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The Journal of Immunology, 2000, 164: 3806-3814.
Copyright © 2000 by The American Association of Immunologists

Cultured Human Fibroblasts Express Constitutive IL-16 mRNA: Cytokine Induction of Active IL-16 Protein Synthesis Through a Caspase-3-Dependent Mechanism1

Daniela Sciaky*, William Brazer{ddagger}, David M. Center{ddagger}, William W. Cruikshank{ddagger} and Terry J. Smith2,*,{dagger}

* Division of Molecular and Cellular Medicine, Department of Medicine, and {dagger} Department of Biochemistry and Molecular Biology, Albany Medical College, and the Samuel S. Stratton Veterans Administration Medical Center, Albany, NY 12208; and {ddagger} Pulmonary Center, Boston University School of Medicine, Boston, MA 02118


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human fibroblasts can express numerous regulatory molecules that influence immune function. IL-16, a ligand for CD4, is a chemoattractant molecule expressed by lymphocytes, eosinophils, mast cells, and lung epithelium. It appears that the sole target for IL-16 is the CD4-bearing cell. Here we demonstrate that fibroblasts from several tissues can express IL-16 mRNA and protein as well as IL-16-dependent chemoattractant activity. The transcript is expressed abundantly under basal culture conditions as a 2.5-kb band on Northern analysis, similar to that observed in lymphocytes. IL-16 protein and activity are undetectable in fibroblast cultures under these same control conditions. However, when treated with proinflammatory cytokines such as IL-1ß, they express very high levels of IL-16 protein and chemoattractant activity, a substantial component of which can be blocked with IL-16-neutralizing Abs. The amount of IL-16 protein released into the medium is 3- to 4-fold greater, on a per cell basis, than that observed in lymphocytes. The induction of IL-16 protein by IL-1ß can be attenuated with specific inhibition of caspase-3, which could be detected in IL-1ß-treated fibroblasts. IL-1ß also induces RANTES mRNA, protein, and activity, and most of the chemoattractant activity released from fibroblasts not derived from IL-16 can be attributed to RANTES. Human fibroblasts appear to be an important source of IL-16 and through expression of this molecule may have key roles in the recruitment of CD4+ cells to sites of inflammation. IL-16 expression and the mechanism involved in its regulation appear to be cell type specific.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Recruitment of lymphocytes and other bone marrow-derived cells to sites of inflammation involves the expression of several types of molecules, including chemoattractants. A group of chemoattractants, the chemokines, are small peptides of 70–80 aa that have characteristic cysteine signatures (1). The arrangement of these cysteines divides chemokines into different classes. Besides the chemokines, other molecules exhibiting chemoattractive activity have been identified. Lymphocyte chemoattractant factor or IL-16 represents a chemoattractant molecule not belonging to the chemokine family (2, 3, 4). IL-16 is synthesized as a precursor molecule of 69 kDa (5, 6), but the secreted, biologically active form has a molecular mass of 56 kDa, as determined by Sephadex chromatography. Secreted IL-16 is thought to be composed of four identical polypeptide subunits, and these chains migrate on SDS-PAGE at 17–20 kDa. IL-16 bioactivity is conferred with noncovalent multimerization (3). This form conveys chemoattractant activity specific for CD4+-bearing T lymphocytes, monocytes, and eosinophils (7, 8, 9). IL-16 release from CD8+ lymphocytes is dependent upon the activity of caspase-3, a cysteine protease (6). IL-16 induces expression of the IL-2R on CD4+ T cells (8) and therefore influences IL-2-dependent activation of these cells. We have recently determined that both CD4+ and CD8+ T lymphocytes express constitutive pro-IL-16 protein. CD8+ T cells, in contrast to CD4+ cells, also produce preformed bioactive IL-16, which is released after activation with serotonin or histamine (10, 11). The cell types known to express IL-16 also include eosinophils (12), mast cells (13), and lung epithelial cells from patients with asthma (14).

Fibroblasts are a diverse population of cells. Substantial evidence supports the concept that fibroblasts from different anatomic regions exhibit distinct phenotypes in culture. Characteristic expression patterns of receptors (15), gangliosides (16, 17), glycosaminoglycans (17, 18, 19, 20, 21, 22), plasminogen activator inhibitor type 1 (23, 24, 25, 26), and PG endoperoxide H synthase-2 (27) have recently been documented in different populations of fibroblasts. Moreover, fibroblasts are capable of responding to their microenvironment in a complex manner. In some tissues, such as the orbit and lung, fibroblasts can be subsetted on the basis of the surface expression of the glycoprotein Thy-1 (28, 29). Despite this heterogeneity, many phenotypic attributes are common to fibroblasts regardless of the tissue of origin. Fibroblasts are capable of expressing multiple small regulatory molecules, including cytokines, prostanoids, and adhesion molecules (28, 30, 31, 32, 33). When activated, they can express chemokines such as IL-8 (31), RANTES (32), monocyte chemotactic protein-1 and -2 (33). Thus, the fibroblast can function as a key orchestrator of diverse aspects of tissue reorganization. We have recently suggested that fibroblasts, by virtue of the diverse array of small molecules that they express, may be involved in the very early molecular events surrounding the recruitment of immunocompetent cells to sites of inflammation (34).

In this paper we report for the first time the results of studies examining the expression of IL-16 in cultured human fibroblasts. Fibroblasts from several anatomic regions express high levels of IL-16 mRNA under basal culture conditions. Unlike lymphocytes, before activation by cytokines fibroblasts fail to express detectable pro-IL-16, mature IL-16 protein, or IL-16-dependent chemoattraction. When treated with proinflammatory cytokines such as IL-1ß, they release substantial levels of chemoattractant activity for human T lymphocytes, a large fraction of which can be attributed to IL-16. Thus, IL-16 expression under inflammatory stress may be a generalized attribute of human fibroblasts and may represent an important signaling mechanism by which fibroblasts participate in the recruitment of CD4-bearing cells to sites of inflammation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Human recombinant IL-1ß and TNF-{alpha} were purchased from BioSource (Camarillo, CA). Leukoregulin, a 50-kDa product of activated T lymphocytes (35), was prepared as described previously (36) and was provided by Dr. Charles Evans, National Cancer Institute( Bethesda, MD). Eagle’s medium, antibiotics, and FBS were supplied by Life Technologies (Grand Island, NY). An affinity-purified polyclonal rabbit anti-rIL-16 Ab was prepared from rIL-16-immunized rabbit serum as described previously (4). Neutralizing anti-RANTES Abs were purchased from R&D Systems (Minneapolis, MN), and RANTES-specific ELISA was obtained from BioSource. Anti-caspase-3 polyclonal Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The specific peptide inhibitors of caspase-3 and -1, Ac-DEVD-CHO (Ac-Asp-Glu-Val-Asp-aldehyde)3 and Ac-YVAD-Ald (acetyl-Tyr-Val-Ala-Asp-CHO), respectively, were purchased from Bachem (Torrance, CA).

Cell culture

Procedures for the culture of primary human fibroblasts have been published previously (19). Briefly, orbital fibroblasts were obtained from individuals undergoing transantral decompressive surgery for severe thyroid-associated ophthalmopathy (TAO) or for some other condition where normal orbital connective tissue was removed. Skin fibroblasts were obtained from punch biopsy or from the edge of surgical incisions of normal-appearing tissue. Thyroid fibroblasts were obtained from normal-appearing tissue in glands without an autoimmune process or from those affected by Graves’ disease. This diagnosis was made on clinical grounds, including high concentrations of free thyroxine, suppressed thyrotropin levels, and the presence of anti-thyroid Abs in the serum. These activities were approved by the institutional review board of the Albany Medical College. Synovial fibroblasts were obtained from diagnostic biopsies of synovial membrane from patients with rheumatoid and osteoarthritis. These were provided by Dr. L. J. Crofford (Ann Arbor, MI). A total of 12 different fibroblast strains were examined in these studies. Tissue explants were placed in culture dishes and minced into small pieces, and fibroblasts were allowed to outgrow the tissue. When fibroblasts outgrew the explants, they were covered with medium containing 10% FBS, L-glutamine, penicillin/streptomycin, and nystatin. For subculture, monolayers were disrupted by gentle treatment with trypsin/EDTA. The fibroblast-like cells failed to express smooth muscle-specific actin, factor VIII, or thyroglobulin, excluding contamination of the cultures with other cell types. All experiments were performed with fibroblasts between the 2nd and 12th passages from culture initiation.

Chemotaxis assay

For chemoattractant assays, fibroblasts were seeded in 24-well arrays and grown to confluence. After rinsing with PBS, the culture monolayers were shifted to medium containing 1% FBS to which nothing (control), IL-1{alpha} (10 ng/ml), IL-1ß (10 ng/ml), TNF-{alpha} (10 ng/ml), or leukoregulin (1 U/ml) was added for the times indicated in the figure legends. At the end of treatment incubation, the culture medium was collected quantitatively and stored at -80°C until use.

Chemotaxis was examined in a modified Boyden chemotaxis chamber using human NWNA-T lymphocytes as the cellular targets, as described previously (4, 37). In brief, 50 µl of a cell suspension (107 cells/ml) was placed in the upper compartments of 48-well microchemotaxis chambers separated from 32 µl of samples by 8-µm micropore nitrocellulose filters (Neuroprobe, Cabin John, MD). These were then incubated at 37°C in 5% CO2 environment for 3 h. Filters were fixed, stained with hematoxylin, dehydrated, mounted on glass slides, and viewed under light microscopy. Lymphocyte migration was quantified by counting the total number of cells migrating beyond a certain depth. This depth was set to routinely identify a baseline migration under control conditions of 10–15 cells/high power field. Five high power fields were counted in duplicate for each sample, and the mean ± SD was calculated and expressed as a percentage of baseline cell migration in control buffer alone (100%). For each set of experimental conditions, at least three separate experiments were performed. The differences between experimental and control conditions were analyzed by Student’s t test using the absolute values obtained for lymphocyte migration, and statistical significance was accepted at the 5% level of confidence. To assess specificity for IL-16, neutralizing experiments were conducted by incubating culture supernatants for 15 min with neutralizing concentrations of anti-IL-16 mAb (clone 14.1; 5 µg/ml), which neutralizes the chemotactic activity of 50 ng/ml rIL-16. Similarly, anti-RANTES mAb (5 µg/ml), with an ND50 of 200 ng/ml for rRANTES, was used to neutralize that cytokine.

Isolation of RNA

For RNA preparation, cultures were treated similarly to those prepared for IL-16 activity studies, except the fibroblasts were allowed to proliferate to confluence in 100-mm plates before treatment with IL-1ß. At harvest, monolayers were washed extensively in PBS. Total cellular RNA was extracted from fibroblasts by the modified method of Chomczynski and Sacchi (38) using guanidium isothiocyanate (Ultraspec RNA isolation systems, Biotecx Laboratories, Houston, TX). RNA was precipitated from the aqueous phase by addition of isopropanol, washed with 75% ethanol, and solubilized in diethylpyrocarbonate-treated water. The integrity of the RNA was established routinely by ascertaining the 260/280 spectroscopic ratio and by staining the electrophoresed sample with ethidium bromide and inspecting it under UV light.

RT-PCR and Northern hybridization

cDNA was prepared by reverse transcribing total fibroblast RNA (1–2 µg) with Superscript (Life Technologies) in the presence of random hexamers (Perkin-Elmer, Norwalk, CT). This cDNA (4–5 µl) was amplified in the presence of IL-16 sense and antisense oligonucleotides essentially as described previously (12). The sequences of these primers, synthesized by BioSource International, were 5'-ATGCCCGACCTCAACTCCTC-3' (sense) and 5'-CTCCTGATGACAATCGTGAC-3' (antisense). The RANTES primers used were 5'-CCATATTCCTCGGACACCACAC-3' (sense) and 5'-AACTCCTGACCTCAAGTGATCCAC-3' (antisense). For Northern blot analysis, RNA samples were electrophoresed in denaturing 1% agarose, formaldehyde gels and transferred to Magna Charge nylon membrane (MSI, Westborough, MA) or ZetaProbe (Bio-Rad, Hercules, CA). The immobilized RNA was allowed to hybridize to a [32P]dCTP-labeled IL-16 cDNA probe generated by random primer synthesis. Radioactive hybrids were visualized by radioautography on X-OMAT film (Eastman Kodak, Rochester, NY) exposed at -70°C.

ELISA analysis of IL-16 and RANTES levels

An ELISA specific for IL-16 was used to assess the chemoattractant protein mass released by fibroblasts as described previously (12), and an ELISA for RANTES was used according to the manufacturer’s specifications (BioSource). Lymphocytes used for these determinations were activated with anti-CD3 as previously reported (39). Recombinant IL-16 and conditioned medium were diluted in PBS to appropriate concentrations. A standard curve was generated using serial dilutions of rIL-16. Samples of the culture medium (100 µl) were incubated in duplicate in a 96-well microtiter plate (Nunc, Naperville, IL) at 37°C for 1 h. The subsequent maneuvers were performed at room temperature. The Ag was removed by washing extensively with a solution of PBS/Tween-20. Nonspecific binding was reduced by blocking with 1% BSA (100 µl) for 1 h. After washing, 100 µl of a rabbit anti-IL-16 polyclonal Ab (10 µg/ml) diluted in PBS containing 0.05% Tween-20 was added to each well. The presence of an IL-16/anti-IL-16 complex was detected by incubation for 1 h with biotinylated goat anti-rabbit IgG (Sigma, St. Louis, MO) diluted 1/500 in PBS.

Immunoprecipitation of IL-16

Newly synthesized IL-16 levels were quantitated by incubating fibroblast culture monolayers in methionine-free medium for 18 h, followed by labeling with [35S]methionine (500 µCi/ml) for 6 h. The medium samples were collected and subjected to immunoprecipitation with anti-IL-16 (clone 14.1; 5 µg/ml) conjugated to protein A beads. The medium sample was incubated with the bead/Ab complex for 1 h at room temperature, and beads were centrifuged down, washed, and counted.

Western analysis of caspase-3 levels

To determine whether cytokine activation of fibroblasts influences the levels of caspase-3 protein, cultures were treated with nothing or IL-1ß (10 ng/ml) for 0–24 h and then removed from substratum with EDTA treatment. Cells (3 x 106) were lysed by sonication in a buffer containing PBS (pH 7.5), 1 mM PMSF, 10 µg/ml apronin, and 10 µg/ml leupeptin and were subjected to electrophoresis through a 15% SDS-polyacrylamide gel. Separated proteins were transferred electrophoretically to nitrocellulose membrane and probed with goat polyclonal anti-caspase-3 raised against the p20 subunit (Santa Cruz Biotechnology). The secondary Ab, an anti-goat Ig labeled with HRP (Santa Cruz Biotechnology) was used at a dilution of 1/5000, and the signal was detected with chemiluminescence (Pierce, Rockford, IL). With regard to caspase-3 inhibition studies, cultures were incubated with either Ac-DEVD-CHO (100 µM), a specific inhibitor of caspase-3, or Ac-YVAD-Ald (100 µM), a caspase-1 inhibitor, for 24 h. The cultures were treated without or with IL-1ß, and then the supernatant was assayed for chemotaxis or IL-16 ELISA, as described above. As a control, the peptides were assayed alone at the same concentration in the chemotaxis buffer, and there was no detectable effect on normal cell migration (data not shown).

Statistical analysis

Data are expressed as the mean ± SD of replicate determinations unless indicated otherwise. Statistical significance was determined by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cultured human fibroblasts exhibit low levels of basal lymphocyte chemoattractant activity that are inducible by a variety of proinflammatory molecules

Fibroblasts derived from several anatomic regions of the human body have been shown to express substantial levels of various chemoattractants (31, 32, 33). Moreover, we have proposed that fibroblasts are important mediators of numerous molecular events associated with tissue remodeling (34). We therefore determined whether cultures of these cells release activity that is chemotactic for T lymphocytes. Because fibroblasts are activated by many proinflammatory molecules, we examined cultures under basal and cytokine-treated conditions. Confluent fibroblasts, in this instance from a thyroid gland affected by Graves’ disease, were treated with nothing (control), IL-1{alpha} (10 ng/ml), IL-1ß (10 ng/ml), TNF-{alpha} (10 ng/ml), or leukoregulin (1 U/ml) for 17 h. Conditioned medium was harvested, and NWNA-T lymphocytes were subjected to these medium samples in a T lymphocyte chemotaxis assay. Fibroblasts release very low level chemoattractant activity into the medium under control culture conditions. As demonstrated in Fig. 1Go, the fibroblasts were capable of expressing substantial lymphocyte migration-stimulating activity following treatment with all cytokines tested. IL-1ß increased activity most robustly (~4-fold above control levels), followed closely by leukoregulin. A substantial fraction of this increase could be neutralized with specific anti-IL-16 Abs (5 µg/ml). These results indicate that multiple cytokines are capable of increasing T lymphocyte migratory activity expressed by human fibroblasts and that a prominent component of this chemoattraction activity is attributable to IL-16.



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  		FIGURE 1. Human fibroblasts in culture express and release substantial IL-16-derived chemoattractant activity for lymphocytes. This activity is elicited by several proinflammatory cytokines. Confluent fibroblasts from a thyroid gland affected by Graves’ disease were shifted to medium containing 1% FBS and were treated with nothing or IL-1{alpha} (10 ng/ml), IL-1ß (10 ng/ml), TNF-{alpha} (10 ng/ml), or leukoregulin (1 U/ml) for 17 h. The medium was collected and assayed without (solid bars) or with neutralizing Abs specific for IL-16 (shaded bars). Lymphocyte migration was assessed by a modified Boyden chamber technique and is expressed as a percentage of a control migration in buffer, which is set at 100%. Results are presented as the mean ± SD of three separate experiments. Differences in cytokine-treated cultures without and with anti-IL-16 Ab are significant to p < 0.001 in each case.

 
Because IL-1ß up-regulated the chemotactic activity attributable to IL-16 robustly, we characterized this response by determining its time dependence. As Fig. 2GoA suggests, at 2, 4, and 8 h after IL-1ß was added to the culture medium, there was very little increase in total or IL-16-dependent chemotaxis. However, by 18 h IL-16 activity was detectable, and at 24 h it had increased to 185% over baseline. The data contained in Figs. 1Go and 2Go also demonstrate that another chemoattractant(s) might be induced in the cytokine-activated fibroblasts. This is represented by the residual activity that remains following the neutralization of IL-16. Earlier studies verified the expression of RANTES in dermal fibroblasts treated with TNF-{alpha} or IL-1 (32). We therefore assessed whether RANTES was contributing to the chemoattraction activity observed in IL-1ß-treated fibroblasts that was resistant to IL-16 neutralization. IL-1ß treatment elicited a rapid increase in chemotactic activity that was in part neutralized with anti-RANTES neutralizing Abs (Fig. 2GoB). The increase was apparent within 6–9 h and was sustained for the duration of the study. The magnitude of the up-regulation was considerably less than that of IL-16. We then assessed whether the activities attributable to IL-16 and RANTES in aggregate could completely account for the lymphocyte chemotaxis released by the activated fibroblasts. In that study cultures treated for 16 h with IL-1ß (10 ng/ml) exhibited a 214 ± 14% increase in chemoattractive activity compared with controls (100 ± 5%; Table IGo). IL-16-neutralizing Abs lowered this activity to 149 ± 18% of baseline, while anti-RANTES Abs decreased the activity to 176 ± 10%. The combination of anti-IL-16 and anti-RANTES Abs blocked activity to 111 ± 12%, which was not statistically different from control values. These data indicate that IL-16 and RANTES are the two principal CD4+ chemoattractants released by IL-1ß-activated human fibroblasts.



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  		FIGURE 2. Time dependence of the induction by IL-1ß of IL-16- and RANTES-dependent lymphocyte chemotaxis in human fibroblasts. Confluent fibroblasts, in this case from normal s.c. connective tissue, were incubated for 24 h in medium supplemented with 1% FBS to which IL-1ß (10 ng/ml) was added for the time intervals indicated along the abscissas. Conditioned medium samples were then subjected to a lymphocyte chemotaxis assay as described in Materials and Methods in the absence or the presence of neutralizing Abs against IL-16 (5 µg/ml; A) or RANTES (5 µg/ml; B). Data are expressed as the mean ± SD of three independent determinations.

 

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  		Table I. Effects of anti-IL-16 and anti-RANTES neutralizing Abs on T lymphocyte chemoattractant activity in IL-1ß-treated fibroblasts

 
We next determined whether the IL-1ß induction of IL-16 activity was peculiar to certain fibroblast types or represents a phenotypic attribute common to human fibroblasts. Chemoattractant activity emanating from different types of fibroblasts was assessed by incubating several strains in culture medium for 16 h without or with IL-1ß (10 ng/ml). As the data contained in Fig. 3Go demonstrate, fibroblasts from skin, thyroid, synovial membrane, and orbital connective tissue all responded to IL-1ß with increases in lymphocyte chemotactic activity, a large fraction (up to 70%) of which could be neutralized with anti-IL-16 Abs. The increases in total activity ranged from 2- to >4-fold. Thus, it would appear that fibroblasts from multiple human tissues can express IL-16 chemotactic activity following treatment with IL-1ß. Moreover, IL-16 synthesis is present whether the fibroblasts derive from normal or diseased tissue.



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  		FIGURE 3. Human fibroblasts derived from several anatomic regions and from normal and diseased tissue express substantial IL-16 chemoattractant activity when treated with IL-1ß. Confluent monolayers of TAO orbital fibroblasts (strain 1), Graves’ thyroid fibroblasts (strain 2), dermal fibroblasts (strain 3), normal thyroid fibroblasts (strain 4), normal s.c. connective tissue fibroblasts (strain 5), and synovial fibroblasts from patients with rheumatoid arthritis (strain 6) or osteoarthritis (stain 7) were shifted to medium containing 1% FBS and were untreated (control) or received IL-1ß (10 ng/ml) for 16 h, and the medium was collected and stored at -70°C until assayed. Aliquots of the medium were then assayed for T lymphocyte chemoattractant activity as described in Materials and Methods. Some of the samples in the lymphocyte migration assay also received neutralizing anti-IL-16 Ab (5 µg/ml). Data are presented as the mean ± SD of three separate experiments. Differences in lymphocyte migratory activity between IL-1ß-treated cultures without and with anti-IL-16 Ab are significant to p < 0.001 in each case.

 
Fibroblasts express high levels of IL-16 protein following treatment with IL-1ß

We quantitated the amount of IL-16 protein released by fibroblasts under basal and IL-1ß-treated conditions. An ELISA specific for IL-16 was used for these measurements. IL-16 protein was not detected in conditioned culture medium from untreated fibroblasts. Moreover, neither pro-IL-16 nor mature IL-16 protein could be found within the unstimulated cells. However, as the data in Table IIGo suggest, fibroblast cultures treated for 16 h with IL-1ß (10 ng/ml) achieved an IL-16 level of 124 ± 32 pg/ml/106 cells (mean ± SD). In contrast, T lymphocytes, maximally stimulated with anti-CD3, expressed an IL-16 protein level of 37 ± 15 pg/ml/106 cells. It would appear, therefore, that fibroblasts express considerably higher levels of IL-16 protein, on a per cell basis, than do lymphocytes following activation, and thus the fibroblast may represent an important source of IL-16 at sites of inflammation. Using a RANTES-specific ELISA, we have determined that IL-1ß treatment resulted in the production of substantial levels of that chemokine as well (28 ± 9 pg/ml/106 cells) compared with the undetectable levels found in control fibroblasts.


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  		Table II. Comparison of IL-16 protein release from activated T lymphocytes and fibroblasts

 
To determine whether the up-regulation of IL-16 protein by IL-1ß in fibroblasts involved the de novo synthesis of protein, confluent monolayers were labeled with [35S]methionine (500 µCi/ml) for 6 h after various treatment intervals with the cytokine. The medium was subjected to immunoprecipitation with anti-IL-16 (5 µg/ml), and radioactivity was determined by scintillation counting. As the data contained in Fig. 4Go suggest, IL-1ß up-regulated the synthesis of IL-16 protein in a time-dependent manner. The increase was 7-fold above baseline at 6 h and reached a peak at 24 h, at which time the synthesis was induced >150-fold. The level of synthesis remained close to this level for the duration of the study (48 h).



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  		FIGURE 4. IL-1ß up-regulates the synthesis of IL-16, as assessed by immunoprecipitation of pulse-labeled protein. Fibroblast monolayers, in this case from normal skin, were treated with nothing (control) or IL-1ß (10 ng/ml) for the times indicated along the abscissa. They were then pulse labeled for 6 h with [35S]methionine (500 µCi/ml). Medium samples were collected and subjected to immunoprecipitation with an anti-IL-16 Ab (clone 14.1; 5 µg/ml; conjugated to protein A beads) as described in Materials and Methods. The data are expressed as the mean ± SD of three replicates.

 
Fibroblasts from several human tissues constitutively express IL-16 mRNA

Because human fibroblasts express high levels of IL-16 protein, and IL-16-dependent chemotaxis appears to be an important component of the lymphocyte signaling emanating from those cells, we examined their pattern of IL-16 mRNA expression. As the data in Fig. 5GoA indicate, when total cellular RNA from several types of fibroblasts was reverse transcribed and subjected to PCR using specific IL-16 primers, the transcript appeared as a 347-bp product, consistent with our earlier studies (12). These fibroblasts were derived from TAO and normal orbital connective tissue, skin, s.c. connective tissue, and Graves’ and normal thyroid. The identity of the PCR product as IL-16 was verified by sequencing.



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  		FIGURE 5. Constitutive expression of IL-16 (A and C) and RANTES (B) mRNA in cultured human fibroblasts. A, RT-PCR was performed on total cellular RNA isolated from untreated fibroblasts derived from a number of normal and diseased tissues. The RNA was reverse transcribed, and the resulting cDNAs were subjected to PCR using the specific IL-16 primer sequences described in Materials and Methods. The product was a 347-bp nucleotide sequence. The fibroblast strains studied were as follows: lane 1, TAO orbit; lane 2, normal orbit; lane 3, dermal; lane 4, TAO orbit; lane 5, s.c. connective tissue; lanes 6–8, Graves’ thyroid; lane 9, normal thyroid. B, RT-PCR analysis of RANTES expression in untreated fibroblasts in Graves’ orbit (lane 1) and normal thyroid (lane 2). A single 440-bp nucleotide product was generated. C, Northern blot analysis of fibroblast mRNA revealed a transcript of 2.5 kb (lanes 2 (orbital) and 3 (thyroid)), consistent with that found in lymphocytes (lane 1). Total cellular RNA was electrophoresed, transferred to a membrane, and subjected to hybridization with a 2-kb IL-16 cDNA probe generated by random primer synthesis.

 
A single, abundant 2.5-kb transcript hybridizing to a probe generated from a 2-kb, nearly complete IL-16 cDNA is expressed by fibroblasts not activated by cytokines on Northern blot analysis as shown in Fig. 5GoC. The size of the mRNA was similar to that detected in T lymphocytes. Because fibroblasts have been shown to produce RANTES, we next assessed the expression of mRNA encoding this c-c type chemokine in our fibroblasts using RT-PCR. As suggested by the data in Fig. 5GoB, we detected the transcript as a 440-bp PCR product in the two fibroblast strains tested under basal culture conditions. Thus, transcripts for both IL-16 and RANTES are readily detectable by RT-PCR in untreated human fibroblasts.

IL-1ß can up-regulate the expression of both IL-16 and RANTES protein and activity in human fibroblasts. We next determined whether the effects of this proinflammatory cytokine on the expression of these chemoattractant molecules in fibroblasts was mediated at the pretranslational level. As the Northern blot analysis shown in Fig. 6GoA suggests, IL-16 mRNA was easily detected in the fibroblast under basal (control) culture conditions, designated time 0 in the figure. IL-1ß (10 ng/ml) treatment failed to influence the relative abundance of this transcript in a time dependence study, ranging from 30 min to 7 h of exposure to the cytokine. This finding was verified in another study in which two different fibroblast strains were examined after a 20-h treatment with IL-1ß. In contrast, the transcript encoding RANTES was highly inducible with IL-1ß (Fig. 6GoB). The basal expression of RANTES mRNA was barely detectable by Northern analysis in untreated fibroblasts, but after 2 h the 1.2-kb transcript was induced 7.4-fold, and by 7 h the levels were 50-fold above baseline. This induction was observed in another study using multiple fibroblast strains treated with IL-1ß for 16–24 h (data not shown). Thus, it would appear that the up-regulation of IL-16 expression in fibroblasts by IL-1ß is mediated through influences exerted on the translation of constitutively expressed mRNA, while that of RANTES is elicited through an induction of steady-state levels of its transcript.



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  		FIGURE 6. Northern blot analysis of the IL-1ß effects on steady state IL-16 and RANTES mRNA levels in fibroblasts. Fibroblasts were allowed to proliferate to confluence in 100-mm diameter plates in medium supplemented with 10% FBS. They were shifted to medium containing 1% serum for 24 h, and then IL-1ß (10 ng/ml) was added for the times indicated. Monolayers were rinsed with PBS, and RNA was extracted as described in Materials and Methods. The immobilized RNA (20 µg/sample) was hybridized with cDNA probes for either IL-16 (A) or RANTES (B). The resulting autoradiograms were analyzed by densitometry, membranes were rehybridized to a probe for GAPDH, and the signals were normalized for variations in loading. The heights of the bars denote the corrected signals.

 
Human fibroblasts treated with IL-1ß exhibit high levels of caspase-3

Caspase-3 is a cysteine protease that represents an important component of the biochemical cascade used in the mediation of cell death (40). Important proteolytic events have been linked to the activation of caspase-3. As demonstrated in Fig. 7Go, fibroblasts cultured under control conditions did not express detectable levels of caspase-3. When treated with IL-1ß (10 ng/ml), these cells expressed, in a time-dependent manner, high levels of immunodetectable caspase-3 that appeared as both a 35-kDa band representing pro-caspase-3 and a 20-kDa band, which is the p-20 subunit of mature caspase-3. The caspase-3 signal in this particular study was near maximal after 12–16 h of cytokine exposure and began to decay at 24 h. Caspase-3 levels in different fibroblast strains were variable.



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  		FIGURE 7. The expression of caspase-3 in IL-1ß-treated human fibroblasts. Fibroblasts were allowed to proliferate to confluence and then they were shifted to medium supplemented with 1% FBS for 18 h. Monolayers were then treated with IL-1ß (10 ng/ml) for the times indicated along the abscissa. Cell proteins were harvested as described in Materials and Methods, and 20 µ of protein per sample were electrophoresed and subjected to Western analysis for caspase-3 using the reagents specified. The bottom panel represents densitometric analysis of the caspase-3 signals from a representative experiment.

 
Expression of active IL-16 protein from cytokine-activated fibroblasts is dependent upon caspase-3 activity

We have demonstrated previously that the cleavage of bioactive IL-16 from the promolecule in lymphocytes transfected with IL-16 cDNA is dependent upon the activity of caspase-3 (6). The pattern of IL-16 expression differs markedly in fibroblasts and lymphocytes. High constitutive levels of mRNA but no detectable protein are characteristic of the former. The mechanism through which IL-1ß could initiate expression of mature IL-16 protein is uncertain. We therefore determined whether caspase-3 played a role in the processing of endogenous IL-16 in cultured fibroblasts. Fibroblasts, in this case those from normal skin, were treated with specific inhibitors of caspase-3 (Ac-DEVD-CHO, 100 µM) or caspase-1 (Ac-YVAD-Ald, 100 µM), and the supernatants were assayed for IL-16-dependent chemotaxis or IL-16 protein by ELISA (Table IIIGo). The differences in activities without anti-IL-16 Abs compared with those in their presence represent IL-16-dependent chemoattraction. As the data in Table IIIGo demonstrate, the caspase-3-specific inhibitor completely blocked detectable IL-16-dependent chemotaxis released from the fibroblasts into the medium following IL-1ß treatment and attenuated the up-regulation of released, immunodetectable IL-16 protein. In contrast, the caspase-1 inhibitor failed to influence the expected up-regulation of either IL-16 activity or protein levels. To determine whether IL-16 was being synthesized but not released from fibroblasts in the setting of this caspase-3 inhibition, intracellular IL-16 was assessed by ELISA (Table IIIGo). As the data suggest, fibroblasts treated with the caspase-3 inhibitory peptide exhibited a greater time-dependent accumulation of IL-16, presumably representing pro-IL-16, than did either those receiving nothing (control) or those receiving the caspase-1 inhibitor. These results are consistent with caspase-3 activity being necessary for the release, but not the synthesis, of mature IL-16. It would appear that the caspase-3 pathway in fibroblasts is critical to the maturation and release of biologically active IL-16 protein.


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  		Table III. Effect of specific caspase-1 and caspase-3 inhibitors on IL-16-dependent chemotaxis, intracellular IL-16, and release from human fibroblasts

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Activated CD4-bearing T lymphocytes as well as other bone marrow-derived cells migrate to sites of inflammation in response to chemoattractant signals. Fibroblasts have been shown previously to express several chemoattractant molecules and thus they may function as initiators of the early events associated with recruitment of bone marrow-derived cells to sites of inflammation (31, 32, 33, 34, 41). We now present data indicating that IL-16 expression and release from fibroblasts are potentially important mechanisms by which T lymphocytes can be recruited to these sites. Our findings are consistent with the fibroblast representing a major site of IL-16 biosynthesis. In fact, the levels of IL-16 released into conditioned medium from activated fibroblasts are substantially greater, on a per cell basis, than those found in either CD4+ or CD8+ lymphocytes. Lymphocytes also express basal IL-16 mRNA, and cell activation does not appear to influence the steady state levels of the transcript (42). Lymphocytes store IL-16 as a precursor molecule, unlike unprovoked fibroblasts, which fail to express detectable precursor or active IL-16 protein under basal culture conditions.

Fibroblasts from certain anatomic regions of the body exhibit phenotypic attributes not shared by fibroblasts derived from other areas (15, 16, 17, 22, 23, 24, 25, 26, 43, 44). Unlike these specialized features, it would appear from our results to date that cells from a wide spectrum of tissues are capable of expressing IL-16. This finding implies that the synthesis and release of substantial amounts of IL-16 may be common to many, if not all, human fibroblast populations. Moreover, given the high levels of IL-16 production achieved, the fibroblast may, in fact, serve as a major cellular mediator of CD4+ lymphocyte recruitment under conditions where they are fully activated. The concept that fibroblasts might assume complex roles in tissue remodeling and repair, apart from the laying down of collagen and other components of the extracellular matrix, has been emerging for some time. By virtue of their extensive biosynthetic repertoire, fibroblasts appear capable of orchestrating the initiation of the inflammatory response and the perpetuation of both proinflammatory and profibrotic events. Besides the synthesis of chemokines and other chemoattractive molecules, fibroblasts can express IL-1 (45, 46, 47, 48), TNF-{alpha} (48), TGF-ß (49), and insulin-like growth factor I (50).

The mechanism through which IL-1ß and the other cytokines tested provoke the expression and release of IL-16 from fibroblasts is not yet fully defined. From earlier work, it is clear that the synthesis of this chemoattractant molecule in response to various signals is cell specific. For instance, in CD8+ T lymphocytes, histamine elicits the release of IL-16 through a mechanism that is post-translational (11). In contrast, the up-regulation of IL-16 gene transcription appears to be crucial to the release of active IL-16 from mast cells treated with PMA or C5a (13). The finding of IL-16 mRNA in untreated mast cells is reminiscent of the current observations in fibroblasts (Figs. 5Go and 6Go), but mast cells store appreciable amounts of preformed IL-16. This would appear not to be the case in fibroblasts, where immunorecognition of IL-16 by indirect immunofluorescence and ELISA failed to detect either precursor or mature polypeptide in untreated cells. IL-1ß induces the translation of preformed IL-16 mRNA and the expression of IL-16 protein. IL-1ß-treated fibroblasts express caspase-3 (Fig. 7Go), and blocking the activity of this enzyme with a caspase-3-specific inhibitory peptide precluded the formation of active IL-16 (Table IIIGo). The role of this enzyme in the processing of mature IL-16 in lymphocytes has recently been established (6). It would appear, therefore, that cytokine activation of mature IL-16 protein expression in fibroblasts involves multiple steps. Moreover, while the patterns of IL-16 mRNA expression and pro-IL-16 protein storage differ among the various cell types shown to synthesize the cytokine, cleavage of active IL-16 seems to use the same proteolytic pathway in IL-16-expressing cells.

From the results shown, it seems that IL-16 and RANTES both represent major lymphocyte chemoattractants expressed by human fibroblasts in culture. The importance of IL-16 expression in fibroblast-like cells in vivo was demonstrated recently in the report by Franz et al. (51), in which high levels of IL-16 were found in the synovial fluid of patients with rheumatoid arthritis, and IL-16 mRNA was easily detected in fibroblast-like synovial membrane cells by in situ hybridization. However, fibroblasts elaborate other chemoattractant signals for CD4-bearing cells, because neutralization of IL-16 failed to block substantial lymphocyte migratory activity in some of the fibroblast strains tested. The majority of this activity, not attributable to IL-16, is likely to emanate from RANTES (Table IGo). Our studies to date demonstrate that RANTES expression and inducibility in the fibroblast are very different from those of IL-16. In the case of RANTES, we were able to detect low levels of mRNA under basal (control) culture conditions. When the cells were treated with IL-1ß, steady state RANTES mRNA levels were induced within 2 h and were elevated several-fold by 7 h of cytokine exposure. RANTES protein and chemotactic activity were observed within 3–6 h of IL-1ß treatment, well in advance of the apparent IL-16 contribution to lymphocyte chemotaxis. Thus, despite constitutive expression of IL-16 mRNA, it would appear that RANTES may represent the earlier chemotactic signal emanating from fibroblasts. This result may mirror the temporal relationship between IL-16 and RANTES in vivo, but confirmation of this must await more extensive kinetic studies in intact experimental animals.

Fibroblasts express a number of other chemoattractant molecules. For instance, IL-8 can be substantially induced in dermal fibroblasts by cytokines (31). Mauviel et al. demonstrated that leukoregulin can substantially up-regulate IL-8 expression (31). We reported recently that orbital and thyroidal fibroblasts display CD40, and that IL-8 as well as IL-6 release from these fibroblasts can be dramatically enhanced by the engagement of CD40 with CD154 (17, 52). Monocyte chemotactic protein-1 and -2 can be expressed at substantial levels by certain fibroblasts (33). Thus, there is abundant reason to believe that cultured human fibroblasts can, upon activation with a variety of proinflammatory molecules, synthesize and release an array of chemoattractant signals. With regard to T lymphocyte trafficking, it would appear from the data presented here that IL-16 constitutes a predominant signal emanating from fibroblasts.

The finding that fibroblasts from several anatomic regions can express and release substantial IL-16 levels and thus activate CD4+-bearing T lymphocytes is of considerable potential importance with regard to our understanding of connective tissue inflammation. The low level of IL-16 release found in cultures not treated with proinflammatory cytokines suggests that, at least in vitro, this chemoattractant does not play a major role in CD4 cell surveillance in physiological states. It is possible, however, that in situ fibroblasts are tonically stimulated by multiple factors emanating from neighboring cells and that IL-16 release is considerably greater, even under nonpathological conditions. IL-1ß exhibited especially robust activity with regard to provoking IL-16 release, and given the ubiquitous nature of both IL-1{alpha} and IL-1ß as proinflammatory cytokines, these may well represent important signals for chemoattractant molecule release from fibroblasts. Moreover, both can be expressed by fibroblasts themselves and thus may have an autocrine function with regard to IL-16 and RANTES synthesis.

Among the fibroblasts included in the current studies were those derived from the orbital connective tissue and the thyroid. Autoimmune processes affecting these tissues are components of Graves’ disease. In that disease syndrome, the orbital contents become infiltrated with activated lymphocytes and mast cells (53, 54). The make-up of the lymphocyte population found in orbital tissue affected in TAO is controversial with regard to whether CD4+- or CD8+-bearing cells predominate (55, 56), but these conflicting data most likely reflect the limitations in access to tissue sampling. The small molecules synthesized by bone marrow-derived cells are currently believed to activate orbital fibroblasts, leading to the aberrant expression of PG endoperoxide H synthase-2 (27) and the overproduction of hyaluronan (22). These inductions we believe underlie the intense inflammation and orbital connective tissue volume expansion, respectively, the two cardinal features of tissue remodeling found in TAO. Thus, our finding that IL-16 is expressed at high levels in orbital and thyroid-derived fibroblasts treated with IL-1ß represents a potentially critical insight into how CD4+ lymphocytes might be recruited to the orbit and thyroid in these processes. The observations reported here have broad biological implications, in that they define a previously unrecognized attribute of human fibroblasts in culture. It would appear that IL-16, in concert with RANTES, constitutes an important chemoattractant signal, emanating from fibroblasts, for CD4+-bearing bone marrow-derived cells. Our findings imply the potential for fibroblast-derived IL-16 participating as an early sentinel in inflammation and define a potentially important target for disease intervention.


   Acknowledgments
 
The expert technical assistance of Mario Roma and Heather Meekins is gratefully acknowledged.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grants EY11708, EY08976, and HL32802 and by a Merit Review award from the Department of Veterans Affairs. Back

2 Address correspondence and reprint requests to Dr. Terry J. Smith at his current address, Division of Molecular Medicine, University of California-Los Angeles, School of Medicine, Harbor-UCLA Medical Center, 1124 West Carson Street, Building C-2, Torrance, CA 90502. E-mail address: Back

3 Abbreviations used in this paper: Ac-DEVD-CHO, Ac-Asp-Glu-Val-Asp-aldehyde; Ac-YVAD-Ald, acetyl-Tyr-Val-Ala-Asp-CHO; TAO, thyroid-associated ophthalmopathy. Back

Received for publication September 27, 1999. Accepted for publication January 14, 2000.


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 Introduction
 Materials and Methods
 Results
 Discussion
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