Human lung fibroblasts release chemokinetic activity for monocytes constitutively

Sekiya Koyama1,2, Etsuro Sato1, Tsuyoshi Masubuchi1, Akemi Takamizawa1, Hiroshi Nomura1, Keishi Kubo1, Sonoko Nagai2, and Takateru Izumi2

1 Shinshu University School of Medicine, First Department of Internal Medicine, Matsumoto 390; and 2 Kyoto University Chest Disease Research Institute, Kyoto 606-01, Japan

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

We determined whether human lung fibroblasts (HLFs) might release mediators that are responsible for monocyte chemokinetic activity (MCA) constitutively. HLF supernatant fluids showed MCA in a time-dependent manner (P < 0.001). Checkerboard analysis of 24- and 72-h supernatant fluids showed that the activity was chemokinetic. Partial characterization of 24- and 72-h supernatant fluids revealed that the mediators released after 24 h were predominantly composed of lipid-soluble activity, and MCA was blocked by lipoxygenase inhibitors. The mediators released after 72 h were predominantly trypsin sensitive and blocked by cycloheximide. Molecular-sieve column chromatography identified four peaks of MCA. A polyclonal antibody to monocyte chemoattractant protein-1 (MCP-1) inhibited MCA by 20% after 24 h and by 40% after 72 h. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor-beta (TGF-beta ) antibodies attenuated MCA released after 72 h by 30 and 10%, respectively. These antibodies inhibited corresponding molecular-weight peaks separated by molecular-sieve column. The concentrations of MCP-1, GM-CSF, and TGF-beta were 4,698 ± 242, 26.8 ± 3.8, and 550 ± 15 pg/ml, respectively. A leukotriene B4 (LTB4)-receptor antagonist attenuated the total MCA and the lowest molecular weight peak of MCA. The concentrations of LTB4 were 153.4 ± 12.4 (24 h) and 212 ± 16.6 (72 h) pg/ml. These findings suggest that HLFs may modulate the recruitment of monocytes into the lung by releasing MCP-1, GM-CSF, TGF-beta , and LTB4 constitutively.

monocyte chemoattractant protein-1; granulocyte-macrophage colony-stimulating factor; transforming growth factor-beta ; leukotriene B4

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE NORMAL LUNG contains a resident population of macrophages within intravascular, interstitial, and alveolar spaces. These cells have a physiological role as part of the host defense of the lung (25, 42). Unlike other residential macrophages, they may be less important as antigen-presenting cells but may retain an important immune function (13, 14, 33). The macrophage may have an influence both as an activating cell of the immune process and as a cell that can terminate inflammatory responses. Although proposed mechanisms responsible for persistent maintenance of these phagocytic cells within the lung include chemotaxis (6, 18), local proliferation (1, 11), and migration inhibition (42), macrophages predominantly originate from peripheral blood monocytes that migrate into the lung (2, 18, 39, 41).

The fibroblast is the principal cell of most connective tissues and is involved in constituting collagenous and noncollagenous components of the extracellular matrix. This synthetic activity serves an important structural function by providing a frame network for organ integrity. In addition to this traditionally accepted function, recent studies have demonstrated that fibroblasts not only serve to maintain the connective tissue but are important participants in the orchestration of acute and chronic inflammation. In this context, fibroblasts released monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and transforming growth factor-beta (TGF-beta ) in response to interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha ), and platelet-derived growth factor (PDGF), suggesting a contribution to certain disease states (22, 23, 26, 36, 46). Therefore, the fibroblast, because of its anatomic location, is in a pivotal position to participate in and to direct bidirectional communications between interstitial and vascular events.

Koyama and colleagues (20, 21) previously reported that type II pneumocytes and bronchial epithelial cells release monocyte chemoattractant activity constitutively, suggesting participation of these cells in monocyte recruitment as an origin of alveolar macrophages in the steady state. Although these epithelial cells may play a role in monocyte migration from the interstitium to the alveolar and bronchial spaces, the underlying mechanisms of monocyte migration from the vascular compartment to the interstitium remains to be elucidated. The role of human lung fibroblasts (HLFs) in monocyte recruitment from the vascular compartment to the interstitium in the steady state is uncertain.

The objective of the present study was to determine whether HLFs may release mediators that are responsible for monocyte chemokinetic activity (MCA) constitutively. The results demonstrated that HLFs released MCA and that MCA involved GM-CSF, TGF-beta , MCP-1, and leukotriene B4 (LTB4). The present investigation suggested that HLFs may contribute to the maintenance of host defense and the lung immune environment by recruiting monocytes into the lung interstitium.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Preparation of HLFs. We used fetal HLFs (diploid, passage 27), which were established as a cell line and commercially available (American Type Culture Collection, Rockville, MD). HLFs were suspended at 1.0 × 106 cells/ml in F-12 medium supplemented with penicillin (50 U/ml; GIBCO BRL, Grand Island, NY), streptomycin (50 µg/ml; GIBCO), Fungizone (2 µg/ml; GIBCO), and 10% FCS (GIBCO). HLF suspensions (3 ml) were added to a 30-mm-diameter tissue culture dish (Corning, Corning, NY) and cultured at 37°C in a 5% CO2 atmosphere. After 4-6 days in culture, the cells had reached confluence, and then the culture medium was replaced with 2 ml of medium supplemented as above and incubated for 1 more day. On the following day, the culture was washed two times; the culture medium was replaced with 1 ml of serum-free F-12 medium with penicillin, streptomycin, and Fungizone; and the culture supernatant fluids were harvested at 12, 24, 48, 72, and 96 h and frozen at -80°C until assayed for MCA. At least six separate HLF culture supernatant fluids were harvested for each experiment.

Monocyte chemotaxis assay. Blood was drawn from healthy adults into heparinized syringes by venipuncture. Mononuclear cells were obtained for monocyte chemotaxis by Ficoll-Hypaque density centrifugation (Histopaque-1077, Sigma) to separate the red blood cells and neutrophils from the mononuclear cells. The mononuclear cells were harvested at the interface, and the red blood cells were lysed by suspending the cells in a solution consisting of 0.1% KHCO3 and 0.83% NH4Cl. The suspension was then centrifuged at 400 g for 10 min and washed three times in Hanks' balanced salt solution (Biofluids, Rockville, MD). The preparation routinely consisted of 30% large monocytes and 70% small lymphocytes as determined by morphology and alpha -naphthyl acetate esterase staining (Sigma), with >98% viability as assessed by trypan blue and erythrocin exclusion. The cells were suspended in Gey's balanced salt solution (GIBCO) containing 2% BSA (Sigma) at pH 7.2 to give a final concentration of 5.0 × 106 cells/ml. This suspension was then used in the chemotaxis assay.

The chemotaxis assay was performed in duplicate in 48-well microchemotaxis chambers (NeuroProbe, Cabin John, MD) as described previously (8). The bottom wells of chambers were filled with 25 µl of fluid containing the chemotactic stimulus. A polycarbonate filter with a pore size of 5 µm (Nuclepore, Pleasanton, CA) was placed over the bottom wells. The silicon gasket and top pieces of the chambers were applied, and the entire assembly was preincubated at 37°C in humidified air for 15 min before the top wells were filled with 50 µl of the cell suspension. The chamber was incubated for 90 min in 5% CO2 in humidified air at 37°C. After incubation, the chamber was disassembled, and the filter was removed. The filter was then fixed, stained with Diff-Quik (American Scientific Product, McGaw Park, IL), and mounted on a glass slide. Mononuclear cells that completely migrated through the membrane were counted in 10 random high-power fields (HPFs; ×1,000). Chemotactic response was defined as the mean number of migrated cells per HPF for each group. F-12 medium supplemented as above but without FCS and incubated identically to the HLF cultures was used to determine background monocyte migration. Formyl-methionyl-leucyl-phenylalanine (10-7 M in supplemented F-12 medium; Sigma) and normal human serum that was activated by incubation with bacterial endotoxin (Escherichia coli lipopolysaccharide 0127:B8; DIFCO, Detroit MI) and diluted 10-fold with F-12 medium were used as positive controls (21).

To determine whether the migration of monocytes was because of movement along a concentration gradient (chemotaxis) or stimulation of the monocytes to randomly migrate (chemokinesis), a checkerboard analysis was performed (47). To do this, various concentrations of HLF supernatant fluid (1:1, 1:4, 1:16, 1:64, and 1:254) were placed with mononuclear cells above the membrane and supernatant fluids with similarly diluted concentrations were placed below the membrane. Thus monocyte migrations were tested by a variety of concentration gradients of supernatant fluid across the membrane.

To ensure that monocytes, but not lymphocytes, were the primary cells that migrated, some membranes were stained with alpha -naphthyl acetate esterase according to the manufacturer's directions (Sigma).

Partial characterization of MCA. Because MCA could be detected in HLF culture supernatant fluid, a partial characterization of the released activity was performed with supernatant fluid harvested at 24 and 72 h. Sensitivity to protease was tested by incubating the HLF culture supernatant fluid with trypsin (final concentration 100 µg/ml; Sigma) for 30 min at 37°C followed by the addition of a 1.5 M excess of soybean trypsin inhibitor to terminate the proteolytic activity before the chemotaxis assay. The lipid solubility of the activity was evaluated by mixing the HLF supernatant fluid two times with ethyl acetate, decanting the lipid phase after each extraction, evaporating the ethyl acetate to dryness, and resuspending the extracted material in the F-12 medium used for cell culture before the chemotaxis assay. Heat sensitivity was determined by heating the culture supernatant fluid at 98°C for 30 min.

To further examine the release of MCA, cycloheximide (10 µg/ml; Sigma) was added to the supplemented F-12 medium without FCS to inhibit protein synthesis (9), and nordihydroguaiaretic acid (NDGA; 100 µM; Sigma), diethylcarbamazine (DEC; 1 mM; Sigma), and AA-861 (100 µM; Takeda Pharmaceutical, Tokyo, Japan) were added to inhibit lipoxygenase-derived arachidonic acid metabolite release (16). The HLF culture was incubated with cycloheximide and the lipoxygenase inhibitors for 24 and 72 h and harvested, and the supernatant fluid was assayed for MCA.

To determine the approximate molecular weight of the released MCA, molecular-sieve column chromatography was performed with Sephadex G-100 (75 × 1.75 cm; Pharmacia, Piscataway, NJ) at a flow rate of 12 ml/h. The HLF cell supernatant fluid was eluted with phosphate-buffered saline, and every fraction after the void volume was evaluated in duplicate for monocyte chemotactic activity.

Effects of LTB4- and platelet-activating factor-receptor antagonists. LTB4-receptor (ONO-4057, Ono Pharmaceutical, Tokyo, Japan) and platelet-activating factor (PAF)-receptor (TCV-309, Takeda Pharmaceutical) antagonists at a concentration of 10-5 M were used to evaluate the responsible chemotactic activity in the supernatant and column chromatography-separated lowest molecular weight peak. These receptor antagonists completely blocked the chemotactic responses of 10-7 M LTB4 and PAF but did not have any influence on the chemotactic response of monocytes to endotoxin-activated serum (data not shown).

Measurement of LTB4 and PAF by radioimmunoassay. The measurement of LTB4 and PAF in HLF supernatant fluid harvested at 24 and 72 h was performed by RIA, as previously described (21), with anti-LTB4 serum, [5,6,8,9,11,12,14,15-3H]- LTB4, and synthetic LTB4, which were purchased from Amersham (Arlington Heights, IL). Briefly, ethanol samples were centrifuged at 5,500 g at 0°C. The supernatants were evaporated under N2 gas at 37°C. To each sample, 10 ml of distilled water were added. These samples were acidified to pH 4.0 with 0.1 N HCl and were applied to Sep-Pak C18 columns (Waters Associates, Milford, MA); the columns were washed with 10 ml of distilled water and 20 ml of petroleum ether and then were eluted with 15 ml of methanol. These eluates were dried with N2 gas at 37°C and redissolved in 20 µl of methanol and 180 µl of RIA buffer [50 mM Tris · HCl buffer containing 0.1% (wt/vol) gelatin, pH 8.6]. [3H]LTB4 was diluted in RIA buffer, and 100-µl aliquots containing 4,000 disintegrations/min were mixed with 100 µl of standard or sample in siliconized tubes. Anti-LTB4 serum diluted in RIA buffer (100 µl) was added to give a total incubation volume of 400 µl, and the mixture was incubated at 4°C for 18 h. Free LTB4 was adsorbed onto dextran-coated charcoal. The supernatant containing the antibody-bound LTB4 was decanted into a scintillation counter after centrifugation at 2,000 g for 15 min. Scintillation fluid was added, and the radioactivity was counted by a scintillation counter (Tricarb-3255, Packard, Downers Grove, IL) for 4 min.

PAF concentration in the supernatant fluid was measured by a scintillation-proximity assay system. This system combined the use of a high specific activity tritiated-PAF tracer with an antibody specific for PAF and a PAF standard, similar to the methods used for the measurement of LTB4.

Effects of regulated on activation normal T cells expressed and secreted, GM-CSF, TGF-beta , and MCP-1 polyclonal antibodies on the released chemotactic activity. The neutralizing antibodies to regulated on activation normal T cells expressed and secreted (RANTES), GM-CSF, TGF-beta , and MCP-1 (purchased from Genzyme, Cambridge, MA) were added to the supernatant fluids harvested at 24 and 72 h at the suggested concentrations to inhibit these cytokines and incubated for 30 min at 37°C. Then these samples were used for chemotactic assay. These antibodies did not influence the chemotactic response to endotoxin-activated serum (data not shown).

Measurement of RANTES, GM-CSF, TGF-beta , and MCP-1 concentrations in the supernatant fluid. The concentrations of RANTES, GM-CSF, TGF-beta , and MCP-1alpha in the supernatant fluids harvested at 24 and 72 h were measured by ELISA according to the manufacturer's directions. GM-CSF and RANTES kits were purchased from Amersham (Amersham, UK), and the minimum concentration detected by these methods was 2.00 pg/ml for GM-CSF and 15.6 pg/ml for RANTES. MCP-1 and TGF-beta kits were purchased from R&D Systems (Minneapolis, MN), and the minimum concentration for MCP-1 and TGF-beta was 31.3 pg/ml and 0.31 ng/ml, respectively.

Statistical analysis. Differences in the chemotactic activity were tested for significance with a two-tailed Student's paired t-test. A P value < 0.05 was considered significant. Data are expressed as means ± SD.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Release of MCA from HLFs. Six different HLF culture supernatant fluids were evaluated. Each culture contained MCA that was significantly increased compared with the supplemented F-12 medium alone (P < 0.001; Fig. 1). MCA was released into the culture medium in a time-dependent manner, reaching a plateau at 72 h (Fig. 2). Checkerboard analysis revealed that supernatant fluids harvested after both 24 and 72 h of culture induced increasing migration with increasing concentrations of supernatant fluid in the absence of a gradient across the membrane (data not shown). Thus MCA in both the 24- and 72-h supernatant fluids was consistent with chemokinetic activity rather than chemotactic activity.


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Fig. 1.   Demonstration that human lung fibroblasts (HLFs) release monocyte chemokinetic activity (MCA). CONT, supplemented F-12 medium alone (control); FMLP, formyl-methionyl-leucyl-phenylalanine. Dots connected by a line are matching monocyte chemotaxis experiments from 6 different HLF culture supernatant fluids.


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Fig. 2.   Time course of MCA release from HLF cultures. Values are means ± SE; n = 7 experiments. MCA increased in a time-dependent manner and reached a plateau at 72 h. * P < 0.05 compared with F-12 medium (0 h).

Confirmation that monocytes were the primary cells attracted was provided by the following three lines of evidence: 1) >90% of the migrated cells appeared to be monocytes by light microscopy, 2) >90% of the migrating cells were esterase positive, and 3) lymphocytes prepared by incubating mononuclear cells recovered by Ficoll-Hypaque density centrifugation on plastic dishes (Costar no. 3424, Costar, Cambridge, MA) for 60 min at 37°C and tested for chemotactic activity as above resulted in 0-20% of the activity of the mononuclear cells not depleted of monocytes.

Partial characterization of MCA. The MCA was heterogeneous in its character. The MCA released at 24 h was predominantly sensitive to heat, extractable into ethyl acetate, and partially digested by trypsin (Fig. 3A). In contrast, MCA released at 72 h was predominantly sensitive to heat, digested by trypsin, and partially extractable into ethyl acetate (Fig. 3B). Incubation of HLFs with cycloheximide inhibited the release of MCA more potently at 72 h than at 24 h. The nonspecific lipoxygenase inhibitors NDGA and DEC and the 5-lipoxygenase inhibitor AA-861 inhibited the release of MCA at 24 h rather than at 72 h (Fig. 4). Cycloheximide, NDGA, DEC, and AA-861 themselves did not inhibit monocyte migration in response to HLF supernatant fluid harvested after 72 h of incubation (data not shown). Thus the effects of these agents were due to the inhibition of the release of MCA.


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Fig. 3.   Partial characterization of MCA release from HLFs. A: supernatant fluid harvested at 24 h of incubation. B: supernatant fluid harvested at 72 h of incubation. F-12, medium alone. Values are means ± SE; n = 6 experiments. * P < 0.01 compared with crude supernatant fluid.


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Fig. 4.   Effects of incubation of HLFs with cycloheximide, nordihydroguaiaretic acid (NDGA), diethylcarbamazine (DEC), and AA-861 on release of MCA. A: supernatant fluid harvested at 24 h of incubation. B: supernatant fluid harvested at 72 h of incubation. Values are means ± SE; n = 7 experiments. * P < 0.01 compared with crude supernatant fluid.

Molecular-sieve column chromatography revealed that MCA was heterogeneous in size (Fig. 5). At least four peaks of MCA were separated by column chromatography, with the estimated molecular weight before that of BSA and slightly before that of cytochrome c (mol wt 12,384) and with an additional peak that eluted near quinacrine (mol wt 565). At 72 h of incubation, the second and third molecular-weight peaks became prominent (Fig. 5B).


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Fig. 5.   Molecular-sieve column chromatography with Sephadex G-100 of HLF supernatant fluid harvested after 24 (A) and 72 h (B) of incubation. Arrows and nos. on top, position and molecular weight, respectively, of markers for BSA (66,000), cytochrome c (12,400), and quinacrine (450). This is a representative profile of 4 experiments.

Effects of LTB4- and PAF-receptor antagonists on MCA and concentrations of LTB4 and PAF in supernatant fluid. LTB4 and PAF were potent chemotactic activators for monocytes; thus the effects of LTB4- and PAF-receptor antagonists were evaluated. LTB4-receptor antagonists inhibited the total MCA to 60% released after 24 h and to 40% released after 72 h in the supernatant fluid (Fig. 6) and also inhibited the column chromatography-separated lowest molecular weight peak of MCA released after 72 h to 60% (34.5 ± 2.4 vs 16.3 ± 3.2 cells/HPF; P < 0.001). The release of LTB4 in the supernatant fluid after 24 and 72 h of incubation was 154.3 ± 12.4 and 212.0 ± 16.6 pg/ml, respectively. These concentrations of synthetic LTB4 induced monocyte migration (10-9 M, 35.4 ± 5.6 cells/HPF; 10-10 M, 27.3 ± 4.3 cells/HPF). In contrast, the PAF-receptor antagonist did not block the chemotactic response, and PAF was not detected in both the 24- and 72-h supernatant fluids.


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Fig. 6.   Effects of leukotriene B4 (LTB4)- and platelet-activating factor (PAF)-receptor antagonists on supernatant fluid harvested at 24 (A) and 72 h (B) of incubation. Values are means ± SE; n = 6 experiments. * P < 0.01 compared with untreated supernatant fluid.

Effects of GM-CSF, RANTES, TGF-beta , and MCP-1 antibodies on MCA. The effects of blocking polyclonal antibodies to GM-CSF, RANTES, TGF-beta , and MCP-1 on MCA released after 24 h revealed that the MCP-1 antibody significantly inhibited MCA in the supernatant fluid (Fig. 7A). The polyclonal antibodies to MCP-1, GM-CSF, and TGF-beta attenuated MCA in 72-h supernatant fluid up to 40, 30, and 10%, respectively (Fig. 7B). In contrast, RANTES antibodies did not show any effects for both the 24- and 72-h supernatant fluids. Furthermore, the corresponding molecular-weight peaks of MCA after 72 h, as separated by a molecular-sieve column, were inhibited by MCP-1, GM-CSF, and TGF-beta polyclonal antibodies up to 60-70%, respectively.


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Fig. 7.   Effects of monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-beta (TGF-beta ), granulocyte-macrophage colony-stimulating factor (GM-CSF), and regulated on activation normal T cells expressed and secreted (RANTES) polyclonal antibodies on released MCA released after 24 (A) and 72 h (B). Values are means ± SE; n = 6 experiments. * P < 0.01 compared with untreated supernatant fluid.

Concentrations of GM-CSF, RANTES, TGF-beta , and MCP-1 in the supernatant fluid. The concentrations of MCP-1, GM-CSF, and TGF-beta evaluated by ELISA in the supernatant fluid after 24 and 72 h of incubation were 1,863 ± 214, 7.3 ± 2.1, and <310 pg/ml (not detected) (24 h) and 4,698 ± 242, 26.8 ± 3.8, and 550 ± 15 pg/ml (72 h), respectively. There was no detectable amount of RANTES in the supernatant fluid harvested at both 24 and 72 h.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The present study demonstrates that HLFs can release MCA. The partial characterization of MCA revealed that the activity was composed of a low- molecular-weight lipid-soluble activity and high-molecular-weight peptides after both 24 and 72 h in culture. The MCA was partly inhibited by MCP-1, GM-CSF, and TGF-beta antibodies and an LTB4-receptor antagonist. The concentration of LTB4 was almost 10-9 M. MCP-1, GM-CSF, and TGF-beta were detected by ELISA of the supernatant fluid. These concentrations were high enough for monocyte migration. These data suggest that lung fibroblasts may modulate their local environment by releasing MCA.

Previous studies addressing monocyte influx into the alveolar space have yielded conflicting results. Collins and Auclair (5), studying unstimulated parabiotic rats, found no evidence for radiolabeled monocyte influx into the lungs of the recipient rat after injecting the donor rat monocytes with tritiated thymidine. Other studies have found no changes in alveolar macrophage cell number in animals with radiation-induced monocytopenia (32), also suggesting that the normal alveolar macrophage population is at least partially independent of circulating blood monocytes. Similar observations have been made in humans with severe monocytopenia (37, 38). In contrast, Blusse Van Oud Albas and Van Furth (2), employing multiple injections of tritiated thymidine into normal mice, observed simultaneous radiolabeling in the blood monocyte and alveolar macrophage populations. This coincident radiolabeling was interpreted to mean that the labeled alveolar macrophages were directly derived from the blood monocytes. Bowden and Adamson (3) radiolabeled normal mice with multiple injections of tritiated thymidine and observed heavily labeled cells among the alveolar macrophages. Furthermore, support for a bone marrow origin of alveolar macrophages has come from cell marker studies in mouse radiation chimeras and from studies in humans after bone marrow and heart-lung transplantation (9, 28, 39). Our study demonstrated that MCA released from HLFs facilitated the monocyte recruitment into the lung, supporting the concept that alveolar macrophages are at least partly derived from circulating blood monocytes.

Macrophages may have an influence as an activating cell of the immune responses by weakly presenting antigens to lymphocytes during the development of specific immunity (31, 40) and releasing IL-1 (13, 14), a mediator that is crucial for the initiation of lymphocyte activation. Previous studies (22, 35, 36, 46) have shown that dermal and synovial fibroblasts can release soluble chemotactic factors that direct the migration of neutrophils and monocytes into the alveolar space in response to TNF-alpha , IL-1, and PDGF, and the present study demonstrated that HLFs also release MCA constitutively. In this context, HLFs may modulate their local immunologic environment by releasing chemotactic activity for both neutrophils and monocytes.

The lung population of macrophages continually exits from the interstitium to the airway (10). This perpetual movement of phagocytic cells from the alveolar lesions cleanses the lung and aids in the maintenance of lung sterility (4, 12). Forces that attract macrophages to the interstitium and direct macrophages from the interstitium to the distal alveolar surface areas and further to the central airways are poorly understood. Type II pneumocytes and bronchial epithelial cells release chemoattractant activity for alveolar macrophages (20, 21), which may serve as one of the cellular sources of lung lining material and may attract alveolar macrophages toward the central airways. In contrast, HLFs may release MCA and may maintain the population of macrophages in the lung interstitium.

The identification of MCA released from HLFs is not complete. However, the trypsin sensitivity of MCA along with the inhibition of the release by cycloheximide treatment suggests that the activity is at least partly dependent on protein synthesis (10). In contrast, extractability of MCA into ethyl acetate along with the inhibition of release by NDGA, DEC, and AA-861 suggests that the activity is composed of lipid. The released activity was attenuated by antibodies to MCP-1, GM-CSF, and TGF-beta . The lipid-extractable activity was inhibited by the LTB4-receptor antagonist. The concentrations of MCP-1, GM-CSF, and TGF-beta in the supernatant fluid reached the concentrations as monocyte chemotactic activity (34, 43, 44). Thus HLFs at least partly released LTB4, MCP-1, GM-CSF, and TGF-beta as responsible MCA.

Production of chemotactic factors for monocytes by cells other than HLFs may also be important in regulating monocyte recruitment to the lung. Numerous cells that produce monocyte chemotactic activity are present in the lung, and a wide variety of molecules including fibronectin fragments (27), elastin fragments (17), collagen and collagen fragments (29), complement-derived chemoattractants (20), and surfactant proteins (15, 45) has been identified that may serve as chemoattractants in certain lung disorders. One cell that can release monocyte chemotactic activity is the alveolar macrophage, which can release PDGF (7, 24), GM-CSF (34), TGF-beta (43), and lipoxygenase products (25). Other cells associated with lung tissues, such as bronchial epithelial cells, type II pneumocytes, and lymphocytes, may also release MCA, including TGF-beta , GM-CSF, MCP-1, RANTES and lipoxygenase products (19-21, 30). Further studies clarifying the role of HLFs and purifying and identifying MCA produced by HLFs should make the relevance of these known chemoattractants clear. Furthermore, the actual migratory responses of monocytes in a complex biological fluid such as lung epithelial lining fluid likely involves complex interactions among factors. It is reasonable to suggest that interactions among chemokinetic, chemotactic, and migration inhibitory factors may determine monocyte movement in vivo (42).

The present investigation demonstrates that HLFs can release MCA constitutively. Although these data are derived from HLFs in in vitro conditions, HLFs may contribute to the maintenance of host defense by modulating the population of lung macrophages.

    FOOTNOTES

Address for reprint requests: S. Koyama, First Department of Internal Medicine, Shinshu Univ. School of Medicine, 3-1-1 Asahi, Matsumoto 390, Japan.

Received 21 July 1997; accepted in final form 27 April 1998.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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