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Division of Rheumatology, Department of Medicine, and
Department of Immunology, University of Colorado Health Sciences Center, Denver, CO 80262
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß,
and a specific receptor antagonist,
IL-1Ra3 (1, 2, 3, 4). While IL-1
and IL-1ß play proinflammatory roles in the pathophysiology of
various human diseases, either endogenously produced or exogenously
administered IL-1Ra is potently anti-inflammatory (5, 6, 7, 8, 9, 10, 11, 12). Three
different isoforms of IL-1Ra have been described, one secreted and two
intracellular (4, 13, 14, 15, 16). Secretory IL-1Ra (sIL-1Ra) and the original
intracellular isoform (icIL-1Ra) are transcribed from the same gene by
alternative RNA splicing. The sIL-1Ra is a major secreted product of
monocytes, macrophages, neutrophils, and other cells, whereas icIL-1Ra
is found constitutively in large amounts in keratinocytes and other
epithelial cells (14, 17). A novel 16-kDa intracellular isoform of IL-1Ra has recently been described by our laboratory (15). We have proposed to name the 16-kDa isoform icIL-1RaII, and the earlier described 18-kDa icIL-Ra isoform icIL-1RaI. The icIL-1RaII is formed by alternative translation initiation from sIL-1Ra mRNA (15). The initial 5' AUG of sIL-1Ra is the translation start site for sIL-1Ra protein. The translation start site consensus sequence surrounding this AUG is relatively weak, and thus the translation apparatus occasionally skips this site, proceeding to the next downstream AUG. Translation beginning at the second AUG results in icIL-1RaII, a 143-amino acid in-frame protein that lacks a leader sequence. The icIL-1RaII binds type I IL-1R less avidly than does sIL-1Ra and icIL-1RaI (15).
The ability of neutrophils and PBMC to transcribe and translate both IL-1ß and IL-1Ra has been demonstrated (18, 19, 20, 21, 22, 23, 24, 25, 26). However, a complete profile of the size and species of IL-1Ra mRNA and protein produced by neutrophils and PBMC has not been elucidated in detail. The present study used RT-PCR to examine sIL-1Ra and icIL-1RaI steady state mRNA levels in LPS-stimulated neutrophils and PBMC. Extracellular and cell-associated IL-1Ra protein production was quantified by ELISA, and the specific IL-1Ra protein species present were determined by Western blot analysis. The subcellular location of the intracellular isoforms was determined after nitrogen cavitation and differential centrifugation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neutrophils were isolated from the peripheral blood of normal human donors using a previously described technique involving dextran sedimentation and Lymphoprep gradient centrifugation (20). All preparations were >99% neutrophils with <0.5% monocyte contamination as determined by modified Wright and nonspecific esterase staining. PBMC were isolated from normal human donors using Lymphoprep gradient centrifugation as previously described (27). Preparations were approximately 25% monocytes and 75% lymphocytes as determined by modified Wright and nonspecific esterase staining. Cell viability in both neutrophils and PBMC was >99% at the initiation of cell cultures as determined by trypan blue dye exclusion.
Neutrophils and PBMC were cultured at 37°C in 5% CO2 at a concentration of 5 x 106 and 3 x 106 cells/ml, respectively, in medium alone or with LPS (Escherichia coli 055:B5, Difco, Detroit, MI) at various concentrations for periods ranging from 1 to 66 h. In preliminary dose-response experiments, maximal stimulation of neutrophils at 22 h was observed with 5 µg/ml LPS, and maximal stimulation of PBMC at 44 h was observed with 0.1 µg/ml LPS; these LPS concentrations were used in all subsequent experiments. All cultures contained RPMI 1640 medium (Mediatec, Herndon, VA), 1 mM L-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin. Additionally, neutrophils and PBMC were cultured in 5 and 2% heat-inactivated, low endotoxin FCS (Summit Biotechnology, Greeley, CO), respectively. Cell cultures for IL-1Ra ELISA, Western blot analysis, and RT-PCR were performed in parallel from the same donors.
IL-1Ra ELISA
Cell-free supernatants were isolated from neutrophils and PBMC cultured in 1-ml volumes in 10 mm wells, and cell lysates were generated by adding 1 ml of fresh medium to the isolated cells followed by three freeze-thaw cycles. IL-1Ra protein concentrations were measured in culture supernatants and cell lysates using a modification of a previously described sandwich ELISA (15, 28). ELISA was performed in triplicate for each supernatant and lysate sample; the range of triplicate values for each sample was <10%. Proteinase inhibitors added to cells before lysis did not alter levels of cell-associated IL-1Ra protein as determined by ELISA.
IL-1Ra Western blot analysis
IL-1Ra Western blot analyses, using a recently described anti-sIL-1Ra mAb, were performed on cell lysates and culture supernatants obtained from neutrophils and PBMC cultured in 5-ml volumes on 60-mm plates (29). Cell lysates were generated using a lysis buffer containing 0.5% Nonidet P-40 in 20 mM Tris, pH 7.5, and the following proteinase inhibitors: 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM PMSF, and 1 mM EDTA. Iodoacetamide (1 mM) and 0.2 mM N-methoxysuccinyl-Ala-Ala-Pro-Val (Sigma, St. Louis, MO) were used as additional proteinase inhibitors in some experiments without any effect on the amount or size of IL-1Ra produced. In some experiments, cell lysates and culture supernatants were treated with N-glycanase according to the manufacturer’s instructions (Genzyme Institute, Cambridge, MA). Cell lysates (15 µg of total protein) and culture supernatants (undiluted) were heated to 95°C for 5 min in the presence of a reducing loading buffer (0.0625 M Tris base, 1.15% SDS, 5% glycerol, 2.5% ß-ME, and 0.05% bromophenol blue, final pH 6.8). Samples were then electrophoresed on 15% polyacrylamide gels with 0.1% SDS followed by electrophoretic transfer to nitrocellulose paper, and the procedure was completed as previously described (29).
RT-PCR of sIL-1Ra and icIL-1RaI mRNA
Total cellular RNA was obtained from neutrophils and PBMC cultured in 10-ml volumes on 100-mm plates by guanidinium isothiocyanate extraction and CsCl2 gradient centrifugation (30). RT-PCR of sIL-1Ra and icIL-1RaI mRNA was performed as previously described (20). Amplification of the synthesized cDNA for sIL-1Ra and icIL-1RaI was performed separately for each sample. As a control, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA was amplified simultaneously within the same reactions as sIL-1Ra and icIL-1RaI cDNA amplification. PCR amplification was performed with the following primers (5' to 3'): 5' sIL-1Ra, GGC CTC CGC AGT CAC CTA ATC ACT CT; 5' icIL-1RaI, CAG GTA CTG CCC GGG TGC TAC TTT AT; 3' common IL-1Ra, TAC TAC TCG TCC TCC TGG AAG TAG AA; 5' G3PDH, AAG GTG AAG GTC GGA GTC AAC G; and 3' G3PDH, CCT TCT CCA TGG TGG TGA AGA C.
Thirty cycles of PCR were performed, each cycle consisting of 94°C for 90 s, 45°C for 90 s, and 72°C for 90 s. The amplified products were then electrophoresed within a 1% agarose gel containing ethidium bromide. DNA bands were visualized by UV fluorescence and photographed. The amplified cDNA products of icIL-1RaI, sIL-1Ra, and G3PDH were 577, 521, and 316 bp in size, respectively.
Subcellular localization of IL-1Ra in neutrophils and PBMC
Subcellular localization of IL-1Ra was performed in freshly isolated and LPS-stimulated neutrophils and PBMC using a modification of a previously described technique (31). Plasma membrane disruption was performed by nitrogen cavitation, which was modified to allow disruption of plasma membranes while leaving nuclear membranes intact. Upon release from the cell disruption bomb (Kontes, Vineland, NJ), the cavitate was collected, and an aliquot was examined by phase contrast microscopy, trypan blue dye stain, and modified Wright stain for efficiency of plasma membrane disruption in the absence of disruption of nuclear membranes. Nuclei, granules, plasma membrane/endoplasmic reticulum, and cytoplasm compartments were isolated by differential centrifugation (31). To determine the efficiency of subcellular separation, assays for lactate dehydrogenase and ß-glucuronidase (Sigma) content were performed within each compartment as markers for the cytoplasm and granule compartments, respectively. Assays were performed according to the manufacturer’s recommendations. Additionally, an assay for DNA content was performed in each subcellular compartment as a marker for the nuclear compartment (32). IL-1Ra protein was examined within each compartment by ELISA and Western blot analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects of culture in LPS or medium alone on neutrophil and
PBMC production of cell-associated and extracellular IL-1Ra as
determined by ELISA of cell lysates and supernatants are shown in
Figure 1
. Freshly isolated neutrophils
contained 0.19 ± 0.04 ng/106 cells (mean ± SEM;
n = 7) of cell-associated IL-1Ra (Fig. 1A).
Total IL-1Ra levels (sum of supernatants and lysates) did not
appreciably change in neutrophils cultured in medium alone for
intervals up to 22 h, although there were small decreases in
cell-associated levels with reciprocal increases in extracellular
IL-1Ra levels. LPS stimulation (5 µg/ml) of neutrophils resulted in a
5.1-fold increase in total IL-1Ra by 22 h of culture;
approximately 37% was cell associated.
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B). LPS stimulation (100 ng/ml) of PBMC resulted in a
22-fold increase in total IL-1Ra by 44 h of culture, approximately
47% cell associated. Neutrophil and PBMC viability were >90% at the
end of 22 and 44 h of culture, respectively, as determined by
trypan blue dye exclusion. Neutrophil and PBMC production of sIL-1Ra, icIL-1RaI, and icIL-1RaII
To determine which isoforms accounted for cell-associated and
extracellular IL-1Ra in neutrophils and PBMC as determined by ELISA,
Western blot analyses of lysates and supernatants from neutrophils and
PBMC cultured in the absence or the presence of LPS were performed
(Fig. 2). Regardless of culture
conditions, the predominant band present within neutrophil lysates was
16 kDa icIL-1RaII (Fig. 2A, upper). The
icIL-1RaII was present in freshly isolated neutrophils, and LPS
stimulation resulted in substantial increases in icIL-1RaII in
neutrophil lysates by 3 h of culture, with minimal further change
through 22 h. Culture in medium alone through 22 h led to no
increase in icIL-1RaII compared with freshly isolated neutrophils. A
small amount of approximately 23-kDa IL-1Ra was also present in lysates
from neutrophils stimulated for 3 and 8 h with LPS and was
determined by N-glycanase treatment to be glycosylated
sIL-1Ra (data not shown). There were no bands detected within
neutrophil lysates that aligned with recombinant 17-kDa sIL-1Ra or
18-kDa icIL-1RaI. To determine whether inadvertent proteolysis of
sIL-1Ra peptide during preparation of neutrophils for Western blot
analysis was responsible for the observed smaller species, multiple
proteinase inhibitors were added to cells before lysis; no differences
were observed in the amount or size of the IL-1Ra present.
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A, lower). This 23-kDa IL-1Ra species was
determined to be glycosylated sIL-1Ra by N-glycanase
treatment, as previously described (20). The 16-kDa icIL-1RaII peptide
was not observed in culture supernatants. Although the IL-1Ra ELISA
cannot distinguish among the three species of IL-1Ra, these results
demonstrate that the species measured by ELISA in neutrophil
supernatants and lysates, respectively, are predominantly sIL-1Ra and
icIL-1RaII.
Although 16-kDa icIL-1RaII protein was not present in all samples of
freshly isolated PBMC by Western blot analysis (Fig. 2B, upper), LPS stimulation resulted in
detectable icIL-1RaII in PBMC lysates by 8 h of culture, which
further increased by 44 h in every experiment (n =
5). A small amount of approximately 23-kDa IL-1Ra was also present in
lysates from PBMC stimulated for 8 to 22 h with LPS and was
determined by N-glycanase treatment to be glycosylated
sIL-1Ra (Fig. 3). An 18-kDa band in PBMC
lysates became evident by 15 h of LPS stimulation and increased in
amount by 44 h. Western blot analysis of culture supernatants from
LPS-stimulated PBMC revealed a predominant band of approximately 23 kDa
in size with a faint band at 18 kDa (Fig. 2B,lower). The larger IL-1Ra species in PBMC supernatants was
previously determined to be glycosylated sIL-1Ra by
N-glycanase studies (33). These results suggest that the
IL-1Ra measured by ELISA in PBMC lysates primarily consists of both
intracellular forms, whereas PBMC supernatants contain mainly
glycosylated sIL-1Ra.
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The sIL-1Ra and icIL-1RaI steady state mRNA levels in neutrophils
and PBMC cultured in the absence or the presence of LPS were determined
by RT-PCR (Fig. 4). Freshly isolated
neutrophils contained relatively low levels of sIL-1Ra mRNA (Fig. 4A, upper). The sIL-1Ra steady state mRNA levels
in neutrophils cultured in medium alone declined by 1 h and
remained low through 44 h of culture. LPS stimulation of
neutrophils resulted in substantial increases in sIL-1Ra steady state
mRNA levels by 1 h of culture, which were maintained through
8 h. By 44 h of culture, LPS-induced sIL-1Ra mRNA levels had
decreased substantially, yet remained greater than those in neutrophils
cultured in medium alone.
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A,bottom). Furthermore, LPS stimulation of neutrophils for intervals
ranging from 1 to 44 h failed to generate detectable levels of
icIL-1RaI mRNA. To determine whether icIL-1RaI transcription was
dependent upon LPS concentration, neutrophils were cultured for 24
h in LPS concentrations ranging from 10 ng/ml to 100 µg/ml; icIL-1RaI
mRNA remained undetectable in each of these conditions. As a control,
the keratinocyte cell line A431 contained readily detectable levels of
icIL-1RaI mRNA.
Freshly isolated PBMC also contained relatively low levels of sIL-1Ra
mRNA (Fig. 4B, upper). The sIL-1Ra steady state
mRNA levels increased slightly by 1 h of culture in medium alone,
falling to baseline levels by 22 h of culture. LPS stimulation
resulted in substantial increases in sIL-1Ra mRNA by 1 h that
remained elevated through 22 h of culture, after which levels
decreased substantially yet remained greater than those in PBMC
cultured in medium alone. The icIL-1RaI mRNA was undetectable in
freshly isolated PBMC and in cells cultured in medium alone (Fig. 4B, lower). However, LPS stimulation of PBMC
resulted in detectable icIL-1RaI mRNA by 8 h of culture,
increasing to maximal levels by 15 h and subsequently falling to
low but still detectable levels by 66 h.
Subcellular localization of IL-1Ra peptide in neutrophils and PBMC
Subcellular localization of cell-associated IL-1Ra in neutrophils
and PBMC was determined by ELISA and Western blot analysis of
subcellular compartments generated by nitrogen cavitation and
differential centrifugation. The subcellular compartments generated by
this procedure were 1) intact nuclei, 2) granules, 3) plasma
membrane/endoplasmic reticulum, and 4) cytoplasm. Phase contrast
microscopic evaluation of nitrogen bomb cavitates revealed <10%
intact cells. Lactate dehydrogenase (cytoplasm), ß-glucuronidase
(granules), and DNA (nuclei and intact cells) assays performed on each
subcellular compartment demonstrated minimal contamination between
compartments (Table I), with the
exception of ß-glucuronidase. This marker was present in the nuclear
as well as the granular compartments, a result also obtained by the
investigators who described this technique (31). The most important
point for our results was that the cytoplasmic compartment demonstrated
low levels of markers for nuclei and granules.
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). When neutrophils were cultured in
the absence or the presence of LPS (5 µg/ml) for 4 h, the
percentage of IL-1Ra present within the granules increased slightly,
but did not change appreciably in other compartments.
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). In freshly isolated PBMC, 100% of
the intracellular IL-1Ra was present within the cytoplasm. Regardless
of PBMC culture condition or duration, >94% of IL-1Ra remained within
the cytoplasmic compartment, with only small amounts (<5%) appearing
within the other compartments.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The biologic role of icIL-1RaII (and icIL-1RaI) is speculative at this
time. icIL-1RaII may function as an intracellular store of IL-1Ra,
being released into the extracellular space upon necrotic cell death
where it may then bind to cell surface receptors and block the
stimulatory effects of IL-1. Alternatively, icIL-1RaII may have
intracellular effects separate from binding to IL-1R. Intracellular
IL-1 has been demonstrated to promote senescence in human
endothelial cells after transport to the nucleus (34, 35). Conceivably,
icIL-1RaII may play a role counter-regulatory to the intracellular
effects of IL-1. Alternatively, icIL-1RaII may also play an
intracellular role similar to that observed for icIL-1RaI involving
alterations in IL-1-induced gene expression (36).
Although the capacity of monocytes to produce sIL-1Ra and icIL-1RaI has previously been demonstrated, this is the first study examining differential regulation of sIL-1Ra and icIL-1RaI transcription and translation in LPS-stimulated PBMC (3, 25). Whereas sIL-1Ra mRNA levels increased within 1 h upon LPS stimulation, icIL-1RaI mRNA was not detectable until 8 h of stimulation. Corresponding with the mRNA results, extracellular glycosylated sIL-1Ra and intracellular icIL-1RaI were detectable at 3 and 15 h of LPS stimulation, respectively. Small quantities of glycosylated sIL-1Ra were also noted within cell lysates at 8 to 22 h of LPS stimulation and probably represent newly translated and glycosylated sIL-1Ra within the secretory pathway. These observations extend earlier morphologic findings of a later cytoplasmic appearance of IL-1Ra protein in monocytes (37). The icIL-1RaI mRNA may be a delayed gene product, requiring de novo synthesis of transcription factors. Alternatively, LPS may be an indirect inducer of icIL-1RaI in monocytes, analogous to TGF-ß induction of IL-1Ra in monocytes through IL-1 (38).
Neutrophils have previously been demonstrated to produce IL-1Ra protein, and our results indicate that these cells transcribe only the sIL-1Ra mRNA. Although no production of icIL-1RaI mRNA or protein by neutrophils was observed in the present study, Muzio et al. concluded that human neutrophils stimulated with TGF-ß1 or IL-13 transcribed and translated icIL-1RaI, as determined by RT-PCR and ELISA of cell lysates (24, 25). Since the IL-1Ra ELISA cannot distinguish among the three forms of IL-1Ra, it is possible that the IL-1Ra assayed by ELISA in neutrophil lysates in the previous studies was the newly described icIL-1RaII and not icIL-1RaI. Furthermore, the degree of monocyte contamination in the previously reported studies was probably greater than that in the present study. Since monocytes produce 100-fold more IL-1Ra per cell than neutrophils, it is possible that the observed icIL-1RaI mRNA levels in the neutrophil preparations in the previous studies may be secondary to monocyte contamination.
Muzio et al. also cloned a unique IL-1Ra cDNA in neutrophils that was larger than icIL-1RaI cDNA and that was concluded to arise by inclusion of a 63-bp segment present within the first intron of the IL-1Ra gene (39). In the present experiments, no IL-1Ra mRNA or intracellular protein corresponding to this species was observed in LPS-stimulated neutrophils or PBMC. In a pattern similar to that in PBMC, small quantities of glycosylated sIL-1Ra were noted within neutrophil lysates at 3 to 8 h of LPS stimulation, probably representing newly translated and glycosylated sIL-1Ra within the secretory pathway.
In summary, the results of this study demonstrate a differential production of the multiple isoforms of IL-1Ra by LPS-stimulated neutrophils and PBMC. The early transcription and translation of sIL-1Ra by both neutrophils and PBMC may play an important role in regulating the cytokine effects of IL-1 in the extracellular environment. Late transcription and translation of icIL-1RaI in PBMC conceivably play an intracellular regulatory role as acute inflammation resolves or becomes chronic. Finally, icIL-1RaII is translated by both neutrophils and PBMC, and potentially may also play an intracellular regulatory role. Further studies are necessary to determine the possible biologic role of both intracellular forms of IL-1Ra in normal cell physiology and in pathophysiologic conditions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. William P. Arend, Division of Rheumatology B-115, University of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262.
3 Abbreviations used in this paper: IL-1Ra, IL-1R antagonist; sIL-1Ra, secretory IL-1R antagonist; icIL-1RaI, intracellular IL-1R antagonist type I; icIL-1RaII, intracellular IL-1R antagonist type II; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; sIL-1RI, soluble type I IL-1R; sIL-1RII, soluble type II IL-1R.
Received for publication January 2, 1998. Accepted for publication April 23, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in rheumatoid arthritis. Arthritis Rheum. 38:151.[Medline]
and ß genes by human blood polymorphonuclear leukocytes. J. Clin. Invest. 87:1312.
, IL-1ß, and IL-1 receptor antagonist by soluble IL-1 receptors and levels of soluble IL-1 receptors in synovial fluids. J. Immunol. 153:4766.[Abstract]
antisense oligomer. Science 249:1570.This article has been cited by other articles:
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