Introduction

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Chronic inflammation and fibrosis are tissue responses that may occur throughout the tracheobronchial tree and the lung parenchyma. Infiltrating cells such as lymphocytes, mast cells, eosinophils, and monocytes are traditionally considered as fully involved in the pathophysiologic events that underlie these responses. In this context, fibroblasts have been regarded as target cells, simply responsible for extracellular matrix protein production and reparative processes. Today, however, a great deal of evidence is available showing that fibroblasts are able to produce cytokines, chemokines, and lipid mediators such as prostaglandins; and, through the release of these soluble signals, they may initiate, amplify, and above all, modulate some of the events related to the inflammatory response (1). We have recently provided evidence that normal human lung fibroblasts, through the release of PGE₂, inhibit, at molecular and cellular levels, the production by peripheral blood monocytes (PBM) of tumor necrosis factor (TNF)-, a cytokine known to affect several fibroblast biologic activities, suggesting a bidirectional and mutual interaction between fibroblasts and monocytes (6). On the other hand, monocytes modulate the inflammatory response by complex and multifactorial processes involving the release of chemokines, growth factors, and a number of cytokines, some of which are crucial for the regulation of the immune response. Among these, interleukin (IL)-10 and IL-12 have a great impact on immune regulation (9). IL-10, by inhibiting interferon (IFN)-, may promote the commitment from the Th0 to the Th2 phenotype. It also suppresses proinflammatory cytokine production, through the selective inhibition of NF-B activation, and reduces the antigen-presenting capacity of monocytes/macrophages and dendritic cells (10). IL-12, on the other hand, promotes growth and activation of natural killer (NK) cells and T-lymphocytes and, by stimulating IFN- secretion, induces the commitment from the Th0 to the Th1 phenotype (13). It appears evident that the homeostatic balance between these two cytokines is critical in the regulation of the immune response. On these bases we thought it important to determine whether lung fibroblasts influence the immune response by regulating the production of IL-10 and IL-12 by monocytes, and whether this activity is modified in fibrotic lung fibroblasts. To address this question, we examined the effect of fibroblast-conditioned medium (FCM) produced by normal (NFCM) and fibrotic (FFCM) human lung fibroblasts on the expression and production of IL-10 and IL-12 by lipopolysaccharide (LPS)-activated monocytes.

Our results indicate that NFCM inhibits IL-12 and strongly stimulates IL-10 release by monocytes, effects that are mediated by prostaglandin (PG)E₂. The increase of IL-10 production, induced by NFCM, is able to affect, in an autocrine way, the antigen-presenting capacity of monocytes via downregulation of the expression of class II major histocompatibility complex (MHC) molecules as well as that of the costimulatory molecule CD40. In addition, our data indicate that fibroblasts coming from fibrotic tissue have a significantly reduced capacity to stimulate the production of IL-10 and, as a consequence, a diminished capacity to downregulate the expression of CD40 on monocytes. The current study demonstrates that fibroblasts, in addition to their traditional role of target cell, play an active role in the modulation of inflammation and, most importantly, in the regulation of the immune response.

	Materials and Methods

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Fibroblast Cultures

Primary lines of normal human adult lung fibroblasts were established by using an outgrowth from explant according to the method described by Jordana and coworkers (14). Fibroblast lines were derived from histologically normal areas of surgical lung specimens from patients undergoing resective surgery for cancer. Their ages ranged from 52 to 61 yr. Five of six patients were men. Lung specimens were chopped into pieces of less than 1 mm³ and washed once with phosphate-buffered saline (PBS) and twice with RPMI containing 10% fetal calf serum (FCS), penicillin 100 U/ml, streptomycin 100 µg/ml, and fungizone 25 µg/ml (supplemented RPMI) (Gibco, Paisley, UK); eight to ten pieces of washed specimens were then plated in a 100-mm polystyrene dish (Falcon, Becton-Dickinson, Lincoln Park, NJ) and overlaid with a coverslip held to the dish with sterile vaseline. Ten milliliters of supplemented RPMI were added and the tissue was incubated at 37°C with 5% CO₂. The medium was changed weekly. When a monolayer of fibroblast-like cells covered the bottom of the dish, usually 5 to 6 wk later, the explant tissue was removed, and the cells were then trypsinized for 10 min, resuspended in 10 ml of supplemented RPMI, and plated in 100-mm tissue culture dishes. Subsequently, cells were split 1:2 at confluence, usually weekly. Aliquot of cells were frozen and stored in liquid nitrogen. Fibrotic lung fibroblast lines were established from histologically proven fibrotic lung tissue of patients with interstitial lung disease and were kindly provided by Dr. Jordana (Department of Pathology, McMaster University, Hamilton, ON, Canada). A characterization of all fibrotic cells lines used in the present study has been previously reported (14). The ages of patients with lung fibrosis ranged from 45 to 55 yr, except one patient aged 12 yr. In the considered parameters, the cell line derived from this patient did not behave differently compared with the other cell lines and for this reason was included in the study. In all experiments we used cell lines at a passage earlier than the tenth.

Monocyte Isolation Procedure

Heparinized venous blood, obtained from healthy donors, was diluted 1:4 with PBS, and 40 ml were then placed on 10 ml of Ficoll-Hipaque (Sigma, St. Louis, MO) for centrifugation at 1,600 rpm for 20 min at room temperature. Mononuclear cells were collected at the interface, washed twice, and resuspended in PBS supplemented with 0.5% bovine serum albumin and 2 mM ethylenediamine tetraacetic acid. Isolation of human monocytes from mononuclear cells was performed by depletion of non-monocytes using a magnetic cell sorting system (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer's instructions. T cells, NK cells, B cells, dendritic cells, and basophils were indirectly magnetically labeled using a cocktail of hapten-coniugated CD3, CD7, CD19, CD45RA, CD56, and anti-IgE antibodies and magnetic microbeads coupled to an anti-hapten monoclonal antibody. The magnetically labeled cells were depleted by retaining them on a column in the magnetic field of the MACS system. Unlabeled cells, representing the enriched monocyte fraction, passed through the column and were collected as effluent.

Monocyte Cultures

PBM were incubated in 24-well tissue culture plates (Falcon, Becton-Dickinson) at a concentration of 2.5 × 10⁵ cells in 1 ml of either FCM or supplemented RPMI. All experiments were performed to make sure that PBM from the same donor were incubated with FCMs from both normal and fibrotic cultures. LPS from Escherichia coli 0.26:B6 (Sigma) was immediately added (1 µg/ml) and the plates incubated in a humidified atmosphere of 5% CO₂ at 37°C. After 18 h supernatants were harvested, centrifuged, stored in 0.5 ml aliquots at 80°C, and then assayed for IL-10 and IL-12. This specific experimental procedure was chosen based on the inconsistent levels of cytokines observed under basal conditions that were either unmeasurable or in the very low range of detection (data not shown). FCM, however, slightly increased the release of IL-10 also from unstimulated monocytes, but did not affect the secretion of IL-12 (data not shown).

Fibroblast-Conditioned Medium

Normal and fibrotic FCM were generated from cultures of 2 × 10⁶ fibroblasts incubated for 24 h in 10 ml of supplemented RPMI. In a number of experiments fibroblasts were cultured in the presence of indomethacin (50 µM) (Sigma). Supernatants were centrifuged and stored in aliquots at 80°C until use.

IL-10 and IL-12 Assay

Concentrations of IL-10 and IL-12 in PBM and fibroblast supernatants were determined by a commercially available enzyme immunoassay (BioSource International, Camarillo, CA). The assay is sensitive to 5 pg/ml of IL-10 and to 1 pg/ml of IL-12. Both assays were performed according to the manufacturer's instructions and all samples were determined in duplicate.

RNA Isolation and Reverse Transcriptase-Polymerase Chain Reaction

Monocytes, purified as described earlier, were plated in a 100-mm tissue culture dish with a ratio of 5 × 10⁵ cells/ml of either FCM or supplemented RPMI. LPS was immediately added (1 µg/ml) and the plates incubated in a humidified atmosphere of 5% CO₂ at 37°C. After 2 h of incubation, total cellular RNA was extracted from PBM with the guanidium isothiocyanate/acid-phenol procedure as previously described (15). The yield and the purity of RNA was measured spectrophotometrically by absorption at 260/280 nm. Total RNA was used for the generation of cDNA. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using the Super preamplification System (Gibco), with some modifications. Briefly, 1 µg of total RNA was reverse transcribed with 200 U of RNase reverse transcriptase (Superscript II RT; Gibco). The reverse-transcribed product (cDNA) was amplified by PCR (Gene Amp PCR System 2400; Perkin-Elmer, Foster City, CA) in the presence of a master mix containing PCR buffer, MgCl₂ (under optimal concentrations), 1.5 U Taq DNA Polymerase Recombinant (Gibco), and 10 mM dNTPs. The following specific primer pairs were used: IL-10 sense 5'-CTG-AGA-ACC-AAG-ACC-CAG-ACA-TCA-AGG-3' and antisense 5'-CAA-TAA-GGT- TTC-TCA-AGG-GGC-TGG-3' (to amplify a 352 bp fragment); p40 subunit of IL-12 sense 5'-CCA-AGA-ACT-TGC-AGC-TGA- AG-3' and antisense 5'-TGG-GTC-TAT-TCC-GTT-GTG-TC-3' (297 bp); -actin sense 5'-TGA-CGG-GGT-CAC-CCA-CAC-TGT- GCC-CAT-CTA-3' and antisense 5'-CTA-GAA-GCA-TTG-CGG- TGG-ACG-ATG-GAG-GG-3' (661 bp). Reactions were performed under the following conditions: IL-10: 60'' 95°C, 45''60°C, 45'' 72°C (30 cycles); IL-12: 45'' 95°C, 60'' 65°C, 105'' 72°C (35 cycles). Final extension was at 72°C for 7 min for both cytokines. PCR-amplified products (10 µl) were electrophoresed through a 1.8% agarose gel (Ambion Inc., Austin, TX) containing 0.5 µg/ml of ethidium bromide and compared with DNA reference markers. Products were visualized by UV illuminations. Polaroid photographs with UV exposure were taken with a 665 Polaroid film (Polaroid, St. Albans, Hertfordshire, UK). Bands were analyzed with the Phoretix 1D version 3.0 (Phoretix International, Newcastle upon Tyne, UK).

Flow Cytometric Analysis of Human Leukocyte-Associated Antigen-DR and CD40

Experiments to determine CD40 and human leukocyte-associated antigen-DR (HLA-DR) expression were performed on monocytes isolated from peripheral blood from healthy subjects, isolated as described above. Monocytes were plated at a concentration of 5 × 10 ⁵ cells/ml with or without NFCM or FFCM. Next, monocytes were treated with LPS (1 µg/ml) and subsequently with 500 U/ml of IFN- (Peprotech LTD, London, UK) to stimulate the expression of HLA-DR and CD40. To investigate the role of FCM-induced secretion of IL-10 from monocytes, some experiments have been performed either in the presence of a neutralizing anti IL-10 antibody (60 µg/ml) (PharMingen International, San Diego, CA) or its relative control (rat IgG₂) used at the same concentration.

Twenty-four hours later monocytes were lightly trypsinized, washed, and resuspended in PBS with 0.1% BSA. The cells were incubated with primary antibodies, anti-CD40 mAb (PharMingen) or anti HLA-DR mAb (PharMingen) for 60 min at room temperature. Following washing, the secondary antibody, fluorescein isothiocyanate (FITC)-conjugated rabbit antimouse IgG, was added for 60 min at room temperature. Controls included omission of the primary antibody and incubation only with the secondary antibody. Samples were analyzed using a Coulter Epics Elite ESP flow cytometer (Coulter Corporation, Miami, FL). At least 10,000 forward and side scatter gated events were collected per specimen. Cells were excited at 488 nm and the fluorescence was monitored at 525 nm. FITC fluorescence was collected using logarithmic amplification.

Immunocytochemistry

Cytocentrifuge slides for immunocytochemistry were prepared with isolated monocyte populations. The slides were allowed to air-dry overnight at room temperature, then fixed in acetone for 20 min at 20°C, and additionally air-dried for 1 h. The slides were then wrapped in pairs in aluminum foil and stored at 20°C before immunostaining. To rehydrate monocyte cytospins, slides were rinsed in PBS for 15 min at room temperature. To block nonspecific binding, slides were preincubated in PBS containing 75% heat-inactivated AB sera for 1 h and then in the same buffer with 25% heat-inactivated normal rabbit serum for 30 min at room temperature. Rat monoclonal antihuman IL-10 (PharMingen) and mouse monoclonal antihuman IL-12 (Bender MedSystems Diagnostics, Vienna, Austria) were diluted in PBS containing 1.0% BSA (Sigma). After brief washing with PBS, slides were incubated with the mAbs overnight at 4°C. Slides were then washed in the same buffer and incubated with rabbit antimouse or rabbit antirat secondary antibodies (Dako S.p.A., Milan, Italy) for 60 min at room temperature and further incubated with mouse or rat alkaline phosphatase anti-alkaline phosphatase (APAAP) complex (Dako) for 60 min at room temperature. Alkaline phosphatase substrate in the presence of Levamisole (Dako) to block endogenous alkaline phosphatase was used to signal detection (New Fuchsin Substrate System; Dako). Counterstaining was performed with Mayer's hematoxylin (Sigma). Negative isotype controls (murine IgG₁ and rat IgG_2a) were included at the same concentration as the primary antibody in each staining run. All slides were coded before evaluation and positive cells were counted blind by two independent observers. At least 1,000 mononucleated cells were counted on each coded slide. Results were expressed as the percentage of positively stained cells.

Statistical Analysis

Statistical comparisons of the levels of both IL-10 and IL-12 produced by LPS-stimulated monocytes with and without FCM were performed by a paired Student's t test. The statistical analysis of the amounts of IL-10 and IL-12 released by LPS-stimulated monocytes with NFCM and FFCM, and expressed as percent variations from control, was performed by a Mann-Whitney U test for unpaired data. The same test was used to evaluate, on LPS/IFN-stimulated monocytes, the difference between the percentage of inhibition of HLA-DR and CD40 expression induced by NFCM and FFCM. A P value of less than 0.05 was considered significant. Results are expressed as means ± SE. For statistical analysis of immunocytochemistry data, a two-way analysis of variance (ANOVA) followed by the Newman-Keuls test for comparisons of specific means was applied. The coefficient of variance for three repeated counts of a single slide was less than 5%.

	Results

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Production of IL-10 and IL-12 by LPS-Activated Monocytes Cultured with NFCM

NFCM were produced by six different cell lines of normal human lung fibroblasts; each NFCM was tested at least three times. Figure 1 shows the effect of NFCM on the production of IL-10 and IL-12 by LPS-activated monocytes. NFCM induced a significant increase (+97.5 ± 12.8%; P < 0.001) on the production of IL-10 by LPS-activated monocytes as compared with controls. On the contrary, the release of IL-12 was significantly reduced (68.7 ± 7.3%; P < 0.01).

View larger version (12K): [in this window] [in a new window]   Figure 1.   Effect of normal human lung fibroblast conditioned medium (NFCM) on the release of IL-10 and IL-12 by LPS-stimulated monocytes. NFCM was collected for 24 h and transferred to monocyte cultures that were then exposed to LPS (1 µg/ml) for 18 h. IL-10 (triangles) and IL-12 (circles) were determined by ELISA. Data are reported as the % variation of cytokine release from LPS-stimulated monocytes in the absence of NFCM. Cytokine release under LPS-stimulated conditions in the absence of NFCM was 393 ± 14.6 and 4,078 ± 539 pg/ml for IL-10 and IL-12, respectively (n = 6). One representative experiment in which six different fibroblast cell lines were used is shown.

Immunocytochemistry

The immunocytochemical staining of untreated monocytes from healthy subjects showed that less then 2% of the cells were IL-10 positive. In LPS-activated monocytes the percentage of IL-10-positive cells augmented to 34.3 ± 5.3% and further increased to 79.4 ± 8.3% in the presence of NFCM. In contrast, when LPS-activated monocytes were incubated in the presence of FFCM the percentage of IL-10-positive cells was 50.3 ± 8.2%, showing a significant reduction as compared with monocytes stimulated in the presence of NFCM (P < 0.01). The percentage of IL-12-positive cells in untreated monocytes was 50 ± 10.3% and increased to 94.8 ± 1.9% in LPS-stimulated monocytes. The immunocytochemical staining of LPS-stimulated monocytes in the presence of NFCM or FFCM showed that the percentage of IL-12-positive cells significantly decreased to 65.6 ± 6.4% and to 76.8 ± 2.6%, respectively, as compared with LPS-stimulated monocytes (P < 0.05) (Figure 2). Negative staining controls using secondary antibody alone were performed for all experimental conditions and resulted in an absence of immunostaining (data not shown).

View larger version (38K): [in this window] [in a new window]   Figure 2.   Immunocytochemical analysis of IL-10 and IL-12 expression in peripheral blood monocytes. IL-10 (A, B) and IL-12 (C, D) immunopositive cells in LPS- (A, C) and LPS/NFCM (B, D)-treated monocytes were visualized by the APAAP complex and counterstained with Mayer's hematoxylin.

Effect of NFCM on IL-10 and IL-12 mRNA Accumulation in LPS-Activated Monocytes

As shown in Figure 3, RT-PCR demonstrated that FCM increased IL-10 transcripts in monocytes cultured with FCM. On the contrary, the FCM inhibited transcription of the p40 subunit of IL-12 in monocytes stimulated with LPS. RT-PCR of the house keeping -actin gene was not affected by FCM treatment, showing that the effects observed were specific for IL-10 and IL-12 mRNA.

View larger version (28K): [in this window] [in a new window]   Figure 3.   Effect of NFCM on the levels of mRNA for IL-10 and IL-12 in LPS-activated monocytes. Monocytes were treated with NFCM and LPS prior to processing for RNA extraction and RT-PCR for IL-10 and IL-12 (p40 subunit). In the upper panel, modifications in the appearance of a 352-bp (IL-10) and a 297-bp (IL-12) band are compared with that of -actin. In the lower panel, the densitometric analysis is shown. Data are from one experiment representative of three in which monocytes from different donors and NFCM from various cell lines were used.

Effect of NFCM Produced by Fibroblasts Treated with Indomethacin and Exogenous PGE₂

To reduce the production of PGE₂ by fibroblasts we also generated NFCM in the presence of indomethacin. Indomethacin treatment reduced the stimulatory and inhibitory effect exerted by NFCM on the production of IL-10 and IL-12 by LPS-activated monocytes. The percentage of stimulation exerted by NFCM (+97.5 ± 12.8%) decreased to +30.3 ± 7.5% with indomethacin (P < 0.01). The percentage of inhibition for IL-12 exerted by NFCM (68.7 ± 7.3%) decreased to 29.7 ± 3.4% when NFCM were produced in the presence of indomethacin (P < 0.01) (Figure 4). We also examined the effect of exogenous PGE₂ on the production of both IL-10 and IL-12. As shown in Figure 5, exogenous PGE₂, in a concentration-dependent manner, stimulated and inhibited IL-10 and IL-12 release, respectively. The concentrations tested were within the range effectively measured in our NFCM (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 4.   Effect of indomethacin on the NFCM-induced changes in IL-10 and IL-12 release by LPS-activated monocytes. Indomethacin (50 µM) was added to fibroblasts during the 24-h incubation time. Data from NFCM ( filled symbols) and indomethacin-treated NFCM (empty symbols) on IL-10 (triangles) and IL-12 (circles) release are reported as % change of cytokine secretion from LPS-activated monocytes without NFCM. Data are from one representative experiment in which six independent fibroblast cell lines were used.

View larger version (11K): [in this window] [in a new window]   Figure 5.   Effect of increasing concentrations of PGE2 on the release of IL-10 and IL-12 from LPS-activated monocytes. Monocytes were exposed to LPS (1 µg/ml) for 18 h in the presence of increasing concentrations of PGE2. Culture medium was collected and assayed for IL-10 and IL-12 as described in MATERIALS AND METHODS. Data represent means ± SE of an experiment run in triplicates.

HLA-DR and CD40 Expression in Monocytes Cultured in the Presence of NFCM

When LPS/IFN--stimulated monocytes were cultured in the presence of NFCM, the levels of HLA-DR and CD40 were respectively 51.8 ± 8.7% and 53.9 ± 11.7% lower compared with monocytes cultured without NFCM (P < 0.05). However, in the presence of a neutralizing anti-IL-10 antibody, the inhibitory effect exerted by NFCM on the expression of both HLA-DR and CD40 was completely abrogated (Figure 6).

View larger version (29K): [in this window] [in a new window]   Figure 6.   Effect of NFCM on the expression of HLA-DR and CD40 by LPS/IFN--stimulated monocytes. In the upper panel, flow cytometry histograms of HLA-DR and CD40 expression by cultured monocytes in the absence (A) or presence (B) of NFCM are shown. Analysis of the data (lower panel), expressed as percent variation of mean fluorescence intensity, reveals that the decrease of the expression of HLA-DR and CD40 induced by NFCM (white bars) is completely prevented by treatment with an anti-IL-10 neutralizing antibody (gray bars) but not by incubation with control rat IgG2 (black bars). Data represent means ± SE of three independent experiments in which six different cell lines were used.

Production of IL-10 and IL-12 and Expression of HLA-DR and CD40 by Monocytes Cultured with FFCM

Figure 7 shows the effect of FFCM on the release of IL-10 and IL-12 by LPS-activated monocytes. The stimulatory activity of FFCM on the production of IL-10 was significantly reduced (48.2 ± 10.3%; P < 0.05) as compared with the effect exerted by NFCM (+97.5 ± 12.8%) . No statistical difference was observed between FFCM and NFCM in terms of IL-12 inhibition (60.7 ± 6%). As shown in Figure 8, FFCM showed a diminished capacity to modulate the expression of both HLA-DR and CD40 as compared with NFCM. The percent inhibition of the expression of HLA-DR (51.8 ± 8.7%) and CD40 (53.9 ± 11.7%) were reduced to 31.9 ± 7.4% and 15.9 ± 7.8%, respectively. The difference of the latter was statistically significant (P < 0.05) compared with the effect induced by NFCM.

View larger version (22K): [in this window] [in a new window]   Figure 7.   Effect of FFCM on the release of IL-10 and IL-12 by LPS-activated monocytes. NFCM and FFCM were transferred to monocytes before exposure to LPS for 18 h. Data are expressed as % change from monocyte cultures not exposed to FCM and represent means ± SE of three independent experiments in which six different cell lines were used. *P < 0.05 versus NFCM.

View larger version (19K): [in this window] [in a new window]   Figure 8.   Effect of FFCM on the expression of HLA-DR and CD40 by LPS/IFN--activated monocytes. Monocytes were exposed to culture medium from normal (NFCM) or fibrotic (FFCM) fibroblast cell lines and assayed for HLA-DR and CD40 expression by flow cytometry after labeling with specific monoclonal antibodies. Data are reported as the % inhibition versus monocytes treated with LPS/IFN- in the absence of FCM and represent means + SE of three independent studies in which six different cell lines were used.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study we have demonstrated that normal human lung fibroblasts strongly stimulate IL-10 mRNA accumulation and protein release by LPS-activated monocytes, whereas IL-12, at the level of both transcription and protein release, is inhibited. The increase of IL-10 production by monocytes, induced by FCM, was able, in an autocrine way, to downregulate the expression of HLA-DR, a class II MHC antigen, as well as the expression of the CD40 costimulatory molecule. The expression of both molecules was in fact completely restored when FCM stimulation of LPS-activated monocytes was performed in the presence of a neutralizing anti-IL-10 monoclonal antibody. In addition, we have shown that fibrotic fibroblasts have a reduced capability to stimulate the production of IL-10 by LPS-activated monocytes and, as a consequence, a significantly reduced capability to downregulate the expression of CD40 on monocytes as compared with normal fibroblasts.

Because it has already been shown that exogenous PGE₂ affect the production of both IL-10 and IL-12 in human monocytes (16, 17) and that fibroblasts release significant amounts of PGE₂ (18), we produced the FCMs in the presence of indomethacin, an agent known to suppress the activity of cyclooxygenase. Indeed, the indomethacin-treated FCMs showed a significantly reduced stimulatory and inhibitory activity on IL-10 and IL-12, respectively. In addition, the effect exerted by the control FCM was mimicked by the addition to monocyte cultures of exogenous PGE₂ within a concentration range similar to that spontaneously produced by our fibroblast cell lines. On the other hand, the possibility that IL-10 produced by fibroblasts interfered with the assay of the cytokine released by monocytes exposed to NFCM was completely ruled out by the undetectable levels of IL-10 in FCM (data not shown).

Therefore, these findings suggest that normal human lung fibroblasts, through the release of PGE₂, are able, as we have previously shown for TNF- (6), to modulate the production of IL-10 and IL-12, whose role is crucial in the regulation of the immune response. IL-12, in fact, through the induction of IFN- secretion, induces the development of Th1-type immune responses. IL-12 is also a costimulus for activated T and NK cells and enhances the cytolytic activity of NK and T cells (13, 19). IL-10, on the other hand, by inhibiting IFN-, indirectly promotes the commitment of Th0 cells to the Th2 phenotype, even though it may also inhibit IL-4 and IL-5, key cytokines of the Th2 pattern (10). IL-10 also suppresses the production of several proinflammatory cytokines, likely through the inhibition of NF-B activation (11). Moreover, IL-10 modulates the immune response through the regulation of the antigen-presenting capacity of monocytes. Human IL-10 in fact strongly inhibits specific T-cell proliferation when monocytes act as antigen-presenting cells. This effect is mainly due to the reduced antigen-presenting capacity of the monocytes, caused by the downregulatory effect of IL-10 on the expression of class II MHC molecules. This is consistent with the observation that endogenously produced IL-10 is responsible for the reduction of HLA-DR expression in monocytes stimulated with LPS (12, 22). In the current study we demonstrate that normal lung fibroblasts strongly stimulate monocytes to produce much higher amounts of IL-10 as compared with monocytes cultured in regular medium and stimulated with LPS. Therefore, the downregulatory effect on the expression of HLA-DR and CD40, exerted in an autocrine way by IL-10 on monocytes, is significantly enhanced. The role of IL-10 in causing this effect is supported by the fact that this downregulatory activity was completely abrogated when FCM stimulation of LPS-activated monocytes was performed in the presence of a neutralizing anti-IL-10 monoclonal antibody.

Based on our findings, human lung fibroblasts modulate the secretion of IL-10 and IL-12 in a different way, although in the same direction from the functional point of view. The inhibitory effect on the release of IL-12 exerted by fibroblasts, and the contemporary stimulation of IL-10 secretion, may represent a mechanism controlling the differentiation of naive CD4⁺ T cells toward Th1 or Th2 cells. Furthermore, the fibroblast-induced release of IL-10, downregulating the expression of HLA-DR and CD40 on monocytes, may affect the antigen-presenting capacity of these cells, as well as the cell contact-dependent activation of T cells and monocytes during antigen presentation. These events may lead to the downregulation of the immune response, contributing to the fine tuning of the balance between stimulatory and inhibitory signals so important in the maintenance of homeostasis. A defective functioning of the cells involved in this homeostatic mechanism could result in an imbalance leading to disease. To demonstrate this hypothesis, we evaluated whether fibroblasts coming from diseased tissue exhibit differential functional features compared with normal cells, with particular regard to the modulatory role we have observed for normal fibroblasts in the context of the immune response. We have already shown that fibrotic fibroblasts have a reduced capability to produce PGE₂, ~ 40% of that produced by normal fibroblasts (data not shown), that is responsible for a reduced inhibitory effect on the production of TNF- by monocytes (8). Similarly, fibroblasts coming from fibrotic tissue show a significantly reduced stimulatory action on the production of IL-10, and consequently a reduced inhibitory effect on the expression of CD40. On the contrary, we have not found any significant difference in the inhibitory effect exerted by normal and fibrotic cell lines on IL-12 production. This discrepancy is likely due to the fact that even the small amounts of PGE₂, still present in FFCM, are able to exert a relevant inhibitory effect, and is also confirmed by the concentration-response curve of exogenous PGE₂ in which a 50% inhibition of IL-12 release is already observed at low PGE₂ concentrations.

This diminished ability of fibrotic cells to interact with monocytes, as regards the production of IL-10 and the regulation of the expression of CD40, may have important implications in a number of pathologic conditions. Particularly, chronic pulmonary inflammation, lung fibrosis, and chronic bronchial inflammation in asthma might be the result of a persistent state of immune activation, possibly due to a reduced production of the anti-inflammatory cytokine IL-10 and to an upregulated expression of costimulatory molecules that might lead to a misregulation of the inflammatory and immune responses. Indeed, in bronchial asthma and chronic obstructive pulmonary disease, IL-10 levels in sputum are reduced and, in an animal model of asthma, IL-10 suppresses allergen-induced airway inflammation and nonspecific airway responsiveness (23). In patients with idiopathic pulmonary fibrosis, measurements of IL-10 protein, in cell-free bronchoalveolar lavage fluid, revealed lower amounts of IL-10 compared with healthy control subjects, although in the alveolar macrophages of the same patients an increased expression of the IL-10 gene has also been observed (25). Several cytokines, whose production is normally inhibited by IL-10, have been found associated with chronic pulmonary inflammation, including IL-1, IL-8, TNF-, and GM-CSF (26). It has also been shown that IL-10 may be crucial for the resolution of pulmonary inflammation promoting apoptosis of neutrophils and eosinophils (28). On the other hand, CD40 is involved in the regulation of several critical aspects of the immune response. In monocytes, CD40 triggering by CD40 ligand rescues these cells from apoptosis, prolonging their survival at the site of inflammation, and stimulates the production of proinflammatory cytokines such as IL-1, IL-6, IL-8, IL-12, and TNF-, and enzymes such as matrix metalloproteinase that are so important in tissue remodeling. In addition, the functional activation of CD40 deeply affects the accessory cell function of monocytes, upregulating the expression of costimulatory molecules such as CD80/B7-1 and CD86/B7-2 (30) crucial for the activation of T-lymphocytes. Several data indicate that in chronic inflammatory diseases monocytes/macrophages are in an immunologically activated state. Enhanced expression of CD40 and other costimulatory molecules has been observed in several inflammatory conditions including sarcoidosis, hypersensitivity pneumonitis, and asthma, suggesting that the upregulated expression of these molecules might cause an excessive and persistent antigen presentation, potentially leading to chronicity and tissue damage (32). In this context, fibroblasts may play an important role. Based on our findings these cells are effective in driving the release of IL-10 and IL-12 by monocytes, and in addition they may regulate the antigen-presenting capacity of these cells as well as their costimulatory function. This strongly suggests that normal human lung fibroblasts actively participate in the control of inflammation and immune regulation, including the dampening of antigen-driven responses. An impairment of these functions may instead contribute to the disruption of this control, leading to the continuation of the events that sustain chronic inflammation and lung damage.