The precursor form of IL-1α is an intracrine proinflammatory activator of transcription

Abstract

Although most cytokines are studied for biological effects after engagement of their specific cell surface membrane receptors, increasing evidence suggests that some function in the nucleus. In the present study, the precursor form of IL-1α was overexpressed in various cells and assessed for activity in the presence of saturating concentrations of IL-1 receptor antagonist to prevent receptor signaling. Initially diffusely present in the cytoplasm of resting cells, IL-1α translocated to the to nucleus after activation by endotoxin, a Toll-like receptor ligand. The IL-1α precursor, but not the C-terminal mature form, activated the transcriptional machinery in the GAL4 system by 90-fold; a 50-fold increase was observed using only the IL-1α propiece, suggesting that transcriptional activation was localized to the N terminus where the nuclear localization sequence resides. Under conditions of IL-1 receptor blockade, intracellular overexpression of the precursor and propiece forms of IL-1α were sufficient to activate NF-κB and AP-1. Stable transfectants overproducing precursor IL-1α released the cytokines IL-8 and IL-6 but also exhibited a significantly lower threshold of activation to subpicomolar concentrations of tumor necrosis factor α or IFN-γ. Thus, intracellular functions of IL-1α might play an unforeseen role in the genesis of inflammation. During disease-driven events, the cytosolic precursor moves to the nucleus, where it augments transcription of proinflammatory genes. Because this mechanism of action is not affected by extracellular inhibitors, reducing intracellular functions of IL-1α might prove beneficial in some inflammatory conditions.

The IL-1 family has 10 known members, and the precursor form of each member lacks a clear signal peptide, suggesting persistence of an early evolutionary role as intracellular proteins (1). Although one form of the IL-1 receptor (IL-1R) antagonist (IL-1Ra) possesses a signal peptide and is secreted by means of the Golgi, the IL-1Ra gene has alternative splice/insertion sites; thus, in nearly all cells, variants of the antagonist cytokine remain intracellular (reviewed in ref. 2). Although recombinant forms of mature IL-1α (mIL-1α), mIL-1β, and mIL-18 are active agonists, the precursor forms of IL-1β and IL-18 (pIL-1β and pIL-18) are inactive. These precursors use the intracellular cysteine protease caspase-1 to cleave the precursor to an active mature form and exit the cell. The precursor form of IL-1α (pIL-1α), in contrast, is fully active when it engages the IL-1 surface receptor. Unlike IL-1β, IL-1α is rarely found in the extracellular compartment but rather is primarily associated with the cell as either an intracellular protein or a membrane form (3, 4). A membrane-associated calcium-dependent protease, calpain, can process pIL-1α to a mature form (5). However, many cells are devoid of this processing ability, leaving the intracellular precursor form in abundance. pIL-1α possesses a KVLKKRR nuclear localization site in its N-terminal propiece (6). In fact, pIL-1α translocates to the nucleus in a variety of cells, and this appears to be necessary for specific downstream events (7). pIL-1α is found in keratinocytes and epithelial cells of healthy subjects, and mice deficient in IL-1α do not exhibit an apparent phenotype (8) unless challenged by disease processes, suggesting that a putative function for the intracellular pIL-1α requires an additional external stimulus. For example, mice deficient in IL-1α are resistant to experimental colitis (unpublished observations), and IL-1α-deficient but not IL-1β-deficient mice fail to prime antigen-specific T cells (9).

The possibility that, upon nuclear localization sequence-dependent nuclear translocation, pIL-1α and propiece IL-1α (ppIL-1α) might exhibit functionality has been the focus of several studies. Indeed, overexpression of the propiece results in transformation to a malignant phenotype (10). The propiece also has been shown to induce apoptosis in some tumor cells by affecting alternate splicing of RNA (11). Antisense directed against IL-1α mRNA reverses the natural senescence of human umbilical vascular endothelial cells (12). Such findings support the hypothesis of a nuclear site of action for IL-1α.

The intracellular accumulation of cytokines like IL-1α lacking a signal peptide likely represent an evolutionary advantage, because readily secreted proteins in aquatic life forms would not be able to function during regenerative processes. An example is the ability of the Asterias forbesi starfish to regenerate a severed arm. The macrophage-like coelomocytes of these animals contain a preformed IL-1-like molecule (13, 14) that is delivered to the site of injury as an intracellular growth factor. If growth factors in starfish were secreted proteins, diffusion into the surrounding seawater would markedly reduce the concentration needed at the site for repair. That IL-1α and other intracellular cytokines persist after the evolution of extracellular receptors in terrestrial life suggests a duality of function, one function being solely in intracellular processes and the second as a mechanism to trigger the extracellular receptor and to initiate signal transduction. The two functions are not necessarily related. In the case of high-mobility group box 1 (HMGB1), one function is to facilitate gene transcription by stabilizing nucleosomes and allowing bending of DNA, and the second function is to trigger extracellular receptors as typical proinflammatory cytokine produced late in endotoxemia (15).

In the present report, we studied a nuclear function of intracellular IL-1α as an activator of transcriptional machinery with its effects on the progression of inflammation. Expression vectors encoding the three forms of human IL-1α were constructed: the full-length pIL-1α (amino acids 1–271), ppIL-1α (amino acids 1–112), and mIL-1α (amino acids 113–271). We also blocked all signal transduction by means of the surface receptors by using saturating concentrations of IL-1Ra. It appears that one of the functions of intracellular IL-1α is to lower the threshold of NF-κB- and AP-1-dependent gene expression to subpicomolar concentrations of inflammatory stimuli. Two obvious implications of these results are priming (memory) and chronicity. Cells previously primed to express IL-1α will hyper-respond to minute concentrations of various inflammatory challenges as compared with previously unprimed cells. In contrast, overexpression of intracellular IL-1α often seen in chronic diseases might play a pivotal role in the pathogenesis and maintenance of chronicity in an IL-1R-independent manner.

Materials and Methods

Cloning of Human IL-1α, IL-18, and IL-1β cDNA. IL-1α and IL-18 were cloned by PCR amplification using a human keratinocyte cDNA library as a template. The primers contained a random pentamer, an endonuclease restriction site, and the first and last 24 bp of the coding region of the gene for the 5′ and 3′ primers, respectively. pIL-1β was amplified by using cDNA derived from human peripheral blood mononuclear cells. Amplified PCR fragments were cloned into pCR3.1 or pcDNA6/His-Myc (Invitrogen). Positive clones were verified by direct sequencing.

Fluorescence Constructs. pECFP-C1 and pEYFP-N1 were purchased from Clontech. IL-1α–enhanced cyan fluorescent protein (ECFP) constructs were generated by inserting a PCR-generated fragment into a BglII and EcoRI sites in pECFP-C1 keeping an ORF from ECFP through pIL-1α coding region. The pEYFP construct was produced by ligating a PCR-generated fragment of pIL-1α synthesized by using a 3′ primer lacking the TAG stop codon. This fragment was ligated to the BamHI/EcoRI sites of the enhanced yellow fluorescent protein (EYFP) vector, creating an ORF from pIL-1α through EYFP.

Fluorescence Microscopy. Cells were observed on cover slips on a DMR fluorescent microscope (Leica, Wetzlar, Germany). CFP and YFP were observed through XF114 and XF104 filters (Omega Optical, Brattleboro, VT), respectively.

GAL4 Constructs. PCR-generated constructs using primers containing a BamHI site following a random pentamer (CGCGG) to ameliorate restriction digestion in the 5′ and a PstI site in the 3′ were inserted into the respective sites in pG5-LUC (Promega). The primers were designed to amplify the coding regions of the entire IL-1α precursor (pIL-1/GAL4), the propiece (ppIL-1/GAL4), and the mature form of IL-1α (mIL-1/GAL4), each with an ORF with the GAL4 DNA binding domain (DBD) coding region (amino acids 1–147). Sequences were verified by direct sequencing at the University of Colorado Health Sciences Center.

Expression Vectors for Human IL-1α, IL-1β, and IL-18. pIL-1α, ppIL-1α, and mIL-1α were constructed by PCR amplification by using 5′ primers containing a random pentamer, an EcoRI site, and a KOZAK sequence upstream of the 5′ 24 bp of the coding region for each form of IL-1α, respectively. 3′ primers contained a random pentamer, a PstI site, and an added TAG stop codon for the ppIL-1 construct. The primers were ligated into pcDNA6-His-myc-B (Invitrogen) previously linearized by using the same restriction endonucleases without keeping an open frame with the His-myc tag. The sequence for each plasmid was confirmed by restriction endonuclease digestion and verified by direct sequencing. pIL-18 and pIL-1β were constructed similarly by inserting a PCR-generated fragment into pcDNA6-His-Myc using primers containing a BamHI and EcoRI in the 5′ and 3′, respectively.

Transfections. Transient transfections were performed by using Lipofectamine 2000 (Invitrogen). MS1, COS-7, NIH/3T3, and RAW264.7 cells were split the day before transfection at numbers sufficient to reach 90% confluence the next day in either 96- or 24-well plates. Transfection was performed by using 0.2 μg of DNA and 0.5 μl of lipid per well for 96-well plates and 0.8 μg of DNA and 2 μl of lipid per well for 24-well plates. DNA consisted of 10% pRL Null-Renilla (Promega), 20% reporter vector (when used), and 70% expression vector. Stable transfectants were performed similarly, except that 3 days posttransfection the cells were trypsinized and plated in 10-cm-diameter plates in the presence of 2 μg/ml Blasticidin (Invitrogen) and maintained in the selection medium for at least 21 days. Cloning, when indicated, was performed by limiting dilution using 25 cells per plate in a 96-well plate.

Luciferase Assays. Luciferase and Renilla were measured by using the dual-luciferase assay kit (Promega) according to the manufacturer's instructions.

Cell Culture and Cytokine Measurements. Murine endothelial MS1, murine fibroblast NIH/3T3, murine macrophage Raw264.7 cells, rhesus COS-7 kidney fibroblast-like cells, and murine embryo fibroblast cells were used in the experiments described. The cells were maintained in DMEM (Cellgro, Waukesha, WI) supplemented with l-glutamine, 10% FCS, 100 units/ml penicillin, and 0.25 μg/ml streptomycin. Recombinant human IL-1Ra was supplied by Amgen Biologicals. Cytokine concentrations in supernatants or cell lysates were measured by liquid-phase electrochemiluminescence assays by using an ORIGEN Analyzer (IGEN, Gaithersburg, MD) (16).

Mouse Embryo Fibroblasts. Mouse embryo fibroblasts were prepared as previously described (17). Cells from passages 4–10 were used in this study.

Statistical Analysis. Cytokine and luciferase were measured in triplicate, and the mean ± SEM of at least three experiments was used in the statistical analysis.

Results

Nuclear Translocation of IL-1α upon Stimulation. Using immunofluorescence and transfections with GFP IL-1α fusion proteins in a variety of cells, we observed that pIL-1α is evenly distributed within the cytoplasm in resting cells but translocates to the nucleus after activation with inflammatory stimuli such as lipopolysaccharide, IL-1, or tumor necrosis factor (TNF). Two vectors encoding pIL-1α tagged to YFP and CFP on the N and C terminus, respectively, were constructed (Fig. 1a). Transfection of both these constructs into NIH/3T3 cells shows an even distribution of both fusion proteins in the cytoplasm of resting cells (Fig. 1 b and c) but nuclear localization in lipopolysaccharide-treated cells (Fig. 1 c–e). In this system, if IL-1α were processed by calpain, ppIL-1α and mIL-1α would be visualized as yellow and cyan, respectively. Both the yellow and cyan fluorescent/IL-1α fusion proteins are evenly distributed in the nucleus, suggesting, in addition, that the translocation is achieved without processing.

Fig. 1.

pIL-1α is translocated to the nucleus upon inflammatory stimuli. NIH/3T3 cells (20,000 per well) were plated on a cover slip placed in six-well plates. Twenty-four hours later, cells were transfected with both the vectors encoding the fusion proteins pIL-1α/EYFP-N1 and pIL-1α/ECFP-C1 (a). Twenty-four hours after transfection, an aliquot of cells were stimulated with lipopolysaccharide (10 μg/ml). Twenty-four hours later, cover slips were observed by fluorescent microscopy. (a) Fusion proteins used (calpain cleavage site marked by arrow). (b and c) Unstimulated cells observed through yellow and cyan filters, respectively. (d–f) Stimulated cells observed through light microscopy and yellow and cyan filters, respectively.

pIL-1α Activates Transcription from a Heterologous Promoter. Because under in vitro inflammatory conditions pIL-1α translocates to the nucleus, it is plausible that pIL-1α exerts its effects through a nuclear mechanism. We used the upstream activator sequence (UAS) of GAL4/GAL4 (UAS/GAL4) system to assess whether pIL-1α participates in transcription machinery. Fig. 2 depicts the construction of the test system in which the various forms of IL-1α are inserted into plasmids containing the DBD of GAL4 creating GAL4-DBD-IL-1 chimeras. As shown in Fig. 3, pIL-1α and ppIL-1α were able to activate transcription of the heterologous promoter by >90- and >50-fold, respectively, whereas mIL-1α was without activity.

Fig. 2.

The UAS/GAL4 system. (Top)The two vectors used in this experiment: IL-1 fused to GAL4-DBD (Top Left) and the GAL4 responsive reporter (Top Right). The yeast GAL4 transcription factor contains a DBD in the N terminus that lacks significant transcriptional activity. Without a transcription activator in close proximity to the DBD, the latter is unable to recruit coactivators and stimulate transcription of the reporter upon binding the enhancer site (Middle). Fusion of an activation domain, in this case forms of IL-1α, to the GAL4-DBD results in an active transcription factor, capable of recruitment of coactivators and transcription of the luciferase reporter driven by UAS/GAL4 (Bottom).

Fig. 3.

IL-1α activates transcription from a heterologous promoter. MS1 cells were transfected with empty plasmid (mock) or plasmids expressing the GAL4 DBD, either alone or upstream of pIL-1α, mIL-1α, or ppIL-1α, represented by pIL-GAL4, mIL-GAL4, and ppIL-GAL4, respectively. Cells were also cotransfected with pG5-LUC containing five copies of the GAL4 enhancer site upstream of the luciferase gene and with pNULL-Renilla to control for transfection efficiency. Data represent the mean ± SEM of three separate experiments, each performed in triplicate.

Functional Role for pIL-1α. If pIL-1α acts through transcriptional activation and is located in the nucleus upon inflammatory stimulus, part of the role of IL-1α in inflammation or in host defense might involve intracellular mechanisms without engaging surface membrane IL-1R. It has been shown that both extracellular pIL-1α and mIL-1α as well as membrane IL-1α are biologically active by means of the engagement of surface IL-1R (3, 4, 18). To demonstrate a functional role of intracellular IL-1α in the absence of triggering the IL-1 surface receptor (because of leakage of IL-1α from dying cells), cells expressing forms of IL-1α intracellularly were cultured in the presence of saturating concentrations of the IL-1Ra at 10 μg/ml. The mouse macrophage cell line Raw264.7 was stably transfected with either pIL-1α or empty vector. Treatment of these cells with the calcium ionophore A23187 activates calcium sensitive calpain and results in the release of active IL-1α (5, 19). As shown in Fig. 4, in the presence of saturating concentrations of IL-1Ra, calcium ionophore-induced macrophage inflammatory protein 2 (MIP-2) expression was similar for pIL-1α-overexpressing cells and controls, whereas vast amounts of the chemokine were present in the supernatants of cells stimulated without IL-1Ra. This indicates that 10 μg/ml recombinant IL-1Ra is sufficient to block downstream effects of IL-1α released from cells in our system.

Fig. 4.

Recombinant IL-1Ra abrogates activity of endogenous IL-1α released to supernatants. Raw264.7 cells were stably transfected with either pcDNA6/His-myc (vector) or pIL-1α expression vector. Cells were plated in the presence of 10 μg/ml IL-1Ra as indicated. On the next day, cells were washed with PBS and replenished with medium containing nothing (control), A23187, IL-1Ra, or both A23187 and IL-1Ra. Twenty-four hours later, medium was removed and concentrations of MIP-2 were measured by ECL.

To test the possibility that intracellular IL-1α activates NF-κB, MS1 cells were transfected with constructs encoding the various forms of IL-1α together with a NF-κB reporter vector. In the presence of saturating concentrations of IL-1Ra, NF-κB was activated by pIL-1α and ppIL-1α (Fig. 5 Upper). A construct of pIL-18 that is structurally related to IL-1β but, like IL-1α, is ubiquitously expressed and found mainly in the cytosol (20), exhibited minimal effects on NF-κB activity. To test whether IL-1α is necessary for NF-κB activation, mouse embryo fibroblast cells derived from either IL-1α-deficient mice or wild-type mice were studied. Cells from IL-1-deficient mice exhibited a reduced ability to activate NF-κB compared with cells from wild-type mice (Fig. 5 Lower).

Fig. 5.

Expression of intracellular IL-1α activates NF-κB in a surface IL-1R-independent manner. MS1 cells were transfected with the empty vector (vector) or with expression vectors encoding pIL-1α, mIL-1α, ppIL-1α, or pIL-18. In each case, cells were cotransfected with a vector containing the NF-κB enhancer upstream of the luciferase gene and pNULL-Renilla. Transfection and culture were performed in the presence of saturating concentrations of IL-1Ra (10 μg/ml) for IL-1 constructs or IL-18BP (1 μg/ml) for the pIL-18 construct. MS-1 WT, cells transfected with the pNFκB-LUC and pNULL-Renilla only (Upper). Mouse embryo fibroblasts from BALB/c and IL-1α-deficient (IL-1α–/–) mice were transfected with pNFκB-LUC and pNULL-Renilla in the presence of IL-1Ra as indicated. On the next day, medium was removed, cells were washed, and fresh medium with or without IL-1Ra was added. Cells were activated with 10 ng/ml TNF-α. Twenty-four hours later, cells were lysed and luciferase was measured (Lower). Data represent the mean ± SEM of three separate experiments performed in triplicate.

AP-1 was activated in a pattern similar to that seen with NF-κB, but again intracellular IL-18 had minimal effect (Fig. 6). To determine whether the presence of intracellular IL-1α forms might sensitize MS1 cells to further stimulation, pooled stable transfected MS1 cells transfected with the various IL-1 forms, vector, or pIL-1β expression vectors, were stimulated with IFN-γ or TNF-α and then secondary cytokines were measured. As shown in Fig. 7, expression of pIL-1α led to sensitization of MS1 cells to stimulation by subpicomolar concentrations of IFN-γ (Upper) or TNF-α (Lower) by releasing increased levels of MIP-2, an NF-κB-regulated chemokine. We further tested the NF-κB-regulated cytokines, IL-6 in NIH/3T3 stable transfectants (Fig. 8 Upper) and IL-8 in COS-7 transiently transfected cells (Fig. 8 Lower). Even under saturating concentrations of IL-1Ra in the cultures, cells expressing pIL-1α produced these NF-κB-regulated cytokines without additional stimuli.

Fig. 6.

Expression of intracellular IL-1α activates AP-1 in a surface IL-1R-independent manner. Experiments were performed similarly to those in Fig. 5 Upper, except using a reporter vector containing the AP-1 enhancer upstream of the luciferase gene. Data represent the mean ± SEM of three separate experiments performed in triplicate.

Fig. 7.

Expression of intracellular IL-1α augments MS1 cells responses to IFN-γ and TNF-α. Stable, pooled MS1 cells transfected with either vector or expression vectors for pIL-1α or pIL-1β were plated in the presence of saturating concentrations of IL-1Ra (10 μg/ml). Twenty-four hours later, cells were washed and treated with IL-1Ra together with either TNF-α (Upper) or IFN-γ (Lower). After 24 h, MIP-2 was measured. Data represent the mean ± SEM of three separate experiments.

Fig. 8.

Stable expression of intracellular pIL-1α provides a complete signal for the synthesis of cytokines independent of the IL-1 surface receptor in different cell lines. NIH/3T3 cells were stably transfected with the various IL-1α constructs or the empty vectors as described in Fig. 2. Clones expressing similar amounts of cytokines were replated in the presence of saturating concentrations of IL-1Ra. After 24 h, the cells were washed and treated again with IL-1Ra for 24 h. On the subsequent day, supernatants were removed and measured by ELISA for murine IL-6 (Upper). COS-7 cells were transiently transfected in the presence of IL-1Ra with the various IL-1 constructs. After 24 h, the cells were washed and replenished with fresh medium containing IL-1Ra. Supernatants were removed on the subsequent day and measured by ECL for human IL-8, which crossreacts with rhesus IL-8 (Lower).

IL-1 is known to up-regulate its own synthesis in an autocrine, receptor-mediated positive-feedback loop (21). Because we have shown above that ppIL-1α activates NF-κB and NF-κB-regulated genes, we assessed whether transfection of ppIL-1α was sufficient to induce synthesis of mouse IL-1α in an intracrine manner. As can be seen in Fig. 9, this is indeed the case.

Fig. 9.

Expression of ppIL-1 α is a sufficient signal for up-regulation of endogenous IL-1α. MS1 cells stably transfected with either the empty vector or an expression vector for human ppIL-1α were plated in the presence of IL-1Ra. The next day, cells were washed and medium containing IL-1Ra was added. Twenty-four hours later, cells were lysed and murine IL-1α was measured by ECL.

Discussion

Our findings indicate that heterologous extracellular stimuli, including cytokines and Toll-like receptor ligands, lead to the translocation of intracellular pIL-1α to the nucleus. There, pIL-1α, in an N-terminal propiece-dependent manner, results in activating transcriptional machinery together with synthesis of proinflammatory cytokines; each process is independent of membrane IL-1R activation. To date, our knowledge of IL-1α biology is based mostly on studies using the 17-kDa recombinant mature form, neutralizing antibodies, or soluble receptors in which the activity of IL-1α follows membrane IL-1R ligation (1). However, in vivo, IL-1α is not readily released from cells even upon activation, and, unlike IL-1β, IL-1α is not detected in the serum of various infectious and inflammatory diseases, with the exception of severe pathological states in which cell death likely results in release of the cytokine (22). In tissues from healthy subjects, pIL-1α is stored intracellularly in a variety of cells (mostly epithelial cells), where it is not readily released in the absence of cell membrane breakdown. As an intracellular molecule, IL-1α has been also shown to be inserted in the membrane and act in a juxtacrine manner (3, 4, 23). However, there is growing evidence for intracellular functions of IL-1α, including promotion of senesence (12), cell growth and differentiation (7) and regulation of gene expression (24).

When NIH/3T3 cells were transfected with pIL-1α fused to fluorescent proteins on either the N terminus or the C terminus, localization of IL-1α was found to depend on cell activation by inflammatory stimuli. In resting cells, pIL-1α was found in the cytoplasm, whereas, upon activation, pIL-1α translocated to the nucleus. To determine whether IL-1α affected transcriptional machinery, the UAS/GAL4 system was used (25). The chimeras of the GAL4 DBD fused to either pIL-1α or ppIL-1α led to activation of transcription from a GAL4-responsive promoter, 90- and 50-fold, respectively. These results must be interpreted carefully. Ruden et al. (26) have shown that ≈1% of sonicated Escherichia coli DNA sequences are able to activate transcription from a LexA- and a GAL4-responsive promoter in a similar assay of UAS/GAL4 in yeast. In contrast, the level of activity of pIL-1α/GAL4 (90-fold) seen in our experiments was similar to that of GAL4 fused to herpesvirus VP16 (data not shown), a construct considered to be of near maximal potency in this system (27). Furthermore, only the chimeras encompassing pIL-1α and ppIL-1α, each of which incorporates a nuclear localization sequence, were active in this system. mIL-1α, which binds to the extracellular IL-1R, was inactive when inserted in the GAL4 vector. This difference indicates a distinction between the functions of mIL-1α compared with intracellular pIL-1α, suggesting evolutionary diversion.

To establish the intracellular role of IL-1α, it was essential to show that the activity of the intracellular IL-1α forms was present in the absence of engagement of its membrane receptor, because both pIL-1α and the calpain cleavage product of pIL-1α, mIL-1α, bind and activate IL-1R (5). Leakage of pIL-1α into the supernatants from transfected MS1, NIH/3T3, or COS-7 cells was below the detection level in our assays. Moreover, saturating concentrations of IL-1Ra were used throughout to further ensure that the intracellular events were independent of activation of the extracellular receptor. The concentrations of IL-1Ra were sufficient to block exogenous as well endogenously produced pIL-1α in overexpressing macrophages activated by the calcium ionophore A23187 (5). We conclude that these studies demonstrate the role of intracellular IL-1α in the absence of receptor triggering.

In our reporter experiments, intracellular overproduction of pIL-1α or ppIL-1α was sufficient to activate NF-κB and AP-1 in nonstimulated MS1 cells. That IL-1α is part of the NF-κB and AP-1 transcription complex remains to be demonstrated by coimmunoprecipitation or two-hybrid experiments. However, IL-1α might act upstream, leading to the activation of NF-κB and AP-1. The fact that intracellular IL-1α activates these transcription factors by an IL-1R-independent mechanism is a previously uncharacterized role for cytokines in general and this cytokine, in particular, in inflammation. Interestingly, pIL-18, which, like IL-1α, is constitutively present in cells from healthy subjects (20) and remains in the cytosol of many epithelial cells (28), did not activate NF-κB and AP-1, using the same experimental procedure.

Only pIL-1α and ppIL-1α stable transfectants of MS1 cells responded to picomolar concentrations of TNF-α and IFN-γ by inducing MIP-2, a known NF-κB-regulated gene, in the absence of surface membrane IL-1R engagement. We have observed the intracellular activation of proinflammatory cytokines by the nuclear localization sequence-containing pIL-1α in a variety of cells, including the two examples in this report of IL-6 in NIH/3T3 cells and IL-8 in COS-7 cells.

Using the yeast two-hybrid system, other investigators have reported that pIL-1α binds to a nuclear protein called necdin, which possesses growth suppressor activity (29). In mammalian cells pIL-1α can associate with necdin to reverse its negative effect on cell growth and production of procollagen (29). Also using the two-hybrid method, ppIL-1α has been shown to associate with HAX-1 (30). HAX-1 contains amino acid motifs for binding to several intracellular proteins including polycystin-2 (a protein linked to polycystic kidney disease), cortactin, and Epstein–Barr virus nuclear antigen leader protein (30). We also reported HAX-1 association with the pIL-1α in the yeast two-hybrid system.§ However, the significance of HAX-1 or necdin to the transcriptional activation of proinflammatory cytokines by the ppIL-1α remains uncertain. Similar to the present studies, others have reported that ppIL-1α activates transcription in the GAL4 system and is increased further by proteins with histone acetyltransferase activity (31).

There are several examples of proteins and peptides that act both through a specific extracellular receptor and also in intracellular compartments; the term “intracrine” was suggested for these proteins. These include insulin, fibroblast growth factors A and B, platelet-derived growth factor, nerve growth factor, epidermal growth factor, growth hormone, prolactin, parathyroid hormone-related protein, angiogenin, IFN-γ, and others (reviewed in ref. 32).

Of relevance to the present study is the example of a well known transcription factor that was rediscovered as a cytokine. HMGB1 is a ubiquitously expressed, highly conserved protein, initially characterized as a DNA binding protein, augmenting gene transcription and stabilizing nucleosome formation (15, 33). However, in searching for late mediators in macrophages exposed to inflammatory stimuli for prolonged periods >8 h, large amounts of HMGB1 were found in the supernatants (15). HMGB1 is a late and essential mediator of endotoxemia in mice, because blockade of HMGB1 by neutralizing antibodies protected mice, whereas injection of recombinant HMGB1 diminished survival from lethal endotoxemia. HMGB1 was shown to activate inflammatory pathways when released from necrotically lysed cells but not apoptotic cells (34). In many ways, HMGB1 is the chronological “other side” of IL-1α, which was initially studied for its activity in triggering its extracellular receptor. In vivo, both molecules appear to act intracellularly by transcriptional modulation. However, they can be released under certain conditions and exert receptor-specific events.

These “dual-function” cytokines likely originated in unicellular organisms as exclusively intracellular mediators, responding to stress by regulating genes. It is possible that these molecules developed from DNA binders through later protein–protein interactions that subsequently evolved ligand receptor binding properties. Despite evolutionary development, the cytokines retained part of their original intracellular roles. In this report we demonstrate that pIL-1α, found in the intracellular compartment of many cells, has a proinflammatory role during transcription in an IL-1R-independent manner. Modalities of treatment based on reduction of nuclear activity of pIL-1α might prove beneficial in diseases hallmarked by elevated intracellular pIL-1α.