In Vivo Detection of a Novel Macrophage-derived Protein Involved in the Regulation of Mucus-like Glycoconjugate Secretion 1 , 2

KIRK SPERBER, EDITH GOLWB,3 SATINDRA GOSWAMI, THOMAS H. KALB, LLOYD MAYER,4 and ZVI MAROM5

Introduction

Pulmonary macrophages have been implicated in the pathogenesis of inflammatory lung diseases like chronic bronchitis and bronchial asthma. It has been demonstrated that there are increased numbers of pulmonary macrophages in the bronchoalveolar lavage fluid (BALF) from patients with chronic bronchitis (1) and increased levels of interleukin-l (IL-l) in the BALF of asthmatic patients experiencing nocturnal symptoms (2). Furthermore, it has been observed that pulmonary macrophage activation can occur via the low-affinity IgE receptor, thus establishing independent monocyte activation by IgE (3). Inflammatory lung diseases are also associated with excessive mucus secretion. Since pulmonary macrophages and peripheral blood monocytes have been implicated as possible contributors to mucus secretion (4-7), we were interested in investigating the role of macrophage-derived proteins in mucus secretion in various disease states. We recently described (4) a novel 68,000 D macrophage-derived mucus secretagogue (MMS-68) that is capable of inducing mucus-like glycoconjugate (MLGC) release from cultured airways, respiratory epithelial cells, and the Ishikawa adenocarcinoma cell line. To be able to measure MMS-68 and assess its role in the regulation of mucus secretion in vitro and in vivo, we generated monoclonal antibodies (mAb) against MMS-68 to detect its presence and measure specific activity in human airways and cultures of peripheral blood monocytes and pulmonary macrophages from both normal subjects and patients with disease states associated with mucus hypersecretion (chronic bronchitis and steroid-dependent asthma). Utilizing an antigen-capture assay, we have demonstrated that MMS-68 is present in cultures of peripheral blood monocytes, pulmonary mac-

SUMMARY We previously described a novel 68,000 D macrophage-derived protein (MMS-68) that can stimulate mucus-like glycoconJugate (MLGC)secretion from cultured human airways, respiratory epithelial cells, and the Ishikawa adenocarcinoma cell line. To better characterize this mucus secretagogue, we generated monoclonal antibodies against MMS-68 by Injecting crushed SDS-PAGE gel slices containing this protein into Balb-C mice followed by fusion with SP2/0, a nonsecretlng mouse myeloma cell line. A panel of monoclonal antibodies was produced that Identified the 68,000 D MMS by Immunoblot analysis and immunopreclpitatlon. The monoclonal antibodies detected MMS68 in normal peripheral blood monocytes and pUlmonary macrophages by cytofluorographlc analysis and in human airways as determined by Immunohistochemistry. Utilizing the monoclonal antibodies, an antigen-capture ELISA assay was developed. Statistically significant elevations in levels of MMS-68 were detected in bronchoalveolar lavage fluid (BALF) of chronic bronchitic SUbjects and cigarette smokers and in monocyte culture supernatants from steroid-dependent asthmatic patients compared to normal control subjects. The 68,000 D MMS is a potent secretagogue and may play an Important role in the regUlation of mucus secretion, especially In chronic bronchitis and sterolddependent asthma. AM REV RESPIR DIS 1992; 146:1589-1597

rophages, and normal lung tissue, is markedly elevated in BALF from cigarette smokers and chronic bronchitic patients, and is also increased in culture supernatants from peripheral blood monocytes of steroid-dependent asthmatic patients. Methods Generation of Human Macrophage Hybridomas Human macrophage hybridomas were obtained by fusing macrophages, obtained by allowing monocytes to mature into macrophages in Teflon®bag culture, with a hypoxanthine-guanine phosphoribosyltransferase(HGPRT)-deficient promonocytic line, U-937, as previously described (8).

Peripheral Blood Monocyte Isolation Monocytes were separated from buffy coats and from study patients by Ficoll-Hypaque'" density centrifugation (Pharmacia, Piscataway,N J) by methods previously described (8). The patients who were studied met the criteria for chronic bronchitis established by the American Thoracic Society (9), and the steroid-dependent asthmatic group met the criteria established by Carmichael and colleagues (10). Freshly isolated peripheral blood monon-

uclear cells (5.0 to 10.0 X 106 ) were incubated in RPMI (GIBCO, Grand Island, NY) supplemented with 10070 fetal calf serum (GIBCO), 1% penicillin-streptomycin (GIBCO), and 2 mM L-glutamine (GIBCO) at 370 C in tissue culture flasks (T-75;Falcon, Oxnard, CA) for 45 min. The nonadherent cells were removed and the adherent cells were extensively washed with sterile phosphate-buffered saline (PBS), harvested by rubber policeman, and stained with monocyte-specific anti-CD14 mAb to assess the purity of the prepara-

(Received in original form November 1, 1991 and in revised form April 29, 1992) 1 From the Divisions of Clinical Immunology and of Pulmonary and Critical Care Medicine, Department of Medicine, Mount Sinai Medical Center, New York, New York. 2 Correspondence and requests for reprints should be addressed to Kirk Sperber, M.D., Division of Clinical Immunology, Box 1089, 1 Gustave Levy Place, New York, NY 10029. 3 Recipient of the New York State Lung Research Award. 4 Supported by National Institutes of Health Grant Nos. CA41583, AI23504, and AI24671 and a recipient of the Irma T. Hirsh! Career Trust Award. s Supported by National Institutes of Health Grant No. NHL-37254 and by the Catherine and Henry Gainsman Foundation.

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tion. The isolated monocytes contained no CD-3+ « 1010), slgG+ « 0.1%), or CD-20+ « 0.1%) cells.

Pulmonary Macrophage Isolation Pulmonary macrophages were isolated from specimens of human lung obtained during surgery for resection of lung cancer by a modification of previously described techniques (6, 7). In brief, fragments (0.5 by 0.5 em) of human lung parenchyma were teased apart, filtered through two layers of sterile wire mesh, and centrifuged at 500 x g for 10 min at 22 0 C. Cell pellets were pooled, layeredonto Ficoll-Hypaque, and centrifuged at 300 x g for 20 min at room temperature. The mononuclear cell preparations were removed from the interface, washed four times in Hanks' balanced salt solution, and resuspended in modified Eagle's medium (MEM) (GIBCO) with penicillin (100 ug/ml), streptomycin (100 ug/ml), and L-glutamine (2 mM/ml) without supplementary serum. The concentration of pulmonary macrophages was adjusted to 1.5 x 106 / ml. Cells were allowed to adhere to plastic dishes for 45 min and then washed vigorously three times with serum-free medium to remove nonadherent celis. Cells were then removed from culture with a rubber policeman and stained according to the nonspecific esterase technique of Burston (11): 95% were positive, and> 96% of the cells were viable by trypan blue exclusion. No attempt was made to separate alveolar macrophages and interstitial macrophages, and the cells were maintained in serum-free medium. Preparation of BALF Fiberoptic bronchoscopy (FOB) with lavage was performed utilizing a fiberoptic bronchoscope as previously described (12). The bronchitic subjects studied met the criteria established by the American Thoracic Society (10). BALF (120to 125 ml) obtained from human subjects during FOB was passed through two layers of sterile gauze and centrifuged at 300 x g for 15 min. The supernatant was then filtered through a YM30 membrane filter (Amicon, Danvers,MA), and the retentate was dialyzed against water containing 0.02% sodium azide at 4 0 C. The dialyzed material was lyophilized and resuspended in PBS, and protein concentration was determined by Lowry's method (13). An aliquot of this processed material was analyzed by ELISA for quantitation of MMS-68 (see later). Pulmonary macrophages from the BALF were isolated as described and cultured in serum-free medium. MMS-68 Generation and Purification Our previously described purification method was used with some modifications (4,5). Supernatants from cultures of clone 63 stimulated with lipopolysaccharide (LPS; 10 IJ,g/ ml), previously shown to secrete large quantities of MMS-68, were first concentrated using a YM30 membrane (molecular weight exclusionof 30,000)(Amicon Corp., Danvers, MA) and the retentate was dialyzed against

SPERBER, GOLWB, GOSWAMI, KALB, MAYER, AND MAROM

PBS at 4 0 C. An aliquot of the dialyzed material was analyzed for biologic activity and another run on a 12.5% sodium dodecyl sulfate-polyacrylamide gelelectrophoresis(SDSPAGE) followed by staining with 1% Coomassie blue. One lane was electrophoretically transferred onto nitrocellulose for immunoblot analysis. The band corresponding to a molecular weight of 68,000 D on the gel was then sliced into small fragments suitable for immunization.

Generation and Screening of anti-MMS-68 Monoclonal Antibodies Monoclonal antibodies were generated against the 68,000 D secretagogue by injecting gel slices (obtained as described earlier) containing the protein into female Balb-C mice, with one booster immunization 3 wk after the initial immunization, followed by fusion to the non-IgG-secretingmyeloma line SP2/0 utilizing methods previously described (14). Immunoblot blot technique (15) wasused to screen the monoclonal antibodies. MMS68 was applied to nitrocellulose membranes using a Bio-dot'" apparatus (Bio-Rad, Richmond, CA) that permitted application of uniform dots. Test supernatants from the fusion were added to the MMS-68 containing nitrocellulose paper in 96-well microtiter plates for 2 h at 25 0 C followed by five PBS washes and the addition of horseradish peroxidase-conjugated goat antimouse antibody (Tago, Burlingame, CA) for 2 h at 250 C. Irrelevant murine monoclonal antibodies of all isotypes were used as specificity controls in each assay. After extensive PBS washing, substrate (3,3'-diaminobenzidine tetrahydrochloride; Pierce Chemical Company, Rockford, IL) was added and the plates read for the appearance of a blue color at 30 min, indicating a positive result. Positive clones were expanded and ascites for one clone of interest, 1-D-1O (selected because of excellent reactivity in the immunoblot assay) was generated by injecting cells (2 x 106 ) into pristaneprimed Balb-C mice by methods previously described (14). The ascites was then passed over a protein G column (Pharmacia, Piscataway, NJ), and fractions of antibody eluted with 0.1 M glycine-HCl buffer (pH 3) and immediately neutralized with 1 M 'Iris (pH 8). The protein concentration of the fractions was determined by absorbance at 280 nM, and the fractions containing the highest concentrations of antibody were pooled. Immunoprecipitation of MMS-68 from Peripheral Blood Monocytes Peripheral blood monoeytes obtained from normal blood donors at the Mount Sinai Medical Center were cultured in cysteine- and methionine-free medium (RPMI 1640,Selectamine" kit; GIBCO) and pulsed for 6 h with [35S]methionine and [35S]cysteine (Translabel®; ICN, Irvine, CA) at 50 IJ,Cil5 x 106 cells as previously described (16).The labeled cells were lysed and the lysates were subjected to immunoprecipitation with monoclonal

antibodies to Class I antigens (W6/32-lgG, ATCC), isotype controls (IgG, and IgG2a antidinitrophenyl (DNP) mAb kindly provided by Dr. J Unkeless) and anti-MMS-68 antibody (l-D-lO) and run on a 12.5% polyacrylamide gel. The gel was then impregnated in 1 M sodium salicylate for 45 min and autoradiographed overnight (XAR-5; Kodak, Rochester, NY).

Isolation of MMS-68 by Affinity Column A 5-cm affinity column was constructed by coupling purified 1-D-1O monoclonal antibody to cyanogen bromide-activated Sepharose® 4B (Pharmacia, Piscataway, NJ) utilizing previously described methods (17). The YM-30 retentate from LPS-stimulated clone 63 supernatant was passed over the column and incubated for 2 h at 25 0 C. The column was then extensively washed with 0.1 M PBS + 0.5 M NaCl buffer to removeunbound material. The bound MMS-68 was then eluted from the column with 0.1 M glycine-HCl buffer (pH 3), and ten 3-ml fractions were collected and immediately neutralized with 1 M Tris buffer (pH 8.0). Individual fractions wereanalyzed (absorbance at 280nm) for biologic activity. The fractions that contained the highest protein concentrations and biologic activity (usually the first five) were run on a 12.5% polyacrylamide gel and then stained with 1% Coomassie blue (described previously). Assay of Cultured MLGC Ishikawa cells (18), which secrete MLGC (5, 19,20), were routinely maintained in MEM (GIBCO) containing Earle's salts, 15% fetal bovine serum, 100 ug/ml of penicillin, 100 ug/ml of streptomycin,and 0.25 ug/ml of amphotericin B. The cells were plated in 35 x 100 mm plastic dishes at a density of lOS cells/ml and incubated for 48 h until the cells reached confluency. The medium was then discarded, and the cells were incubated for 24 h in MEM medium (GIBCO) containing antibiotics and ['H]glucosamine (1 IJ,Cilml) (Amersham, Arlington Heights, IL), which becomes incorporated into newlysynthesized and secreted glycoconjugate. After the initial 24 h incubation period, the labeling medium was discarded, the cells were washed in fresh MEM medium without serum, and ['H]glucosamine was added. The plates werethen incubated for two 1 h periods, PD I and PD II, in MEM medium containing antibiotics and aprotinin (10ug/ml) and phenylmethy1sulfonyl fluoride, 0.5 mmol/L (Sigma Chemical Company, St. Louis, MO), which by themselves did not have a modulating effect on these cells and wereadded as protease inhibitors to prevent possible degradation of MMS68. The biologic effect of MMS-68 was quantified by establishing a secretory index (SI) for the experimentally manipulated cultures compared with the control cultures as previously described (4, 19, 20). The effect of MMS-68 was assessed by relating its SI to the average SI for the corresponding set of con-

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DETECTION OF A MACROPHAGE REGULATING MUCUS SECRETION

trois and expressing the result as percentage change, namely, 100 x (SI experimental/average SI control - 1). Percentage changes were then averaged over experiments and tested for significance with a one-sample t test (t, mean and standard error of the mean) (5, 21, 22). A P value of < 0.05 was considered statistically significant. We have expressed MMS-68 bioactivity in units defined as follows. The amount of MMS-68 tested capable of causing a 30070 increase in glycoconjugate release above control from human airway explants or 2 X 106 secretory epithelial cells in 60 min (PD II) is defined as 1 unit. MMS-68 biologic activity is always compared with a known secretagogue carbachol (positive control), which at concentrations of 10-5 mol/L causes a 30% increase of MLGC releaseabove control (= 1 unit).

Intracytoplasmic Staining of Peripheral Blood Monocytes and Pulmonary Macrophages Peripheral blood monocytes (1 X 106 ) , clone 63 (l X 106 ) , or pulmonary macrophages (l x 106 ) were fixed with 70% ethanol for 30 min at 4 0 C. The cells were then washed three times with PBS and stained with monoclonal I-D-IO, followed by affinity-purified FITC-conjugated F(ab~2 goat antimouse IgO antibody (Tago, Burlingame, CA). W6/32 (anti-Class I) was used as the positive control and Ig02Qanti-DNP was used as the negativecontrol. The cellswerethen analyzed by cytofluorographic analysis (EPICSall C; Coulter, Hialeah, FL) as previously described (5). Detection of MMS-68 In Situ Frozen sections 4 urn thick were obtained from thoracotomy specimens from patients undergoing lung resection for cancer. All airway specimens were obtained from surgical resection proximal to tumor foci. Staining was performed with I-D-IO (anti-MMS-68) monoclonal antibody, mAb P-9 (anti-CD-I4), and W6/32 (anti-HLA, Class I) using an avidin/ biotin/peroxidase sandwich technique (Veetastain, Burlingame, CA). In each experiment, isotype-matched controls wereutilized to control for nonspecific Fc receptor (FcR) binding (murine IgO, and Ig02Qanti-DNP). Sections were counterstained with hematoxylin and examined by light microscopy by two observers (23). Generation of Alkaline Phosphatase-conjugated Polyclonal Guinea Pig Anti-MMS-68 Serum MMS-68 generated and purified from YM30retentate of clone 63 as previously described (4) was injected intramuscularly with complete Freund's adjuvant into two guinea pigs with two booster injections (Pocono Rabbit Farms, Stroudsberg, PA). Preimmune serum was obtained at Day O. Polyclonal antiserum was then prepared from two guinea pigs and pooled. IgO was purified by protein A-Sepharose as previously described (24). Ouchterlony immunodiffusion in gels was used to test antiserum generated against

MMS-68. Immunoelectrophoresis was performed in 2% sodium dodecylsulfate in barbital buffer, pH 8.6, which contained 0.01 mol/L of ethylenediaminetetraacetic acid. Ouinea pig antisera raised against purified MMS-68 developed as a single band in Ouchterlony immunodiffusion. The antiserum was then conjugated to bovine alkaline phosphatase (Sigma, St. Louis, MO) (25). Electron microscopy-grade glutaraldehyde (25%; Sigma) was added to obtain a ratio of 1:3 antibody to alkaline phosphatase to achievea final concentration of 0.2% glutaraldehyde. The reaction was allowed to proceed for 2 h and was stopped by adding PBS containing 100mM lysine and 100mM ethanolamine. The reaction mixture was dialyzed against PBS for 48 h to remove unbound alkaline phosphatase and the conjugated antibody assayed for enzymatic activity by adding p-nitrophenylphosphate disodium (Substrate 104;Sigma). This antibody has been previously described (4) and can identify MMS-68 on immunoblot analysis and block MMS-68 biologic activity.

Antigen-Capture ELISA to Detect MMS-68 in Biologic Fluids An antigen-capture assay was constructed using the I-D-IOmonoclonal antibody as the capture antibody and the alkaline phosphatase-conjugated polyclonal guinea pig antiMMS-68 sera as the developing antibody. The 96-well microtiter plates were coated with 5 ug/ml of purified I-D-IOmonoclonal antibody in borate buffer, pH 8, for 16 h at 4 0 C. The plates were then washed five times with PBS and 0.05% Tween'" 20 (Sigma) and blocked with PBS and 0.05% Tween 20 and 0.25% bovine serum albumin (BSA) for 1 h at 37 0 C. Dilutions of duplicate test samples were incubated in blocked, precoated 96microwell plates for 2 h at 37 0 C. The plates wereextensivelywashed and incubated for 2 h at 37 0 C with the alkaline phosphataseconjugated polyclonal guinea pig antibody followed again by five washes. p-Nitrophenylphosphate disodium (Substrate 104, Sigma) was added for 30 min at 25 0 C, and the plates were read on an ELISA reader at 405 nm. Standard curves of known concentrations of purified 68,000 MMS obtained from clone 63 were generated for each assay. Patient Population A group of 41 volunteers recruited to participate in the clinical study for the detection of MMS-68 underwent FOB. Their BALF was processed and assayed for MMS-68 as described. There were20 smokers (without bronchitis), 15 males and 5 females, with an age

Fig. 1. Monoclonal antibody 1-0-10 identifies MMS-68 on immunoblot analysis, representative of five experiments. Equal quantities of MMS-68 are detected in supernatants of either 1 x 10· pulmonary macrophages or clone 63.

range of 23 to 61, mean age 42, with an average 23 pack-year smoking history; 10 nonsmokers (normal volunteers), 6 males and 4 females, with an age range of 24 to 37, mean age 31; 7 ex-smokers (had stopped smoking at least 6 months before the study), 4 males and 3 females, with an age range of 25 to 43, mean age 32;and 4 patients with chronic bronchitis, with an age range of 24 to 39, mean age 35. Peripheral blood monocytes from 5 female and 3 male asthmatic patients requiring 20 mg prednisone daily for 6 wk, age range between 22 and 57, were isolated and assayed for MMS-68 as described.

Statistical Analysis of the Antigen-Capture Assay of BALF and Monocyte Culture Supernatant Nonparametric analysis was performed using the SAS statistical program package (22) and analyzed by methods previously described (21). Data were compared using a two-tailed Student's t test. A p value < 0.05 was considered statistically significant. Results

I-D-JO Antibody Detects MMS-68 by Immunoblotting and Immunoprecipitation and Can Be Used to Isolate Biologically Active MMS-68 by Affinity Chromatography I-D-lO identified a 68 kD protein obtained from supernatants of LPS (10 ug/ml) stimulated pulmonary macrophages and clone 63 on immunoblot analysis as illustrated in figure 1. As seen in figure I, comparable amounts of MMS-68 were detected from equal numbers of pulmonary macrophages (1 x 106 ) and clone 63 (1 x 106 ) cells. Bovine serum albumin served as the negative control. I-D-lO also identified a 68,000 D protein by immunoprecipitation from [35S]cysteine- and [35S]methionine-labeled peripheral blood monocytes (figure 2). Isotype-matched controls failed to demonstrate similar bands. The 68,000 D protein was then purified by the I-D-lO mAb affinity column chromatography. As illustrated in figure 3, the first five fractions eluted from the I-D-lO affinity column demonstrated bands with a molecular weight of 68,000 D, corresponding to the molecular weight of MMS-68. MMS-68 biologic activity was detected in each fraction, causing a dose-dependent release of MLGC from

BOVINE SERUM ALBUMIN PULMONARY MACROPHAGES CLONE 63

1592

SPERBER, GOLWB, GOSWAMI, KALB, MAYER, AND MAROM

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Fig. 2. Demonstration of the 68,000 D MMS-68 by immunoprecipitation from peripheral blood monocytes. The 1-D· 10 monoclonal antibody (anti·MM8-68) was used after labeling human peripheral blood monocytes with [35Sjcystei ne and [35Sjmethionine. This is representative of three experiments.

43

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• ['H]glucosamine-Iabeled Ishikawa cells were used as described in METHODS. Carbachol, 10-' M, was used as a positive control for mucus secretion and showed an increase of 30 ± 2% in MLGC secretion above control, which is defined as 1 unit. The concentrations shown represent the linear portion of the dose-response curve. Activity plateaus at 30 ~g/mg protein (high) and 1 ~g/ml (low). This is a representative axparlment repeated five times. Each experimental point represents data from three culture dishes.

97 68

29

MMS-68 Biologic Activity

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Fig. 3. An affinity column utilizing the 1-D·10 monoclonal antibody was constructed. YM·30 retentate obtained by concentrating 1 L of supernatant from LPS-stimulated (10I1g/ml)clone 63 was passed over the column. Bound material was eluted with 2 mM glycine, and the fractions collected were analyzed by 8DS-PAGE (15%). This is representative of three experiments. A band with a molecular weight of 68,000 D is demonstrated in all eluted fractions.

the Ishikawa adenocarcinoma cell line (table 1). MMS-68 at a concentration of 10 ug/rnl stimulated an increase in MLGC release of 129 ± 2070 (4.3 units) above control, which was four times greater than carbachol 10-5 M (30% = 1 unit) (positive control). The specificity ofthe 1-D-lOantibody was established by the ability of this mAb to block MLGC release from Ishikawa cells mediated by MMS-68 but not carbachol (10-5 M) (table 2). Furthermore, the I-D-1O antibody did not react with the previously described 2000 D MMS (5).

MMS-68 Can Be Detected by Intracytoplasmic Immunofluorescence in the Cytoplasm of Peripheral Blood Monocytes and Pulmonary Maerophages To determine the source of MMS-68, we isolated peripheral blood monocytes and pulmonary macrophages. Staining by the I-D-lO mAb was observed in unstimulated cells (figure 4C), and LPS stimulation (figure 4D) markedly enhanced MMS-68 production in peripheral blood monocytes. No staining was observed with an isotype-matched control subject mAb (figure 4A). MMS-68 was similarly demonstrated in pulmonary macrophages (figure 5) in isolated macrophages from four separate individuals. A peak channel shift corresponding to the presence of baseline intracytoplasmic MMS68 is seen in figure 5C and could be upregulated with pretreatment with lipopolysaccharide (10 ug/ml) (figure 5D). Again, no staining was observed with the IgG isotype control mAb. Monoclonal I-D-lOdid not stain T cells or B cells (data not shown). MMS-68 Can Be Detected in Human Airways Frozen sections of human airways were

1593

DETECTION OF A MACROPHAGE REGULATING MUCUS SECRETION

TABLE 2 MLGC SECRETAGOGUE ACTIVITY OF MMS-68 CAN BE BLOCKED BY 1-D-10' MMS-68 Biologic Activity (Units)

Condition

0.0 0.0

Media control IgG2 • anti-DNP antibody alone Carbachol (10-' M) + 1-D-10 MMS-68 (6 ~g/ml) MMS-68 + 192• Anti-DNP antibody MMS-68 + 1-D-10

1.0 2.7 2.1

0.0

• ['Hlglucosamine-Iabeled Ishikawa cells were used as described in METHODS. The nonspecific IgG,. anti-DNP monoclonal antibody as well as the 1-0-10 anti-MMS-68 monoclonal antibody were incubated with MMS-68 for 30 min before the assay. The data are expressed in units as previously described. This is a representative experiment repeated five times. Each experimental point represents data from three culture dishes. As can be seen, the 1-0-10 antibody but not the IgG,. antibody blockad MLGC release activity by MMS·68, which was statistically significant using a nonparametric analysis (p < 0.03).

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Panel A-Control

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Fig. 5. Demonstration of MMS-68 in pulmonary macrophages. As with peripheral blood monocytes, baseline production of MMS-68 was detected (C), which can be markedly enhanced by LPS (10 ~g/ml) stimulation (0). This experiment was repeated on macrophages from four separate individuals.

obtained and stained with 1-D-1O. 1-D10staining exhibited a subbronchial distribution (figure 6C) similar to the distribution of staining with the macrophagespecific mAb P-9 (anti-CD-14) in figure 6D. No staining was seen with the isotype control (figure 6A).

Determination of MMS-68 Levels in BALF and Peripheral Blood Monocyte and Pulmonary Macrophages Culture Supernatants An antigen-capture ELISA was constructed to measure MMS-68 levels in biologic fluids. As seen in figure 7, the lowest level of detection by the ELISA of purified MMS-68 was 10ng/ml. Utilizing this assay we measured MMS-68 levels in BALF and in monocyte culture supernatants. As seen in figure 8, the patients with chronic bronchitis had the highest levels of MMS-68 (441J.g MMS68/mg protein) in BALF followed by active smokers (26 IJ.g MMS-68/mg protein) and ex-smokers (7 IJ.g MMS-68/mg protein). Non-smokers had the lowest detectable amounts of MMS-68 (1.1 IJ.g MMS68/mg protein). The levels of MMS-68 were significantly higher in patients with bronchitis (p < 0.001) compared with all other groups. Smokers and ex-smokers also displayed significantly higher levels of MMS-68 (p < 0.001) compared with non-smokers. We then assayed for spontaneous release of MMS-68 from pulmonary macrophages in vitro and compared smokers with non-smokers. In the smokers (n = 12), the amount of MMS-68 secreted by the pulmonary macrophages was significantly higher (1.3 ug/mg protein) than in the non-smokers (0.087 ug/mg protein; n = 6) (p < 0.001). To determine whether this increased secretion related to the locale of the macrophage, we assayed for MMS-68 secreted by peripheral blood monocytes in vitro. As seen in figure 9, monocytes from steroid-dependent asthmatic patients secreted 9.590 ± 6.361J.g MMS-68/mg protein (n = 8); monocytes of chronic bronchitic subjects, 2.97 ± 1.90 IJ.g MMS68/mg protein (n = 3); monocytes from septic patients, 1.770 ± 1.273 IJ.g MMS68/mg protein (n = 2); and monocytes of normal volunteers 0.975 IJ.g MMS-68/ mg protein ± 1.298 (n = 4). We analyzed the data from the four groups using the Kruskal-Wallis test (20,21). This nonparametric analysis for intergroup differences yielded a statistically significant (H = 9.64; P < 0.05) result. There was also a statistically significant difference between control and steroid-dependent

1594

SPERBER, GOLWB, GOSWAMI, KALB, MAYER, AND MAROM

Fig. 6. Demonstration of MMS-68 by immunohistochemistry in sections of human lung after hematoxylin and eosin staining. This is a representative study of multiple repeated frozen sections. (A) Isotype control, anti-DNP = the negative control; (B) anti-Class I W6I32 = positive control. MMS-68 has a subbronchial distribution (e). A similar pattern of staining is observed for P-9 (CD-14, a pan-monocyte marker) (D). Original magnification: x40.

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DETECTION OF A MACROPHAGE REGULATING MUCUS SECRETION

MMS·68 Antigen Capture ELISA

3-r------------------, Fig. 7. An antigen-capture ELISA was constructed to measure MMS-68 levels in biologic fluids. The ELISA measures MMS-68 from a concentration of 10 to 1,000 ng/ml.

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MMS·68 from Fig. 8. MMS-68 was quantified (ELISA) from BALF of normal subjects and patients. A total of 41 SUbjects were studied: 21 smokers, 10 non-smokers (normal VOlunteers), 7 ex-smokers, and 4 patients with chronic bronchitis. MMS-68 levels were higher in patients with bronchitis (p < 0.001)compared with all other groups. Smokers and ex-smokers also displayed elevated levels of MMS·68 (p < 0.001)compared with non-smokers.

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asthmatic groups (p < 0.01) (see subsequent discussion). Thus monocytes from patients with steroid-dependent asthma secrete more MMS-68 compared with control subjects.

non-smokers

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Discussion

To study the role of inflammation in respiratory mucus secretion, we investigated mediators secreted by pulmonary macrophages and peripheral blood mono-

MMS-68 Release from Monocytes in Vitro 20 - , . - - - - - - - - - - - - - - - - - - .

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; steroid dependent asthma

Fig. 9. Measurement of MMS-68 in supernatants from peripheral blood monocytes of patients with steroid-dependent asthma, bronchitis, and sepsis, as well as normal control SUbjects. "Spontaneous" MMS-68 production was measured. MMS-68 production was highest from the monocytes of patients with steroid-dependent asthma and bronchitis compared with normal and septic patients. Only the comparison between the steroid-dependent asthmatic and control groups reached statistical significance (p < 0.01).

cytes to see whether these products might be involved in the regulation of mucus secretion. To facilitate macrophage availability, our laboratory generated a series of human macrophage hybridomas that were used to study the regulation of a previously described 2,000 D monocyte-macrophage-derived mucus secretagogue (5, 6). One clone that produces the 2,000 D secretagogue (clone 63) was also found to produce large amounts of a novel 68,000 D protein (MMS-68), which causes mucus release from cultured airways, respiratory epithelial cells, and the Ishikawa adenocarcinoma cell line (4). To better characterize this high-molecular-weight mucus secretagogue, we generated a panel of monoclonal antibodies both to detect MMS-68 in peripheral blood monocytes, pulmonary macrophages, and airways and to measure levelsin BALF and monocyte culture supernatants from both normal subjects and patients who have diseases associated with mucus hypersecretion (chronic bronchitis and steroiddependent asthma). We generated the monoclonal antibody, I-D-lO which was specific for MMS-68 biochemically (immunoblot analysis (figure 1), by immunoprecipitation (figure 2) and biologically (isolation of biologic active MMS-68 by a I-D-IO affinity column) (table 1) keep and by specific blockade of biologic activity (table 2). Utilizing this monoclonal antibody we demonstrated, in multiple experiments with different patients, that MMS-68 was constitutively present in peripheral blood monocytes (figure 4) and pulmonary macrophages (figure 5). Synthesis by either monocytes or macrophages could be markedly upregulated by stimulation with LPS (10 ug/ml) (figures 4 and 5). Furthermore, MMS68 is specific for monocytic cells and is not present in either T cells or B cells (data not shown), and its presence was also demonstrated in vivo by immunohistochemical staining of surgical specimens of patients undergoing lung resection for cancer (figure 6C). The co-localization of MMS-68 and macrophages was confirmed by similar staining patterns (figure 6D) with the anti-CD-14 monoclonal antibody (P-9). The finding of MMS-68 in the alveolar macrophages is not surprising since alveolar macrophages are exudative macrophages and are most probably derived from peripheral blood monocytes (26). The availability of both a monoclonal antibody and a polyclonal anti-MMS-68 guinea pig antiserum [previously de-

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scribed (4, 5)] allowed the construction of a crude antigen-capture assay ELISA (figure 7) to measure MMS-68 levels in BALF and in both monocyte and macrophage culture supernatants. Significant increases in MMS-68 were seen in BALF from cigarette smokers in general and in cigarette smokers with bronchitis. This increase in MMS-68 associated with cigarette smoking and bronchitis is consistent with previous data demonstrating the activation and accumulation of pulmonary macrophages in smokers (27). Spontaneous MMS-68 production by peripheral blood monocytes is increased in steroid-dependent asthma compared with normal subjects, septic patients, and chronic bronchitic patients. To define group differences, we performed the Mann-Whitney test (21, 22), comparing each of the four groups with each other. Only comparison of the control with the steroid-dependent asthmatic group achieved statistical significance (Z = 2.548, p < 0.01). Thus MMS-68 levels were significantly greater in the steroiddependent asthmatic than in the control group. The number of observations among bronchitic subjects and patients with sepsis did not permit us to say with confidence that there was or was not any difference between these and control groups and would not support any definite conclusion regarding the role of MMS-68 in the pathogenesis of mucus hypersecretion seen in steroid-dependent asthma. This finding, however, is consistent with other data demonstrating the role of monocytes and pulmonary macrophages in steroid-dependent asthma (28, 29), which is defined by Carmicheal and colleagues (10) as asthma requiring at least 20 mg prednisolone daily for 6 wk. Several reports have observed increased production of IL-1 (2), arachidonic acid metabolites (30), and neutrophil chemotactic factors (31)by the alveolar macrophage in these patients. In one recent publication (32), a novel 3,000 0 peptide that causes neutrophil chemotaxis was produced by monocytes of steroid-dependent asthmatic patients. The increased spontaneous production of MMS-68 from the monocytes from our steroid-dependent asthmatic group and the previous report of the 3,000 0 neutrophil chemotactic factor strongly suggests that the monocytes are "preactivated" or poorly regulated in steroiddependent asthma. The increased production of MMS-68 may therefore contribute to the hypersecretion of mucus

SPERBER, GOLLUB, GOSWAMI, KALB, MAYER, AND MAROM

that is characteristic of this disease (33). Monocytes from septic patients failed to secreteMMS-68. However,not all monocyte activation results in increased MMS68 production. In contrast to steroid-dependent asthma, spontaneous MMS-68production by monocytes in chronic bronchitis appeared to be less (figure 9), suggesting that monocytes in this disease may not be preactivated. Activation would have to occur in the lungs of these patients. This was suggested by the studies (figure 8) in which levels of MMS-68 were markedly higher in BALF of cigarette smokers and patients with chronic bronchitis than in the culture supernatants of the peripheral blood monocytes, This increased MMS-68 in the BALF reflects increased production by pulmonary macrophages and suggests that MMS-68 production is markedly upregulated when monocytes are activated and mature into macrophages when they enter the lung. This activation may be due to an exposure to cigarette smoke or cigarette smoke byproducts. We did not yet measure MMS68 levels in BALF from steroid-dependent asthmatic patients or from monocytes of smokers without bronchitis. The relationship between MMS-68 and the low-molecular-weight MMS (2,0000) is of interest. One polyclonal antibody generated against the 2,000 0 MMS reacted with MMS-68 on immunoblot and one polyclonal antibody generated against MMS-68 also reacted with the 2,0000 MMS. The 1-0-10 monoclonal antibody did not react with the 2,000 0 MMS. These findings point to a close relationship between the two molecules and is the current focus of active investigation in our laboratory. Taken together, these data strongly suggest that MMS-68 plays an important role in the regulation of mucus secretion. We have demonstrated its presence in normal peripheral blood monocytes, pulmonary macrophages, and normal airways. The increased levels of MMS-68 observed in BALF in cigarette smokers may contribute to the hypersecretion of mucus that is seen in these patients, some of whom complained of mucus hypersecretion and cough. The increased spontaneous production of MMS-68 from peripheral blood monocytes seen in steroiddependent asthma may represent the preactivation state of monocytes in this subgroup of asthma. Further studies are required to delineate the role of MMS68 in airway inflammation.

Acknowledgment We are also grateful for the excellent technical help of Josephina Hinojosa and Andrew Pizzimenti.

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