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The Journal of Immunology, 1999, 163: 3969-3975.
Copyright © 1999 by The American Association of Immunologists

Intracellular IL-1 Receptor Antagonist Is Elevated in Human Dermal Fibroblasts That Overexpress Intracellular Precursor IL-1{alpha}1

Gloria C. Higgins2,*, Yong Wu{dagger} and Arnold E. Postlethwaite{dagger},{ddagger}

* Department of Pediatrics, Division of Clinical Immunology, Crippled Children’s Foundation Research Center at LeBonheur Children’s Medical Center, Memphis, TN 38103; {dagger} Department of Medicine, Division of Connective Tissue Diseases, University of Tennessee, Memphis, TN 38163; and {ddagger} Department of Veterans Affairs Medical Center, Memphis, TN 38104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cultured dermal fibroblasts from systemic sclerosis patients express higher levels of intracellular IL-1{alpha} than fibroblasts from healthy controls. In this study, we found that systemic sclerosis dermal fibroblasts also express higher levels of the intracellular isoform of IL-1 receptor antagonist (icIL-1Ra) than normal fibroblasts after stimulation with IL-1ß or TNF-{alpha}. A possible relationship between elevated precursor IL-1{alpha} (preIL-1{alpha}) and elevated icIL-1Ra was investigated by transducing normal dermal fibroblasts to overexpress preIL-1{alpha}, preIL-1ß, or icIL-1Ra. Fibroblasts that overexpressed icIL-1Ra did not have elevated levels of IL-1{alpha}. On the other hand, fibroblasts that overexpressed preIL-1{alpha} had at least 4-fold higher basal levels of icIL-1Ra than control fibroblasts and 4-fold higher levels of icIL-1Ra after induction with IL-1ß or TNF-{alpha}. Fibroblasts overexpressing preIL-1ß did not exhibit elevated icIL-1Ra. The differences in icIL-1Ra protein levels were reflected in differences in mRNA. In contrast, IL-1-stimulated levels of MCP-1 and IL-6 were not different in control and preIL-1{alpha}-transduced fibroblasts. Addition of neutralizing anti-IL-1{alpha} Abs to fibroblast cultures did not diminish basal or stimulated levels of icIL-1Ra in the preIL-1{alpha}-transduced cells, supporting an intracellular site of action of preIL-1{alpha}. This is the first report of an association between intracellular levels of these IL-1 family members. We hypothesize that intracellular preIL-1{alpha} participates in the regulation of icIL-1Ra.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin 1{alpha} is synthesized as a biologically active precursor, preIL-1{alpha},3 which is proteolytically processed during secretion to cause release of the carboxyl-terminal "mature" cytokine (1). Fibroblasts appear to be deficient in their ability to process and secrete IL-1{alpha} and to retain the precursor form intracellularly when stimulated to produce IL-1 (2, 3). The IL-1 receptor antagonist (IL-1Ra) exists as at least three isoforms. Secreted IL-1Ra (sIL-1Ra) and intracellular IL-1Ra (icIL-1Ra) are transcribed from the same gene from two separate promotors (4, 5) and are differentially expressed in various cell types and tissues (4, 5, 6). The mRNAs for these forms encode different amino termini (7), so that icIL-1Ra shares 152 amino acid residues with sIL-1Ra but has a short hydrophilic amino terminus instead of a leader sequence. Another isoform of the intracellular IL-1 receptor antagonist (icIL-1RaII) has been described, which is encoded by the same gene but contains a 21-amino acid insert encoded by an additional exon (8). Keratinocytes and other epithelial cells constitutively produce icIL-1Ra (9). It was previously reported that normal fibroblasts lack constitutive production but can be stimulated by IL-1, TNF-{alpha}, and mitogens to produce high levels of icIL-1Ra (10).

Numerous endogenous and exogenous substances induce or regulate the synthesis of IL-1 family members. A large body of knowledge exists concerning the complex autoregulatory loops involving secreted forms of IL-1{alpha} and ß, sIL-1Ra, and cell membrane IL-1 receptors. For example, binding of IL-1 to cell surface receptors results in induction of more IL-1 synthesis (11, 12) and induction of sIL-1Ra synthesis (13, 14, 15). Secreted IL-1Ra blocks both the activity and the synthesis of IL-1{alpha} and ß (16, 17, 18). In contrast, there is little insight into the biological roles of the intracellular forms of IL-1 family cytokines.

Altered responses of fibroblasts to cytokine stimulation are likely to be important in the pathogenesis of systemic sclerosis (SSc) (19, 20). In agreement with previous reports (21, 22), we have observed that cultured dermal fibroblasts from patients with SSc have elevated levels of intracellular IL-1{alpha} compared with dermal fibroblasts from matched controls. In this report, we present data demonstrating that icIL-1Ra is also elevated in stimulated SSc fibroblasts compared with controls. To understand the relationship between these observations, we transduced normal infant foreskin fibroblasts with retroviral vectors to obtain cells that constitutively overproduce preIL-1{alpha}. These transduced fibroblasts exhibited marked up-regulation of basal icIL-1Ra levels, as well as an accentuated response of icIL-1Ra to induction by exogenously added cytokines IL-1ß and TNF-{alpha}. Our results support the idea that, analogous to the case for their secreted counterparts, intracellular preIL-1{alpha} participates in the regulation of icIL-1Ra.


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

Dermal fibroblasts from involved skin of four SSc patients and four controls, all women between the ages of 28 and 68 years, were kindly supplied by Dr. Barbara White, University of Maryland, Baltimore, MD. These cells were cultured in complete maintenance medium consisting of RPMI 1640 (Life Technologies, Gaithersburg, MD) containing 100 U/ml penicillin, 100 µg/ml streptomycin, 10 µg/ml gentamicin, 10 mM HEPES, 1 mM sodium pyruvate, 100 µM nonessential amino acids, 2 mM L-glutamine, and 10% FCS. Low passage (5th to 10th passage) cells were used. The human dermal fibroblasts used for transduction were grown from foreskins of newborn males after elective circumcision, as described previously (23). These fibroblasts were maintained in Eagle’s MEM with Earle’s salts (BioWhittaker, Walkersville, MD) containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FCS. All media supplements were from Life Technologies. Fibroblasts were passaged by treatment with 0.25% trypsin in Hanks’ balanced salt solution (Life Technologies) and replating.

Transduction of fibroblasts

Inserts for the expression vectors were obtained as follows: The cDNA for preIL-1{alpha} was purchased from American Type Culture Collection (ATCC, Rockville, MD). The cDNA for preIL-1ß was a gift from Upjohn Pharmaceuticals (Kalamazoo, MI). These cDNAs were used as templates for PCR, with 5'- and 3'-primers containing the desired terminal restriction endonuclease sites. To produce cDNA for icIL1-Ra, poly(A)+ RNA was isolated from THP-1 monocytic cells (ATCC) stimulated with 1 µg/ml LPS and 100 ng/ml PMA (4), and cDNA was produced by reverse transcription (First Strand DNA Synthesis Kit, Pharmacia, Piscataway, NJ). Total cDNA was used as a template for PCR, with 5'- and 3'-primers corresponding to icIL-1Ra sequence as reported by Haskill et al. (7) and incorporating desired terminal restriction sites. Correct sequences of all PCR products were verified by automated dye terminator cycle sequencing (ABI PRIZM Kit, Perkin-Elmer, Foster City, CA) at the University of Tennessee Molecular Resources Center. The sequence of the cloned icIL-1Ra cDNA corresponds to the isoform designated icIL-1RaI (8, 24). The cDNA inserts encoding preIL-1{alpha}, preIL-1ß, and icIL-1Ra were each cloned into the replication defective retroviral vector pLXSN, kindly supplied by Dr. A. D. Miller, Fred Hutchinson Cancer Research Center, Seattle, WA. Unmodified vector served as a control. Amphotrophic viral particles were produced as previously described (25, 26). Infant foreskin fibroblasts at the fourth passage were transduced with retroviral particles by incubation with PA317 culture media as described and selected with 500 µg/ml G418 (Life Technologies). Low passage transduced fibroblasts (eight or fewer passages after selection) were used for all experiments. Cells used for each experiment were all derived from the same donor and transduced at the same time.

Preparation of fibroblasts for experiments

Fibroblasts were harvested from confluent cultures by trypsin treatment, adjusted to the same concentration, and plated at the appropriate subconfluent density on multiwell (12- or 24-well) plates. Cells were grown 72 h to achieve confluence, as assessed visually, before changing to medium containing 5% FCS with or without the desired cytokine at the indicated concentration. Duplicate wells were set up for each assay condition. Replicate wells were also set up for manual cell counting. Neutralizing, affinity-purified goat anti-IL-1{alpha} and anti-IL-1Ra (R&D Systems, Minneapolis, MN) were added to cultures in some experiments.

Measurement of cytokines

Culture media were harvested and treated by addition of protease inhibitors (25 mM benzamidine, 1 mM PMSF, 10 mM N-ethylmaleimide, 1 mM EDTA, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µg/ml pepstatin). Except as described below, fibroblasts were solubilized directly on multiwell plates by one of two methods that yielded equivalent results in direct comparisons. Normal (NL) and SSc dermal fibroblasts were lysed by sonication in ice-cold PBS containing 2% FCS and protease inhibitors as above. Transduced dermal fibroblasts and vector controls were lysed by incubation for 30 min at 4°C with 50 mM Tris, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Sigma, St. Louis, MO), 0.1% Nonidet P-40, pH 7.5, containing protease inhibitors as above. Culture media and cell lysates were cleared by centrifugation at 18,000 x g for 30 min at 4°C, and then 0.1 volume of 1 M NaCl was added to the cleared lysates. Samples were stored in aliquots at -70°C.

There was not enough cell-associated IL-1{alpha} in the unstimulated NL and SSc fibroblasts to permit accurate measurement in cells lysed directly on multiwell plates as described above. Therefore, for these experiments only, the fibroblasts were grown to confluence on 10-cm dishes, harvested by treatment with trypsin, collected by centrifugation in the presence of serum, washed, counted, and disrupted by sonication. Culture media and lysates were treated as described above.

IL-1{alpha}, IL-1ß, and IL-1Ra were measured by ELISA (R&D Systems). PreIL-1ß was measured by a specific ELISA (Cistron Biotechnology, Pine Brook, NJ) which does not recognize mature IL-1ß. Samples were diluted when necessary with fresh culture media or lysis buffer. Neither buffer used for cell lysis interfered with measurements of these cytokines (not shown). Each well was assayed in duplicate. Except where otherwise indicated, results (picograms or nanograms per ml) were converted to picograms or nanograms per 2 x 105 cells with the manual cell count. MCP-1 and IL-6 were measured in culture medium by ELISA (R&D Systems). ELISA results are expressed as mean concentration ± SE.

RNase protection assay (RPA)

cDNA for secreted (s)IL-1Ra was purchased from the ATCC. Partial cDNAs, obtained by restriction endonuclease digestion or PCR, were cloned into pTRI-kan vectors (Ambion, Austin, TX) to serve as templates for 32P-labeled riboprobes. For the riboprobe designated IL-1{alpha}pro, the insert consisted of base pairs 1–336 of precursor IL-1{alpha}. For the probe designated IRAs/ic, the insert corresponded to base pairs 9 to 224 of sIL-1Ra cDNA coding sequence. Whereas sIL-1Ra mRNA complements the entire 216-base sequence, icIL-1Ra mRNA complements only 160 bases. Under the conditions of probe excess used for these assays, there is no loss of sensitivity for either isoform of IL-1Ra when mRNAs for sIL-1Ra and icIL-1Ra are mixed in various proportions. The pTRI-GAPDH vector was purchased from Ambion. [{alpha}-32P]UTP-labeled riboprobes were prepared from linearized templates by transcription with T7 RNA polymerase (Maxi-Script Kit, Ambion) and purified by gel electrophoresis.

Poly(A)+ RNA was isolated from equal numbers of cells of each type by alkaline detergent lysis and affinity chromatography on oligo(dT) latex beads (Oligotex, Qiagen, Santa Clara, CA). For RPA, (RPA II kit, Ambion), the RNA was hybridized to the 32P-labeled probes, the product was digested with RNases A and T1, and the protected fragments were resolved by electrophoresis on 4% acrylamide-urea gels. Bands were visualized and quantitatively compared with a Bio-Rad Model GS-505 phosphor imager (Bio-Rad, Hercules, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Basal cell-associated IL-1{alpha} in SSc fibroblasts

To confirm previous reports of elevated intracellular IL-1{alpha} in SSc fibroblasts, we measured intracellular IL-1{alpha} protein and preIL-1{alpha} mRNA in fibroblasts derived from involved skin of four female SSc patients, from four female controls of similar age, and from one male control (see Materials and Methods). Extracts were analyzed for IL-1{alpha} content by ELISA. Extracts of unstimulated normal fibroblasts contained a mean of 8 ± 2 pg IL-1{alpha}/106 cells (1.6 pg IL-1{alpha}/2 x 105 cells), whereas extracts of SSc fibroblasts contained a mean of 28 ± 5 pg/106 cells (5.6 pg IL-1{alpha}/2 x 105 cells), significantly different at p < 0.05.

Cell-associated IL-1{alpha} and IL-1Ra in SSc fibroblasts after IL-1ß or TNF-{alpha} stimulation

Dermal fibroblasts from SSc patients and normal controls were treated with IL-1ß or TNF-{alpha} for 48 h (Fig. 1GoA). Cells were disrupted directly on multiwell plates as described in Materials and Methods. The mean levels of cell-associated IL-1{alpha} were significantly greater for stimulated SSc fibroblasts than for the stimulated normal controls. We also measured the levels of cell-associated IL-1Ra in SSc and normal control fibroblasts before and after stimulation. These levels were in the same range (tens to hundreds of nanograms per 106 cells) as those previously detected in human fibroblasts (10), keratinocytes (7), and ovarian carcinoma cells (27). As shown in Fig. 1GoB, the mean levels of cell-associated IL-1Ra were significantly greater for the SSc fibroblasts than for the controls after treatment with IL-1ß or TNF-{alpha}.



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  		FIGURE 1. Cell-associated IL-1{alpha} and IL-1Ra in stimulated fibroblasts. NL and SSc fibroblasts were grown just to confluence and then stimulated with 125 pg/ml IL-1ß or 1 ng/ml TNF-{alpha} for 48 h. Control samples (-) were treated with vehicle alone. Cell extracts were prepared as described in Materials and Methods, and IL-1{alpha} (A) and IL-1Ra (B) were measured by ELISA. Results are represented as mean ± SE for four SSc patients and five normal controls. SSc different from NL at p < 0.05 *, p < 0.01 **, and p < 0.001 ***. Unstimulated cells are not represented in A because in this experiment, cell-associated IL-1{alpha} in this small number of unstimulated cells was at or below the detection limit of the assay.

 
IL-1{alpha} was not detected by ELISA (<4 pg/ml) in culture media of normal or SSc fibroblasts, with or without stimulation. We were also unable to visualize IL-1{alpha} in extracts of NL or SSc fibroblasts or in immunoprecipitates of culture medium by Western blotting with a highly sensitive chemiluminescent substrate. This was not unexpected, because the ELISA results presented above predicted that the IL-1{alpha} content of these samples would be below the threshold of detection by Western blotting (100 pg/lane, not shown.) Although commercially available ELISAs do not distinguish between precursor and mature IL-1{alpha}, it is most likely that the cell-associated IL-1{alpha} was in the precursor form, based on the absence of detectable IL-1{alpha} in the culture media in our experiments and on previous reports demonstrating lack of processing and secretion of IL-1{alpha} by fibroblasts (2, 3).

PreIL-1{alpha} and icIL-1Ra mRNA in NL and SSc fibroblasts

Preliminary results from a quantitative reverse transcription-PCR procedure (28) demonstrated a severalfold increase in preIL-1{alpha} and icIL-1Ra mRNA levels in SSc fibroblasts compared with normal fibroblasts after 48 h of stimulation with IL-1ß (not shown). To study the time course of mRNA induction, the RNase protection assay was used. Replicate cultures were treated with IL-1ß and harvested by trypsin treatment at various times after stimulation. Poly(A)+ RNA prepared from these cells was hybridized to 32P-labeled riboprobes complementary to preIL-1{alpha} and GAPDH mRNAs and to the riboprobe IRAs/ic, which is complementary to 216 bases of sIL-1Ra and to 160 bases of icIL-1Ra mRNA. Fig. 2Go shows representative results from one SSc patient and one matched control. PreIL-1{alpha} and icIL-1Ra mRNA were easily detected in the SSc fibroblasts at 8, 12, and 16 h, in amounts greater than in the control as indicated by band volumes. In the SSc fibroblasts, the preIL-1{alpha} mRNA peaked by 8 h and subsequently declined. Intracellular IL-1Ra mRNA was first detectable at 8 h in both normal and SSc fibroblasts but remained elevated for at least 16 h in SSc fibroblasts. sIL-1Ra mRNA was not detected.



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  		FIGURE 2. Time course of preIL-1{alpha} and icIL-1Ra mRNA induction in SSc and normal fibroblasts. RPA was performed on poly(A)+ RNA prepared from equal numbers of normal and SSc fibroblasts stimulated for the indicated times with 250 pg/ml IL-1ß. Left, probes, with > denoting the migration of full-length probe at 1/50 dilution. Right, Position of the fragments protected from RNase digestion: <{alpha} = preIL-1{alpha} (336 bases), <ic = icIL-1Ra (160 bases), and <G = GAPDH. Band volumes for the protected probe fragments, normalized to GAPDH, are shown below the lanes. Because of differences in abundance of mRNAs, exposure times for these gels were different: the top two were exposed for 20 h; whereas the GAPDH gel was exposed for 1 h.

 
Retrovirally transduced human dermal fibroblasts

To investigate a possible relationship between increased intracellular IL-1{alpha} and icIL-1Ra levels, we transduced infant foreskin fibroblasts to constitutively overproduce preIL-1{alpha}, preIL-1ß, or icIL-1Ra. The human dermal fibroblasts (HDF) so treated are designated HDF-preIL-1{alpha}, HDF-preIL-1ß, and HDF-icIL-1Ra, respectively. Fibroblasts transduced with unmodified vector, expressing only vector-derived neomycin acetyltransferase permitting selection in G-418, are designated HDF-vec. These cells all exhibited normal morphology and growth characteristics (not shown).

Unstimulated, transduced HDFs were shown by RPA to constitutively produce the appropriate retrovirally encoded IL-1 family member mRNA transcript, whereas none was detected in unstimulated control HDF-vec (not shown). Synthesis of protein products was verified by ELISA. The IL-1{alpha} and IL-1Ra ELISAs may underestimate the absolute amounts produced, because these Abs and standards are designed to measure mature IL-1{alpha} and sIL-1Ra instead of preIL-1{alpha} and icIL-1Ra.

IL-1 family cytokine expression by HDF-vec, HDF-preIL-1{alpha}, and HDF-icIL-1Ra

As shown in a representative experiment in Table IGo, cell-associated IL-1{alpha} was detected in extracts of unstimulated HDF-preIL-1{alpha}, but the IL-1{alpha} content of the culture medium was below the limits of the assay. No IL-1{alpha} was detectable in extracts or in medium from unstimulated HDF-vec. HDF-preIL-1{alpha} expressed greater basal cell-associated IL-1Ra than HDF-vec, and small amounts of IL-1Ra were detected in culture medium after a 24-h incubation. In contrast, HDF-icIL-1Ra did not express greater basal IL-1{alpha}.


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  		Table I. Basal cytokine levels in transduced fibroblasts1

 
The basal preIL-1{alpha} content of HDF-preIL-1{alpha} was 2–3 times that found in SSc fibroblasts (12.7 pg vs 5.6 pg per 2 x 105 cells). To test whether these cells show responses similar to those of SSc fibroblasts, we stimulated them with IL-1ß or TNF-{alpha} and measured IL-1Ra in cell extracts. Like SSc fibroblasts, HDF-preIL-1{alpha} synthesized significantly more cell-associated IL-1Ra than the control HDF-vec in response to either IL-1ß (Fig. 3Go) or TNF-{alpha} (not shown). The peak of IL-1Ra production was at 24 h, but significant differences persisted for at least 48 h in other experiments. In dose-response experiments, a plateau of response was achieved at 125 pg/ml IL-1ß or greater (not shown).



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  		FIGURE 3. Time course of IL-1Ra production in stimulated transduced fibroblasts. Confluent fibroblasts were treated with 250 pg/ml IL-1ß. At the indicated time points, medium was removed, and cells were lysed with detergent buffer containing protease inhibitors. Lysates were cleared by centrifugation before IL-1Ra measurement by ELISA. Results are expressed as the mean ± SEM for triplicate determinations from the same cell extracts.

 
Analysis of protein products by biosynthetic labeling and immunoprecipitation (29) verified a 33-kDa product immunoprecipitated by anti-IL-1{alpha} Abs in lysates of HDF-preIL-1{alpha}, with no detectable processed 17.5-kDa mature IL-1{alpha} in cell extracts or culture medium (not shown). Similarly, the expected protein migrating at ~22-kDa was immunoprecipitated by anti-IL-1Ra from detergent extracts of unstimulated HDF-icIL-1Ra, stimulated HDF-preIL-1{alpha}, and stimulated HDF-vec (Fig. 4Go). This result is in agreement with previous reports showing an apparent molecular mass of 22 kDa for icIL-1Ra, which is larger than expected based on amino acid sequence alone (8, 10). A fainter, more rapidly migrating band, which may represent sIL-1Ra, was detected in stimulated HDF-vec and HDF-preIL-1{alpha}.



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  		FIGURE 4. Immunoprecipitation of icIL-1Ra from [35S]methionine-labeled fibroblasts. Confluent fibroblasts in 10-cm dishes were cultured for 8 h with and without 250 pg/ml IL-1ß, washed, and then cultured an additional 2 h in methionine-deficient medium containing 100 µCi/ml [35S]methionine. The cells were solubilized directly on the plates using a detergent solution with protease inhibitors (29 ). Immunoprecipitation was conducted as previously described (29 ), using rabbit anti-IL-1Ra IgG (R&D Systems). icIL-1Ra was resolved on a 13% polyacrylamide-SDS gel and visualized by autoradiography. The position of the 21-kDa molecular mass marker is indicated on the left. The biosynthetically labeled cells were as follows: Lane 1, unstimulated HDF-icIL-1Ra; lane 2, unstimulated HDF-vec; lane 3, stimulated HDF-preIL-1{alpha}; lane 4, stimulated HDF-vec.

 
icIL-1Ra expression by HDF-preIL-1ß

Using transduced cells prepared from five different donors at different times, we consistently found markedly higher basal and stimulated icIL-1Ra in HDF-preIL-1{alpha} than in HDF-vec. We never found the converse, higher preIL-1{alpha} in HDF-icIL-1Ra than HDF-vec, under any condition. To further confirm that the elevation of icIL-1Ra was specifically associated with preIL-1{alpha} expression, fibroblasts were transduced to overexpress preIL-1ß. This IL-1 is similar in size and partially homologous to preIL-1{alpha} (30) and is not cleaved or secreted by fibroblasts (2, 31). Unlike preIL-1{alpha}, preIL-1ß is biologically inactive until it is enzymatically cleaved to the mature cytokine (32, 33). Extracts of HDF-preIL-1ß contained 800-1300 pg preIL-1ß per 2 x 105 cells, and no IL-1ß was detected in culture medium. As shown in Table IIGo, which is representative of three experiments, HDF-preIL-1ß, before or after stimulation with recombinant IL-1ß, did not have increased intracellular IL-1Ra compared with HDF-vec.


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  		Table II. Cell-associated IL-1Ra in unstimulated and stimulated fibroblasts1

 
IL-1Ra mRNA production by transduced fibroblasts

In normal and SSc fibroblasts, icIL-1Ra mRNA induction by IL-1ß was evident at 8 h, but elevation of icIL-1Ra mRNA lasted longer in SSc fibroblasts (Fig. 2Go). The time course of icIL-1Ra mRNA accumulation in IL-1ß-stimulated HDF-vec and HDF-preIL-1{alpha} had a similar pattern as shown in Fig. 5Go. This message peaked at 8 h in both kinds of fibroblasts, but a more pronounced response was observed in HDF-preIL-1{alpha}, with larger relative band volumes (normalized to GAPDH) than HDF-vec for all time points. The mRNA for sIL-1Ra was barely detectable in some samples.



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  		FIGURE 5. Time course of mRNA production. RPA was performed on poly(A)+ RNA prepared from HDF-vec and HDF-preIL-1{alpha} stimulated for the indicated times with 125 pg/ml IL-1ß. Protected fragments of the 32P-labeled probes were analyzed as for Fig. 3Go. On the left, > denotes the migration of full-length probe IRAs/ic at 1/50 dilution. Right, Positions of the protected fragments. <s = sIL-1Ra, <ic = icIL-1Ra, and <G = GAPDH. Band volumes for the probe fragments protected by icIL-1Ra mRNA, normalized to GAPDH, are shown below the lanes. Because of differences in abundance of mRNAs, exposure times for these gels were different: the IRAs/ic gel was exposed for 8 h, whereas the GAPDH gel was exposed for 1 h.

 
Effect of anti-IL-1{alpha} Abs on icIL-1Ra expression

Although we did not detect IL-1{alpha} in media of transduced fibroblasts, it remained possible that small amounts of preIL-1{alpha} released into the culture media might be responsible for increased icIL-1Ra in HDF-preIL-1{alpha}. To test this hypothesis, we treated HDF-preIL-1{alpha} with neutralizing Abs to human IL-1{alpha}. Freshly plated cells were cultured for 1 wk with daily additions of anti-IL-1{alpha}, harvested and replated for the experiment, grown to confluence with daily additions of anti-IL-1{alpha}, and stimulated with IL-ß in the presence of anti-IL-1{alpha} Ab. The amount of Ab (0.1 µg/ml) added daily to these cultures was 100 times the amount required for 50% inhibition of 50 pg/ml IL-1{alpha} according to manufacturer’s specifications (R&D Systems). In separate experiments (not shown), 0.1 µg/ml of this Ab completely inhibited the biological activity of 250 pg/ml recombinant human IL-1{alpha}. As shown in Table IIIGo, anti-IL-1{alpha} Ab had a negligible effect on basal or IL-1ß-stimulated icIL-1Ra production by HDF-preIL-1{alpha}. Anti-IL-1Ra, or the combination of anti-IL-1{alpha} and anti-IL-1Ra, added daily in the same manner, also failed to significantly affect icIL-1Ra levels in HDF-preIL-1{alpha}. These results are representative of two experiments, and are in contrast to a recent report of Muzio et al. (34) that neutralizing anti-IL-1{alpha} Ab inhibited the up-regulation of IL-8 in a fibrosarcoma cell line transduced to make excess preIL-1{alpha}.


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  		Table III. Effect on neutralizing Abs on icIL-1Ra levels1

 
Synthesis of non-IL-1 cytokines by transduced fibroblasts

In cells with elevated levels of preIL-1{alpha}, the augmented induction of icIL-1Ra by IL-1ß appeared to be a specific phenomenon, rather than a global increase in responsiveness to exogenous stimulation. As shown in Table IVGo, basal secretion of two other cytokines, MCP-1 and IL-6, was elevated in HDF-preIL-1{alpha} as compared with HDF-vec. However, the secreted levels of these cytokines by the two types of cells after stimulation by IL-1ß were similar. These results are representative of three experiments.


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  		Table IV. Cytokine production by unstimulated and stimulated transduced fibroblasts1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our observation of increased levels of cell-associated IL-1{alpha} and enhanced induction of IL-1Ra in SSc fibroblasts, as compared with normal controls, led us to investigate the relationship between intracellular preIL-1{alpha} and icIL-1Ra. Using a nontransforming retroviral vector, we transduced primary infant foreskin fibroblasts to overexpress precursor IL-1{alpha} and found that basal and cytokine-stimulated icIL-1Ra levels were markedly increased in these cells as compared with vector controls. In contrast, fibroblasts overexpressing icIL-1Ra did not produce increased amounts of preIL-1{alpha}. Because the transduced cells are not cloned, they represent a mixed population of cells with multiple random sites of insertion of the coding sequence for preIL-1{alpha}. The same results were obtained in five separate sets of transductions with different donor fibroblasts, so the possibility of artifacts caused by inadvertent clonal selection is virtually nil. Fibroblasts transduced to overexpress inactive precursor IL-1ß did not have up-regulated icIL-1Ra production. Therefore, the observed up-regulation of icIL-1Ra is likely to be a result of elevated levels of preIL-1{alpha}.

PreIL-1{alpha} has IL-1 receptor-binding affinity and biological activity very similar to that of mature IL-1{alpha} (32). Fibroblasts are deficient in the ability to process and secrete IL-1{alpha} (2, 3), and we did not detect IL-1{alpha} in culture media of human fibroblasts. Nonetheless, it is possible that very small amounts of preIL-1{alpha} could be continuously released into the culture media, bind cell surface IL-1 receptors, and induce icIL-1Ra. This possibility is rendered unlikely by the absence of an effect of excess neutralizing anti-IL-1{alpha} Ab on icIL-1Ra levels. It is also conceivable that cell-associated preIL-1{alpha} could be expressed on the outer surface of the fibroblasts and engage IL-1 receptors on cell-to-cell contact. PreIL-1{alpha} is myristoylated within the propeptide region, which would favor its targeting to membranes (35), and surface IL-1{alpha} has been demonstrated on B lymphocytes and monocytes by immunofluorescence (36, 37). A functional membrane-bound form of IL-1 was detected on macrophages, endothelial cells, and fibroblasts (38, 39), although others have suggested that this activity may have been an artifact of cell fixation (40). We cannot completely exclude the possibility in our experiments that membrane preIL-1{alpha} may have been, in some manner, unavailable to anti-IL-1{alpha} Ab added to the culture media but still able to bind IL-1 receptors on adjacent cells.

Although preIL-1{alpha} and icIL-1Ra have been shown to accumulate in a variety of cells, the biological functions of intracellular IL-1 family members are poorly understood. PreIL-1{alpha} has a nuclear localization sequence in its propeptide (amino-terminal) region (41) and has been shown to localize in cell nuclei (42). This information has led to speculation of a possible nuclear site of action of preIL-1{alpha} (41, 42). Elevated production of preIL-1{alpha} has been implicated in senescence and growth inhibition of fibroblasts and endothelial cells (42, 43, 44). In contrast to these reports, we did not observe any growth inhibition of fibroblasts transduced to overexpress preIL-1{alpha} compared with control, even in cells carried for >25 passages (data not shown). In fibrosarcoma cells, a direct correlation between reduced tumorigenicity and constitutive expression of IL-1{alpha} has been observed (45). Others have shown that transfection of murine EL4 thymoma cells with a nonsecreted mature IL-1{alpha} fusion protein resulted in constitutive production of IL-2 without addition of exogenous IL-1 and a decrease in expression of type I IL-1 receptors on the cell surface (46). Earlier reports have provided conflicting evidence for the presence or absence of intracellular IL-1 receptors in the EL4 T cell line (47, 48) but, to our knowledge, intracellular IL-1 receptors have not been demonstrated in human fibroblasts.

Similarly, little is known about the biological actions of icIL-1Ra. When purified natural or recombinant icIL-1Ra is applied exogenously to cells, it has biological activity similar to that of sIL-1Ra (7, 8). However, icIL-1Ra differs from the secreted isoform in two important respects: 1) its amino terminus lacks a leader sequence, with the result that the protein is retained intracellularly rather than being secreted (7); and 2) its synthesis is regulated by a separate promoter (5). Intracellular IL-1Ra is made in abundant quantities by human fibroblasts (10), neutrophils (8), keratinocytes (7), and ovarian carcinoma cells (27). Little or no IL-1Ra is found in culture media of unstimulated keratinocytes (7) or fibroblasts (10). Because a 10- to 100-fold molar excess of sIL-1Ra over IL-1{alpha} or IL-1ß is required for inhibition of signal transduction (8, 49), it is unlikely that sufficient quantities of icIL-1Ra are present extracellularly to functionally antagonize receptor binding of extracellular IL-1 to these cells. All the types of cells listed that synthesize icIL-1Ra are capable of responding in vitro to exogenous IL-1 in usual dose ranges. In addition, it was recently reported that fibrosarcoma cells transduced to make excess icIL-1RaI or icIL-1RaII had normal IL-1 responsiveness (34). Thus, there is no evidence that icIL-1Ra has a physiological role as a competitive inhibitor of extracellular IL-1. Although the intracellular isoform was first described 8 years ago (7), to date only a single biological function has been demonstrated for this cytokine. Watson et al. (27) reported that icIL-1Ra decreased the stability of mRNA for the chemokine GRO in transduced ovarian carcinoma cell lines, resulting in impaired GRO synthesis after stimulation by IL-1ß.

Although it was previously shown that cell-associated IL-1Ra in fibroblasts increased after stimulation with exogenous IL-1, TNF-{alpha}, LPS, and PMA (10), our study is the first one that describes a relationship between levels of intracellular preIL-1{alpha} and icIL-1Ra induction. Dinarello (50) and others (46, 47) have postulated the existence of intracellular IL-1 receptors, and a previous report has presented evidence for activation of cells by intracellular IL-1{alpha} (46). Our results support the idea that elevated intracellular preIL-1{alpha} results in elevated icIL-1Ra. We hypothesize that intracellular regulatory loops may exist that modulate the expression and activities of the intracellular IL-1 family members, analogous to the case for the secreted IL-1s. Because SSc fibroblasts express elevated basal preIL-1{alpha} and induction of icIL-1Ra compared with normal fibroblasts, it is interesting to speculate that these intracellular cytokines may play a role in the pathogenesis of scleroderma.


   Acknowledgments
 
We thank Carolyn Fields, Patricia Wheller, and Diane Weisfeld for technical assistance and John L. Foster for suggestions and critical reading of the manuscript.


   Footnotes
 
1 This work was supported by a research grant from the Scleroderma Foundation/United Scleroderma Federation (G.H.), a Cannon Award from the Crippled Children’s Foundation Research Center at LeBonheur Children’s Medical Center (G.H.), a Biomedical Science Grant from the Arthritis Foundation (A.P., G.H.), and a merit review grant from the Department of Veterans Affairs (A.P.). Back

2 Address correspondence and reprint requests to Dr. G. Higgins, Ohio State University Department of Pediatrics, Division of Rheumatology, Children’s Hospital, 700 Children’s Drive, Columbus, OH 43205. E-mail address: Back

3 Abbreviations used in this paper: preIL-1{alpha}, precursor IL-1{alpha}; preIL-1ß, precursor IL-1ß; IL-1Ra, IL-1 receptor antagonist; icIL-1Ra; intracellular IL-1 receptor antagonist; sIL-1Ra, secreted IL-1 receptor antagonist; SSc, systemic sclerosis; HDF, human dermal fibroblasts; -vec, vector; RPA, RNase protection assay; NL, normal; MCP-1, monocyte chemoattractant protein-1. Back

Received for publication November 10, 1998. Accepted for publication July 19, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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