Pulmonary Fibroblast Expression of Interleukin-8: A Model for Alveolar Macrophage-derived Cytokine Networking Mark W. Rolfe, Steven L. Kunkel, Theodore J. Standiford, Stephen W. Chensue, Ronald M. Allen, Holly L. Evanoff, Sem H. Phan, and Robert M. Strieter Departments of Internal Medicine and Pathology, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical School, Ann Arbor, Michigan

The pulmonary fibroblast's (PF) unique location allows it to communicate in a bidirectional fashion between the vascular compartment and alveolar airspace, placing it in a strategic position for the elicitation of inflammatory leukocytes into the lung. In this study, we demonstrate that PF may contribute to pulmonary inflammation through the production of a potent neutrophil chemotactic factor, interleukin (IL)-8. PF-derived IL-8 expression was dependent upon stimulation by either tumor necrosis factor (TNF) or IL-1 but not lipopolysaccharide (LPS). Both TNF and IL-1 stimulation of PF resulted in a time- and dosedependent expression of steady-state levels of mRNA, antigen, and specific chemotactic activity consistent with IL-8. Because it was apparent that cytokine networking may exist in the lung between alveolar macrophage (AM)-derived cytokines and the production of PF-derived IL-8, we next examined an in vitro model of cellular communication within the lung. We determined that LPS-stimulated AM-conditioned media induced significant levels of PF-derived IL-8 mRNA, which was inhibited by preincubation with specific neutralizing TNF and IL-1{j antibodies. Furthermore, when AM were directly co-cultured with PF and stimulated with LPS, the kinetic analysis of PF-derived antigenic expression of IL-8 was shifted toward the right. This suggested that PF-derived IL-8 expression in co-culture was first dependent upon activation of the AM by LPS and subsequent elaboration of macrophage inflammatory mediators. These data provide evidence that cytokine networking between AM and PF may be operative in the lung, culminating in the generation of IL-8 and elicitation of inflammatory leukocytes.

The principal immune and nonimmune cells of the lung that function in either host defense or in maintaining the connective tissue stromal framework of the lung are the alveolar macrophage (AM) and pulmonary fibroblast (PF), respectively. The PF is an essential cell in the production of collagenous and noncollagenous components of the extracellular matrix, whereas the AM is the sentinel cell for pulmonary immunomodulation during inflammation. Recent investigations, however, have expanded the function of the fibroblast to include the synthesis of various soluble mediators, such as prostaglandin E2, interleukin (IL)-l, IL-6, monocyte chemoattractant peptide (MCP-1), and colony-stimulating factor (1-6). Thus, the activated PF, in its anatomicalloca-

(Received in original form March 11, 1991 and in revisedform May 2,1991) Address correspondence to: Robert M. Strieter, M. D., Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, Box 0360, University of Michigan Medical Center, 3916 Taubman Center, Ann Arbor, MI48109-0360. Abbreviations: alveolar macrophage(s), AM; bronchoalveolar lavage, BAL; conditioned medium, CM; enzyme-linked immunosorbent assay, ELISA; formylmethionylleucylphenylalanine, FMLP; interleukin, IL; lipopolysaccharide, LPS; peripheral blood monocytes, PBM; phosphate-buffered saline, PBS; pulmonary fibroblast(s), PF; tumor necrosis factor-a, TNF. Am. J. Respir. Cell Mol. BioI. Vol. 5. pp. 493-501, 1991

tion, is in a pivotal position to communicate with either the alveolar airspace or the vascular compartment via the release of these soluble mediators. . Our laboratory and others have shown that lipopolysaccharide (LPS)-stimulated AM are capable of producing both tumor necrosis factor-a (TNF) and IL-1 (7-11). These cytokines have pleiotropic and overlapping biologic activity on a variety of cell types essential to the propagation of inflammation, yet they fail to induce a direct chemotactic influence on neutrophils (12-14). Recently, however, a cytokine has been isolated, purified, cloned, and expressed from peripheral blood monocytes (PBM) that has significant chemotactic and activating activity for neutrophils (12-15). Our laboratory and others have shown that this chemotactic/activating peptide, known as IL-8, is produced not only by PBM but also by human AM, dermal fibroblasts, synovial fibroblasts, epithelial cells, hepatocytes, and endothelial cells (16-23). Stimulus specificity for the elaboration of this cytokine has been demonstrated using either exogenous or endogenous stimuli. AM, PBM, and endothelial cells have the ability to produce IL-8 in response to either LPS, TNF, or IL-1 (15-18, 23). In contrast, a host-generated stimulus, such as TNF or IL-1, is required for the production of dermal fibroblast or epithelial cell-derived IL-8 (19, 21). This dichotomy in signal specificity exemplifies the importance of

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991

cytokine networking that may be instrumental to the interaction between AM and PF leading to the elicitation of neutrophils into the lung. In this study, we first establish that TNF and IL-1 stimulation, but not LPS, results in PF-derived production of a neutrophil chemotactic factor with mRNA, antigenic, and biologic activity specific for IL-8. Next, we demonstrate a model of cellular communication between AM and PF, whereby LPS-stimulated AM induce PF-derived IL-8 mRNA and antigenic expression, which is primarily due to AMderived TNF and IL-1.

Materials and Methods Reagent Preparation Human recombinant IL-1/3 with a specific activity of30 D/ng and murine anti-human monoclonal IL-{3 antibodies were the generous gift of the Upjohn Co. (Kalamazoo, MI). Human recombinant TNF-a with a specific activity of 22 U/ng was kindly provided by the Cetus Corp. (Berkeley, CA). Human recombinant IL-8 was the generous gift of Sandoz Pharmaceutical Co. (Hanover, NJ) or purchased from Pepro Tech Inc. (Rocky Hill, NJ). Polyclonal anti-human TNF and IL-8 antiserum were produced by immunization of rabbits with recombinant TNF-a or IL-8 in multiple intradermal sites with complete Freund's adjuvant (Sigma Chemical Co., St. Louis, MO). IL-8 antisera, in concentrations used in the enzyme-linked immunosorbent assay (ELISA) or immunohistochemical localization of antigenic IL-8, reacted with recombinant IL-8 in Western analysis and failed to crossreact with CTAP-III, NAP-2, {3-thromboglobulin, GRO/ MGSA, or platelet factor 4. Polyclonal anti-human TNF used in this study was capable of neutralizing 10 U ofrecombinant TNF-a at a dilution of 1:25,625, whereas 1 jlg of the murine antihuman monoclonal IL-1{3 antibody was capable of inhibiting 95 % of the IL-1{3-induced production of IL-2 by lA5 murine T cells. The efficacy of neutralizing antihuman IL-8 antibodies has been previously reported (16). Serial dilutions of pyrogen-free cytokines were prepared in Eagle's minimal essential medium (Whitaker Biomedical Products, Walkersville, MD) containing 1 mM glutamine, 25 mM Hepes, 100 D/ml penicillin, 100 ng/ml streptomycin, and 1% nonessential amino. acids (GIBCO, Grand Island, NY) (complete media). Stock LPS (Escherichia coli 0111:B4; Sigma) was prepared at a concentration of 200 jlg/ml in complete media. Preparation of CCO-18Iu, CCO-16Iu, and CCO-8Iu Pulmonary Fibroblasts Human PF (CCD-18Iu, CCO-16Iu, and CCD-8Iu; American Type Culture Collection, Rockville, MD) are three nontransformed PF cell lines isolated from patients without associated pulmonary disease post mortem. All three human PF cell lines were used before passage 20 and grown to confluency on 100-mm petri dishes (Corning Glass Works, Corning, NJ) in complete MEM plus 10% fetal calf serum (FCS). On the day of use, fibroblast monolayers were washed free of FCS with complete media, and either cytokines or LPS were added for the specified times and concentrations. Cell-free supernatants were collected, and total cellular RNA was extracted as described below,

Recovery and Isolation of Human AM and Generation of Conditioned Media Three normal, nonsmoking volunteers agreed to undergo flexible fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) by standard techniques (9, 16). The subjects had no evidence of recent respiratory tract infection in the preceeding 6 wk and were not taking any medications. The recovered BAL fluid/cell suspension was filtered through gauze and centrifuged at 600 X g. The cells were resuspended in complete media and washed 3 times. Total cell count per BAL was 42.3 ± 1.2 x 106 cells, and cell differential revealed 95 ± 1.8% AM, 2.3 ± 1.3% neutrophils, and 2 ± 1.6% lymphocytes. Lavaged cells were> 95 % viable as assessed by trypan blue exclusion. Freshly isolated human AM were plated on 100-mm culture plates at a concentration of 107 AM/plate. LPS at a concentration of 1 jlg/ml was added to some plates, while the remainder were unstimulated. Conditioned media was collected after a 24-h incubation period at 37° C. Twenty-four-hour AM-conditioned media (LPS-stimulated and unstimulated) were placed over PF monolayers in 60-mm culture plates. Total PF RNA was isolated at 8 h, and Northern blot analysis for IL-8 mRNA was performed. In neutralizing studies, LPS-stimulated AM-conditioned medium was preincubated for 30 min with either: (1) polyclonal rabbit anti-human TNF antiserum or control polyclonal rabbit preimmune serum (1:100 dilution), (2) murine monoclonal anti-human IL-1{3 antibody or murine IgG (Sigma) (10 jlg/ml), or (3) both anti-TNF and IL-l/3 antibodies or the combination of controls. Antibody-treated conditioned medium was placed over PF monolayers, and total cellular RNA isolated at 8 h as described subsequently. Northern Blot Analysis Total cellular RNA from PF was isolated using a modification of Chirgwin and co-workers (24) and Jonas and associates (25). Briefly, PF monolayers were scraped into a solution containing 25 mM Tris (pH 8.0), 4.2 M guanidine isothiocyanate, 0.5 % Sarkosyl, and 0.1 M 2-mercaptoethanol. After homogenization, the above suspension was added to a solution containing an equal volume of 100 mM Tris (pH 8.0), 10mM EDTA, and 1.0% sodium dodecyl sulfate. The mixture was then extracted with chloroform-phenol and chloroform-isoamyl alcohol. The RNA was alcohol-precipitated, and the pellet was dissolved in DEPC H2O. Total RNA was separated by Northern blot analysis using formaldehyde, 1% agarose gels, transblotted to nitrocellulose, baked, prehybridized, and hybridized with a 32P_5' end-labeled oligonucleotide probe. A 30-mer oligonucleotide probe was synthesized using the published eDNA sequence for human-derived IL-8 (14). The probe was complementary to nucleotides 262 through 291 and had the sequence 5'GTT-GGC-GCA-GTG-TGG-TCC-ACT-CTC-AAT-CAC-3'. Blots were washed, and autoradiographs were quantitated using laser densitometry (Ultrascan XS; LKB Instruments, Houston, TX). Equivalent amounts of total RNA/gel were assessed by monitoring 28S and 18S rRNA. Immunohistochemistry Immunolocalization of IL-8 was performed as previously described (16). Briefly, PF and AM mono layers were fixed in

Rolfe, Kunkel, Standiford, et al.: Pulmonary Fibroblast Expression of IL-8 by AM-derived Cytokines

4 % paraformaldehyde in phosphate-buffered saline (PBS) for 10 min, then rinsed twice with PBS. Before staining, the slides were again fixed for 15 min in 1:1 absolute methanol and 3 % H20;!, rinsed in PBS, then nonspecific binding sites were blocked with a 1:50 dilution of normal goat serum. Normal serum was removed, followed by the addition of a 1:2,000 concentration of either control (rabbit), or rabbit anti-human IL-8 serum. After 15 min of incubation at 37° C, the slides were rinsed with PBS, overlaid with biotinylated goat anti-rabbit IgG (1:200; Vector Laboratories, Burlingame, CA), incubated 15 min, and rinsed 2 times with PBS. The slides were treated with streptavidin conjugated to peroxidase for 15 min at 37° C, rinsed 3 times, overlaid with substrate chromogen (3-amino 9-ethylcarbazole) for 7 min at 37° C to allow color development, and rinsed with distilled water. Mayer's hematoxylin was used as a counterstain. To demonstrate antibody specificity, in competitive inhibition studies, immunostaining for human IL-8 showed 100% inhibition by the addition of exogenous recombinant IL-8. Immunohistochemical localization of antigenic IL-8 was quantitated by counting either the number of AM or PF positive for IL-8 antigen at 400 x and expressing this result as a percentage of either the total AM or PF per high power field. IL-8 ELISA Antigenic IL-8 was quantitated using a modification of a double ligand method as previously described (26). Briefly, flat-bottomed, 96-well microtiter plates (Nunc ImmunoPlate; Vanguard International, Neptune, NJ) were coated with 50 j.tl/well of rabbit anti-IL-8 antibody (1 ng/j.tl in 0.6 M NaCI, 0.26 M H3B04 , and 0.08 N NaOH [pH 9.6]) for 16 h at 4°C and then washed with PBS (pH 7.5) and 0.05% Twcen'" 20 (wash buffer). Microtiter plate nonspecific binding sites were blocked with 2 % bovine serum albumin in PBS and incubated for 90 min at 37° C. Plates were rinsed 4 times with wash buffer and diluted (neat, 1:5, and 1:10). PF-derived conditioned media (50 j.tl) in duplicate were added, followed by incubation for 1 h at 37° C. Plates were washed 4 times with wash buffer, then 50 j.tl/well of biotinylated rabbit anti-IL-8 (3.5 ng/j.tl in PBS [pH 7.5], 0.05% Tween 20, and 2 % FCS) was added, and plates were incubated for 30 min at 37° C. Plates were washed 4 times, streptavidin-peroxidase conjugate (Bio-Rad, Richmond, CA) was added, and the plates were incubated for 30 min at 37° C. Plates were washed 4 times, and chromogen substrate (Bio-Rad) was added. The plates were incubated at room temperature to the desired extinction, and the reaction was terminated with 50 j.tl/well of 3 M H2S04 solution. Plates were read at 490 nm in an ELISA reader. Standards were 1:2 log dilutions of recombinant IL-8, from 1,000 ng to 1 pg/ml (50 j.tl/well). This ELISA method consistently detected IL-8 concentrations in a linear fashion greater than 10 pg/ml. Chemotactic Assay Human neutrophils were prepared from peripheral blood by Ficoll-Hypaque density gradient centrifugation followed by sedimentation in 5% dextran in 0.9% saline (Sigma). Neutrophils were-separated from erythrocytes by hypotonic lysis and then suspended in Hanks' balanced salt solution (HBSS)

495

with calcium/magnesium (GIBCO) at 2 x 1()6 cells/ml with greater than 95 % viability by trypan blue exclusion. Chemotaxis was performed as previously described (16, 19). Briefly, 150 j.tl of diluted (1:1) supernatant specimen, 10-7 M formylmethionylleucylphenylalanine (FMLP; Sigma), or HBSS alone was placed in duplicate bottom wells of a blindwell chemotaxis chamber. A 3-j.tm pore size polycarbonate filter (polyvinylpyrrolidone-free; Nucleopore Corp., Pleasanton, CA) was placed in the assembly, and 250 j.tl of neutrophil suspension was placed in each of the top wells. Chemotaxis chamber assemblies were incubated at 37° C in humidified 95 % air/5 % CO 2 for 1 h. The filters were removed, fixed in absolute methanol, and stained with 2 % toluidine blue (Sigma). Neutrophils that had migrated through to the bottom of the filter were counted in 10 high power fields (400 x) using a Javelin chroma chip camera (Javelin Electronics, Torrance, CA) attached to an Olympus BH-2 microscope interfaced with a MacIntosh II computer containing an Image Capture 2 frame grabber (Scion Corp., Walkersville, MD) and image 1.29 software (NIH Public Software, Bethesda, MD). Chemotactic bioactivity was standardized to and expressed as, the percentage of the positive control (10-7 M FMLP). In neutralization studies, PF 24-h-conditioned media was incubated with 1:1,000dilution of either control (rabbit preimmune serum) or neutralizing rabbit anti-human IL-8 antiserum for 30 min at 37° C and then assayed for chemotactic activity. Statistical Analysis Unless otherwise indicated, the individual experiments described were performed on each of the three PF cell lines (CCD-18Iu, CCD-16Iu, and CCD-8Iu) and the results composited. To accommodate for variability in the absolute response of each of the cell lines to the various experimental conditions, before compositing the results they were expressed as a percentage of the maximal response during each of the individual experiments. Data were analyzed by MacIntosh II computer using the Statview II statistical software package (Abacus Concepts, Inc., Berkeley, CA). Data are expressed as mean ± SEM. Data that appeared statistically significant were compared by Student's t test (twotailed) for comparing the means of multiple groups and considered significant if P values were < 0.05.

Results PF-derived Neutrophil Chemotactic Activity Initial studies were performed to assess the ability of TNF, IL-II3, and LPS to induce PF-derived neutrophil chemotactic bioactivity in a time-dependent manner. Confluent PF were stimulated with 20 ng/ml of either TNF or IL-113 or I j.tg/ml of LPS. At specific time intervals, PF-derived supernatants were harvested and assessed for neutrophil chemotactic bioactivity. As shown in Figure 1, IL-II3-treated PF-derived neutrophil chemotactic activity was significant by 4 h (32.4 ± 2.3%) and plateaued by 8 h (61.1 ± 4.4%), with continued chemotactic activity for the next 16 h (75.6 ± 2.9%). In a similar fashion, TNF-induced PF-derived neutrophil chemotactic activity was significant by 4 h (38.5 ± 3.7%), with continued increase over the next 20 h (65.5 ± 3.6%). IL-II3-induced PF-derived neutrophil chemotactic activity

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991

tivity was attributable to IL-8. The first level of assessment was whether PF would express IL-8 mRNA in response to either LPS, IL-II3, or TNF, and whether this response was similar to our previous observations from activated dermal fibroblasts (19). PF confluent monolayers were stimulated in either a time- or concentration-dependent manner. As shown in Figure 2, PF were stimulated at time 0 with either LPS (1 Itg/ml) , IL-1{3 (20 ng/ml), or TNF (20 ng/ml) , and at specific time intervals total cellular RNA was isolated and extracted. IL-l{3-stimulated PF expressed significant steadystate levels ofIL-8 mRNA by 1 h, peaking by 8 h and declining to 62 ± 3 % of the maximal steady-state levels of IL-8 mRNA by 24 h. The expression was assessed by laser densitometry. In a similar manner, TNF treatment paralleled the observed response of IL-ll3 stimulation of PF, with TNFtreated PF expressing significant steady-state levels of IL-8 mRNA by 1 h, peaking by 8 h, and declining to 65 ± 2 % of the maximal response to TNF at 24 h. Although the PF response to either IL-ll3 or TNF was similar in terms of a kinetic expression of IL-8 mRNA, the degree of stimulation from TNF was significantly less than that from IL-l{3 after 8 h of culture (P == 0.004) . In contrast, stimulation of PF with LPS failed to induce steady-state levels of IL-8 mRNA over the entire time course of the experiment. Subsequently, confluent monolayers of PF were stimulated with graded doses of either LPS (1 ng/ml to 10 Itg/ml), IL-l{3 (20 pg/rnl to 20 ng/ml), or TNF (20 pg/rnl to 20 ng/ml), then PFderived total RNA was isolated and extracted at 8 h. As shown in Figure 3, both IL-l{3 and TNF stimulated PFderived steady-state levels ofIL-8 mRNA in a concentrationdependent manner, with 50% of the maximal IL-8 expression seen at concentrations of 200 pg/ml and 2 ng/ml for IL-1{3 and TNF, respectively. Maximal steady-state levels of IL-8 mRNA were achieved with 20 ng/ml stimulation with either IL-l{3or TNF ; concentrations above these levels failed

Figure 1. Time course of pulmonary fibroblast (PF)-derived neutrophil chemotaxic activity post-stimulation by either lipopolysaccharide (LPS), tumor necrosis factor (TNF), or interleukin (IL)1{3. Chemotactic bioactivity is expressed as a percentage of the positive control (10- 7 M formylmethionylleucylphenylalanine) .

was significantly greater than TNF-treated PF-derived neutrophil chemotactic bioactivity by 8 h (P == 0.02), with IL-1{3-induced chemotactic activity persistently greater than TNF-treated PF by 24 h (P == 0.006) . In contrast, LPS treatment failed to induce PF-derived neutrophil chemotactic activity that was significantly greater than unstimulated 24 h control. Neither LPS, TNF, or IL-1{3 were directly chemotactic for neutrophils in our bioassay (data not shown) . PF-derived Gene Expression of IL-8 The observation that IL-1{3-treated or TNF-treated PF generated significant neutrophil chemotactic bioactivity led to subsequent studies to determine if this chemotactic bioac-

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to induce further expression of IL-8 mRNA (data not shown) . In contrast, LPS (1 ng/ml to 10 1!g/ml) failed to induce significant steady-state IL-8 mRNA over unstimulated control. PF-derived Antigenic IL-S Next, we examined whether the PF-derived expression of IL-8 mRNA was associated with antigenic presence of extracellular IL-8 protein. PF were stimulated with either LPS, TNF, or IL-113 in either a time- or concentration-dependent fashion . PF were initially stimulated with either LPS (l ug/ml), IL-I{3 (20 ng/ml), or TNF (20 ng/ml) in a kinetic manner, with PF-derived supernatants harvested at specific time intervals. As shown in Figure 4, both IL-I,B and TNF induced significant antigenic IL-8 by 4 h, with a progressive increase in IL-8 antigenic activity to maximal levels by 24 h (100% and 58 ± 7 % for IL-l{3and TNF stimulation, respectively). In contrast, LPS failed to induce significant antigenic IL-8 over unstimulated control, which was similar to the results of LPS-induced IL-8 mRNA. The dose-dependent production ofPF-derived IL-8 antigenic activity at 8 h paralleled steady-state levels ofIL-8 mRNA. As shown in Figure 5, 200 pg/ml and 20 pg/ml were concentrations of TNF and IL-l{3 to induce significant antigenic production of PFderived IL-8, respectively. TNF (20 ng/ml) and IL-I,B (2 ng/ml) induced equivalent levels of antigenic IL-S (P = 0.5) , while IL-l{3 (20 ng/ml) stimulation resulted in the maximal production of IL-8 antigenic activity. Quantitative antigenic IL-S (ng/ml) production from each of the three PF cell lines

Figure 4. Time course of PF-derived antigenic IL-S expression post-stimulation by either LPS, TNF, or IL-l{1. IL-S antigenic activity was measuredby a specific IL-S enzyme-linked immunosorbent assay (ELISA).

after stimulation with either LPS (1 1!g/ml), IL-I,B (20 ng/ml), or TNF (20 ng/ml) are shown in Table 1. Inhibition of PF-derived Neutrophil Chemotactic Activity by Anti-human IL-S Antibodies To determine the relative contribution of PF-derived IL-8 to PF-derived neutrophil chemotactic activity, 24-h Pf-conditioned medium was harvested from LPS-, TNF-, and IL113-stimulated and unstimulated PF and preincubated with either control or immune anti-human IL-8 serum. As shown in Thble 2, preincubation with IL-8 antiserum resulted in a significant reduction in both IL-l,B-induced and TNF-induced neutrophil chemotactic activity to levels equivalent to unstimulated control, whereas LPS-treated PF-derived chemotactic activity was not influenced by either control or IL-8

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991

TABLE 1

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antiserum and was no different from unstimulated control. Neither control serum or IL-8 antiserum altered the chemotactic influence of 10-7 M FMLP in our chemotactic bioassay (data not shown). LPS-stimulated AM Induce PF-derived mRNA and Antigenic Activity for IL-8 The above experiments demonstrated that PF display signal specificity with regards to production of IL-8. Both hostderived cytokines TNF and IL-l{1 resulted in expression and production of mRNA, antigen, and chemotactic bioactivity specific for IL-8, whereas an exogenous stimulus, LPS, failed to induce PF-derived IL-8. Because the AM, during inflammation, may have a paracrine influence on PF through the release of both TNF and IL-l, we next examined the role of AM-induced PF-derived IL-8. First, AM were stimulated in culture in the presence or absence of LPS (l J,tg/ml) for 24 h. This AM-conditioned medium (CM) was then preincubated in the presence or absence of either anti-TNF antibodies, anti-IL-l{1 antibodies, combination of anti-TNF and anti-Ils-lf antibodies, or combination of control serum and control murine IgG. The various AM -CM were then overlaid on PF for 8 h, and the total PF-derived cellular RNA was isolated. As shown in Figure 6, LPS-stimulated AM-CM resulted in significant expression of PF-derived steady-state levels of IL-8 mRNA. The addition of either anti-TNF or anti-Ib-Id antibodies resulted in a 37 ± 10% and 38 ± 20% reduction in maximal steady-state levels of PF-derived IL-8 mRNA, respectively. The combination of anti-TNF and anti-

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IL-l{1 antibodies resulted in a 69 ± 11% inhibition of LPSstimulated AM-CM-induced PF-derived IL-8 mRNA, which was no different than 8 h unstimulated control (P = 0.1). Finally, because it was apparent that PF respond to LPS-stimulated AM-CM, we next evaluated the response ofPF in direct co-culture with freshly isolated AM. PF were grown to approximately 50% confluence on Labtek (Nunc) multiwell slides, with lQ5 AM added to each well. The combination of PF and AM were then cultured in the presence or absence ofLPS (l J,tg/ml). At specific time points, the cells were fixed in 4 % paraformaldehyde and assessed for immunoreactive IL-8. The photomicrograph of the kinetic immunohisto-

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with LPS-stimulated, alveolar macrophage (AM)-derived conditioned media (CM). Panel A: Representative Northern blot of CCD-16Iu PF. PanelB: Results of the composite laser densitometry from all three PF lines in the presence of either unstimulated media (A) or LPS-conditioned AM media in the presence of both neutralizing antiserum/antibodies to TNF and IL-l~ (B), neutralizing antibodies to IL-l~ (C), neutralizing antiserum to TNF (D), or control antiserum/antibodies (E). Panel C: Corresponding 28S and 18S rRNA for the Northern blot in panel A.

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chemical localization of AM- and PF-derived IL-8 antigen is shown in panels A through D of Figure 7, while the quantitative kinetic expression of IL-8-positive antigen present in either AM or PF is demonstrated in panel E of Figure 7. Panels A, B, C, and D represent 1, 4, 8, and 24 h after stimulation with LPS, respectively. As shown in panel E, AM demonstrated a very rapid expression of IL-8 antigen by 2 h after treatment with LPS, with immunostaining plateau ing over the next 22 h. In contrast, PF showed a time-dependent expression of IL-8 antigen that was shifted toward the right as compared with AM-derived antigenic IL-8. PF-derived antigenic IJ,,-8 showed a more gradual increase in IL-8, which was maximal by 24 h, with 86 ± 6% of the PF staining positive for antigenic IL-8.

I 2 Time

16

20

24

( h rs)

Discussion The elicitation and accumulation of inflammatory leukocytes in the lung constitutes an essential element of the inflammatory response that is important in host defense. Although their presence may be important in healing and repair, they are nevertheless often associated with immunopathology of the lung (27, 28). The migration of inflammatory leukocytes from the pulmonary vascular compartment to the interstitium and alveolar airspace is a complex process that requires initial leukocyte margination and adherence, followed by directed diapedesis along established chemotactic gradients. Historically, a number of mediators have been identified that possess potent chemotactic activity: A number of these chemotactic factors have been shown to be locally

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991

produced in the lung, including lipids leukotriene B4 (18, 29, 30) and platelet-activating factor (29, 31) and proteins C5a (29, 31), platelet-derived growth factor (31, 32), and fibronectin (31). These chemotactic factors are potent for the recruitment of neutrophils and/or monocytes. Previous investigations have focused on the AM as the principle cell for the pulmonary origin of chemotactic factors (29, 31). Recently, however, leukocyte chemotactic activity has been determined to be produced by a number of nonimmune cells that may be analogous to the cellular constituents of the alveolar-capillary wall (19, 23). Thus, the events leading to the elaboration of chemotactic factor(s) and leukocyte migration into the lung may not be dependent upon a single cellular response but contingent upon a dynamic multicellular reaction. Historically, the participation of the PF in acute or chronic inflammation has been regarded as essential only for the process of modeling and remodeling the connective tissue stroma of the lung. It is becoming increasingly apparent that fibroblasts, when stimulated with specific cytokines TNF or IL-I, can potentially be a dynamic cellular participant in inflammation. Both TNF and IL-I induce fibroblast proliferation (33, 34) and enhanced production of prostaglandin E2 (1), collagenase (35), gelatinase (35), glycosaminoglycan (36), monocyte chemoattractant/activating peptide (3, 4), colony-stimulating factor (5), and IL-I and IL-6 (2, 3). The activation of fibroblasts by these cytokines and the release of inflammatory mediators exemplifies the ability of this cell to respond in a diverse fashion and influence the outcome of the inflammatory response. In this study, we demonstrate that PF may contribute to pulmonary inflammatory events through the production of a neutrophil chemotactic factor compatible with IL-8. This PF-derived IL-8 expression and synthesis was dependent upon stimulation by either TNF or IL-I{3 but not by LPS. Stimulation ofPF with IL-I and TNF resulted in a time- and dose-dependent expression of mRNA, antigenic, and chemotactic activity consistent with IL-8. In contrast, LPS stimulation over a broad concentration range and time course failed to induce PF-derived IL-8 at either mRNA, antigenic, or chemotactic levels. In specificity studies using neutralizing IL-8 antibodies, the entire portion ofTNF-induced or ILI{3-induced PF-derived neutrophil chemotactic bioactivity was attributable to IL-8. Other investigators have found that long-term cultured dermal fibroblasts (1 to 8 days) constitutively produce a < IO,OOO-D neutrophil and monocyte chemotactic factor(s) (37). Our data support a cytokine-mediated production of PF-derived IL-8 that was not detectable from 24-h unstimulated PF-derived CM. These observations corroborate our previous findings in regard to signal specificity for the induction of dermal fibroblast-derived neutrophil chemotactic activity compatible with IL-8 (19). In contrast, monocyte, AM, and endothelial cell production ofIL-8 does not display the same signal specificity, as these cells will respond to either LPS, TNF, or IL-I stimulation. These findings suggest that an initial host response is required to generate the appropriate signals prior to PF production of IL-8. These data further establish the PF as a cell situated in the interstitium well suited for participation in bidirectional cellular communication between the vascular and alveolar airspace compartments.

The observation that PF produce IL-8 only in response to specific cytokines, and that these cytokines can be produced by LPS-stimulated AM, suggested that the close proximity of the AM to the PF may be critical to the induction of PFderived IL-8. To substantiate this hypothesis, we found that LPS-stimulated AM-CM induced steady-state levels of PFderived IL-8 mRNA. This response was partially inhibited by neutralizing antibodies directed against either AM-CMderived TNF or IL-l. In addition, the presence of both neutralizing antibodies resulted in the most significant reduction of PF-derived IL-8 mRNA; however, steady-state levels of IL-8 mRNA were still apparent, suggesting other AMCM-derived agonists may be potentially important for the induction of PF-derived IL-8. Furthermore, when AM were co-cultured directly with PF, LPS stimulation resulted in both AM- and PF-derived antigenic IL-8 expression by immunohistochemical analysis. During co-culture, the shift toward the right for the antigenic expression of PF-derived IL8 as compared with the AM-derived IL-8, suggests that the expression of PF-derived IL-8 is dependent upon activation of the AM. This is supported by the observation that: (1) LPS-stimulated AM can produce both IL-I and TNF, and (2) significant inhibition of AM-CM-induced PF-derived IL-8 mRNA occurs in the presence of both neutralizing antibodies to TNF and IL-l{3. These data establish cell-to-cell communication links between AM and PF that may be operative in vivo culminating in the elaboration of a potent chemotactic factor, IL-8. IL-8 is a recently described cytokine that previously was referred to as monocyte-derived neutrophil chemotactic factor (MDNCF) (14), neutrophil-activating factor (NAF) (15), neutrophil chemotactic factor (NCF) (16, 19,23), and more recently IL-8 (38). IL-8 is an 8,000-D peptide that is initially synthesized as a 99....:amino acid precursor with a characteristic leader sequence of 22 amino acids (39, 40). In addition to being a potent chemoattractant and activating cytokine for neutrophils (13, 14), it has a 10- to IOO-fold increase in potency as a lymphocyte chemotaxin (41). IL-8 belongs to a unique supergene family that includes murine macrophage inflammatory peptide-2, (MIP-2), platelet factor-4 (PF-4), platelet basic protein (PBP) and its cleavage products (CTAPIII, (3-thromboglobulin [{3-TG] , and neutrophil-activating peptide-2 [NAP-2]), chicken v-src-inducible protein (9E3), interferon-inducible protein ('Y-IP-IO), growth-regulated gene product (GRO)/melanoma stimulating activity (MGSA) (15, 39,40). A number of cell types produce IL-8, including monocytes and AM, which appear to be the predominant cellular sources of IL-8 (13, 14, 16-18). IL-8 maintains its biologic activity in the presence of significant changes in pH and resists mild proteolytic degradation as compared with other known chemotactic factors (13, 15). This suggests that the production of IL-8 at in vivo sites of inflammation may have prolonged biologic activity for the recruitment of inflammatory leukocytes. The production of IL-8 by PF and other cells that comprise the alveolar-capillary wall may account for the establishment of a chemotactic gradient and the subsequent rapid recruitment of neutrophils to the lung in such diverse pulmonary diseases as the adult respiratory distress syndrome (42), idiopathic pulmonary fibrosis (27, 28), and asbestosis (43). The AM as a sentinel cell appears to playa central role

Rolfe, Kunkel, Standiford, et al.: Pulmonary Fibroblast Expression of IL-8 by AM-derived Cytokines

in the recruitment of neutrophils to the lung. It can respond to either an exogenous stimulus (LPS) or autocrine stimuli such as TNF and IL-l, leading to the production of AMderived IL-8. Perhaps more importantly, the AM through the generation of TNF and IL-l can act in a classic paracrine fashion to induce the expression of PF-derived IL-8. This cytokine networking between immune and nonimmune cells of the alveolar-capillary wall would establish a chemotactic gradient favoring the diapedesis of neutrophils from the vascular compartment to the alveolar airspace. Further investigations are needed to establish the role of IL-8 in mediating pulmonary diseases associated with a significant neutrophilic infiltration. Acknowledgments: This research was supported in part by Grants HL-02401, HL-316~3, HL-35276, an? I?K-38149 from the National Institutes of Health, by an Amencan Lung Association Research Grant, and by the Council for Tobacco Research. Dr. Strieter is an RJR Nabisco Research Scholar.

References J. ~., K. Gustilo, W. Baeder, and B. Freundlich. 1987. Synergistic stimulation of fibroblast prostaglandin production by recombinant interleukin 1 and tumor necrosis factor. J. Immunol. 138:3812-3816. 2. Le, J., D. Weinstein, V. Gubler, andJ. Vilcek. 1987. Induction ofmembrane associated interleukin-I by tumor necrosis factor in human fibroblasts. J. Immunol. 138:2137-2142. 3. Larson, C. G., C. O. Zachariae, J. J. Oppenheim, and K. Matsushima. 1989. Production of monocyte chemotactic and activating factor (MCAF) by human dermal fibroblasts in response to interleukin I or tumor necrosis factor. Biochem. Biophys. Res. Commun. 160:1403-1408. 4. Yoshimura, T., and E. J. Leonard. 1990. Secretion of human fibroblasts of monocyte chemoattractant protein-L, the gene product of gene JE. J. Immunol. 144:2377-2383. 5. Rollins, B. J., P. Stier, T. Ernst, and C. G. Wong. 1989. The human homolog of the JE gene encodes a monocyte secretory protein. Mol. Cell. Bioi. 9:4687-4695. 6. Zucalli, J. R., H. E. Broxmeyer, M. A. Gross, and C. A. Dinerallo. 1988. Recombinant human tumor necrosis factors a and {3 stimulate fibroblasts to produce hemopoietic growth factors in vitro. J. Immunol. 140:840-844. 7. Beutler, B., and A. Cerami. 1986. Cachectin and tumor necrosis factor as two sides of the same biological coin. Nature 320:584-589. 8. Le, 1., and 1. Vilcek. 1987. Tumor necrosis factor and interleukin-l: cytokines with overlapping biological activities. Lab. Invest. 56:234-248. 9. Strieter, R. M., D. G. Remick, 1. P. Lynch, III, R. N. Spengler, and S. L. Kunkel. 1989. Interleukin-2-induced tumor necrosis factor-alpha (TNFa) gene expression in human alveolar macrophages and blood monocytes. Am. Rev. Respir. Dis. 139:335-342. 10. Stri~ter, R. M., D. G. R~mick, 1. P. Lynch, III etal. 1989. Differential regulati?n of tumor necrosis factor-alpha in human alveolar macrophages and penpheral blood monocytes: a cellular and molecular analysis. Am. J. Respir. Cell Mol. Bioi. 1:57-63. 11. Monick, M., 1. Glazier, and G. W. Hunninghake, 1987. Human alveolar macrophages suppress interleukin-l (IL-l) activity via the secretion of prostaglandin E 2 • Am. Rev. Respir. Dis. 135:72-77. 12. Yoshimura, T., K. Matsushima, 1. 1. Oppenheim, and E. 1. Leonard. 1987. N~utrophil chemotactic factor produced by lipopolysaccharide (LPS)stimulated human blood mononuclear leukocytes: partial characterization and separation from interleukin-l (IL-l). J. Immunol. 139:788-793. 13. Peveri, P., A. Walz, B. DeWald, and M. Baggiolini. 1988. A novel neutrophil-activating factor produced by human mononuclear phagocytes. 1 Exp. Med. 167:1547-1559. 14. Matsushima, K., K. Morishita, T. Yoshimura et al. 1988. Molecular cloning of human monocyte-derived neutrophil chemotactic factor MDNCF and induction of MDNCF by interleukin-l and tumor necrosis factor. J. Exp. Med. 167:1883-1893. 15. Baggiolini, M., A. Walz, and S. L. Kunkel. 1989. Neutrophil-activating peptide-l/interleukin-S, a novel cytokine that activates neutrophils. J. Clin. Invest. 84:1045-1049. 16. Strieter, R. M., S. W. Chensue, M. A. Basha et al. 1990. Human alveolar ~acrophage gene expression of interleukin-8 by tumor necrosis factor-a, hpopolysaccharide, and interleukin-l{3. Am. J. Respir. Cell Mol. Bioi. 2:321-326.. 17. Sylvester, I., 1. A. Rankin, T. Yoshimura, S. Tanaka, and E. Leonard. 1990.

1.

Eli~s,

501

S:cretion of neutrophil attractant/activation protein by lipopolysaccharidestimulated lung macro phages determined by both enzyme-linked immunosorbent assay and N-terminal sequence analysis. Am. Rev. Respir. Dis. 141: 683-688. 18. Rankin,1. A., I. Sylvester, S. Smith, T. Yohimura, and E. 1. Leonard. 1990. Macrophages cultured in vitro release leukotriene B4 and neutrophil attractant/activation protein (interleukin 8) sequentially in response to stimulation with lipopolysaccharide and zymosan. J. Clin. Invest. 86: 1556-1564. 19. Strieter, R. M., S. H. Phan, H. 1. Showell et al. 1989. Monokine-induced neutrophil chemotactic factor gene expression in human fibroblasts. 1 Bioi. Chem. 264:10621-10626. 20. Watson, M. L., G. P. Lewis, and 1. Westwick. 1988. Neutrophil stimulation by recombinant cytokines and a factor produced by IL-I treated synovial cell culture. Immunology 65:567-572. 21. Elner, V. M., R. M. Strieter, S. G. Elner, M. Baggiolini, I. Lindley, and S. L. Kunkel. 1990. Neutrophil chemotactic factor (IL-8) gene expression by cytokine-treated retinal pigmented epithelial cells. Am. J. Pathol. 136:745-750. 22. Thorton, A. 1., R. M. Strieter, I. Lindley, M. Baggiolini, and S. L. Kunkel. 1990. Cytokine gene expression of a neutrophil chemotactic factor/IL-8 in human hepatocytes. J. Immunol. 144:2605-2613. 23. Strieter, R. M., S. L. Kunkel, H. 1. Showell et al. 1989. Endothelial cell gene expression of a neutrophil chemotactic factor by TNF, IL-l{3, and LPS. Science 243:1467-1469. 24. Chirgwin,1. M., A. E. Przybyla, R. 1. MacDonald, and W. 1. Rutter. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299. 25. Jonas, E., T. D. Sargent, and I. B. Davis. 1985. Epidermal keratin gene expressed in embryos of Xenopus laevis. Proc. Natl. Acad. Sci. USA 82:5413-5416. 26. B~c~n,.~. B., J. Westwick, and R. D. R. Camp. 1989. Potent and specific inhibition of IL-8, IL-la and IL-l{3-induced in vitro human lymphocyte migration by calcium channel antagonists. Biochem. Biophys. Res. Commun. 165:349-354. 27. Hunninghake, G. w., 1. E. Gadek, T. V. Lawley, and R. G. Crystal. 1981. Mechanisms of neutrophil accumulation in the lung of patients with idiopathic pulmonary fibrosis. 1 Clin. Invest. 68:259-269. 28. Hunninghake, G. w., K. L. Garret, H. B. Richardson et al. 1984. Pathogenesis of granulomatous lung diseases. Am. Rev. Respir. Dis. 130:476-496. 29. F~ls, A. D., and Z. A. Cohn. 1986. The alveolar macrophage. 1 Appl. Physiol. 60:353-369. 30. Samuelsson, B. 1983. Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation. Science 220:568-571. 31. Nathan, C. E 1987. Secretory products of macrophages. 1 Clin. Invest. 79:319-326. 32. Momex,1. E, Y. Montinet, K. Yamauchi etal. 1986. Spontaneous expression of the c-cis gene and release of a platelet-derived growth factor-like molecule by human alveolar macrophages. J. Clin. Invest. 78:61-66. 33. Vilcek, 1., V. 1. PaImombella, D. Henrickson-Destefano etal. 1986. Fibroblast growth enhancing activity of tumor necrosis factor and its relationship to other polypeptide growth factors. J. Exp. Med. 163:632-643. 34. Schmidt, 1. A., S. B. Mizel, D. Cohen, and I. Green. 1982. Interleukin-l, a potent regulator of fibroblast proliferation. 1 Immunol. 128:2177-2182. 35. Nakagawa, H., H. Kitagawa, and Y. Aikawa. 1982. Thmor necrosis factor stimulates gelatinase and collagenase production by granulation tissue in culture. Biochem. Biophys. Res. Commun. 142:791":'797. 36. Elias, 1. A., R. C. Krol, B. Freundlich, and P. M. Sampson. 1988. Regulation of human lung fibroblast glycosaminoglycan production by recombinant interferons, tumor necrosis factor and lymphotoxin. J. Clin. Invest. 81:325-333. 37. Sobel,1. D., and 1. I. Gallin. 1979. Polymorphonuclear leukocyte and monocyte chemoattractants produced by human fibroblasts. 1 Clin. Invest. 63:609-618. 38. Westwick, 1., S. W. Li, and R. D. R. Camp. 1989. Novel neutrophil stimulating peptides. Immunol. Today 10:146-147. 39. Wolpe, S. D., and A. Cerami. 1989. Macrophage inflammatory proteins 1 and 2: members ofa novel super family ofcytokines. FASEBJ. 3:2565-2573. 40. Matsushima, K., and 1. 1. Oppenheim. 1989. Interleukin 8 and MCAF: novel inflammatory cytokines inducible by IL-l and TNF. Cytokine 1:2-13. 41. Larsen, C. G., A. O. Anderson, E. Apella, 1. 1. Openheim, and K. Matsushima. 1989. Neutrophil activating protein (NAP-I) is also chemotactic for T-Iymphocytes. Science 243:1464-1467. 42. Parsons, P. E., A. A. Fowler, T. M. Hyers, and P. M. Henson. 1985. Chemotactic activity in bronchoalveolar lavage from patients with adult respiratory distress syndrome. Am. Rev. Respir. Dis. 132:490-493. 43. Schoenberger, C. I., G. W. Hunninghake, 0. Kawanami, V. 1. Ferrans, and R. G. Crystal. 1982. Role of alveolar macrophages in asbestosis: modulation of neutrophil migration to the lung after acute asbestos exposure. Thorax 37:803-809.