comments of Dr. John Bienenstock are much appreciated. This work was supported by MRC Canada and the Council for Tobacco Research Inc. USA. Gerard Cox is a Research Fellow of the Canadian Lung Association. Manel Jordana is a Research Fellow of the Parker B. Francis Foundation (USA). References

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