Induction of an Interleukin-1 Receptor (IL-1R) on Monocytic Cells

Primary human monocytes and the human monocytic cell line THP-1 were induced to express receptors for interleukin-1 alpha (IL-1 alpha) and IL-1 beta. Treatment of primary monocytes with dexamethasone resulted in a 10-fold increase in receptor number over untreated cells, to approximately 2,000 receptors/cell. Treatment of THP-1 cells with phorbol ester followed by prostaglandin E2 and dexamethasone resulted in the expression of approximately 30,000 receptors/cell. Competitive binding assays on THP-1 cells showed that both IL-1 alpha and IL-1 beta bind to the same receptor. The monocyte IL-1R is significantly smaller (63 kDa) than the T cell IL-1R (80 kDa) and is immunologically distinct. However, induction of monocytes and monocytic cell lines leads to the appearance of an abundant mRNA of approximately 5,000 bases which hybridizes to a cDNA probe from the T cell-type IL-1R. Sequence data obtained from a cDNA clone of this mRNA indicate that the message is identical to the T cell IL-1R mRNA throughout the coding region. A smaller mRNA, also homologous to the T cell IL-1R mRNA, accumulated in induced THP-1 cells and has a shorter 3'-untranslated region than the larger. Data are presented which suggest that neither form of this message encodes the 63-kDa IL-1R, but rather that this protein is the product of a separate nonhomologous mRNA.


Primary
human monocytes and the human monocytic cell line THP-1 were induced to express receptors for interleukin-la (IL-la) and IL-10. Treatment of primary monocytes with dexamethasone resulted in a lofold increase in receptor number over untreated cells, to -2,000 receptors/cell. Treatment of THP-1 cells with phorbol ester followed by prostaglandin Ez and dexamethasone resulted in the expression of -30,000 receptors/cell. Competitive binding assays on THP-1 cells showed that both IL-la and IL-l@ bind to the same receptor.
The monocyte IL-1R is significantly smaller (63 kDa) than the T cell IL-1R (80 kDa) and is immunologically distinct. However, induction of monocytes and monocytic cell lines leads to the appearance of an abundant mRNA of -5,000 bases which hybridizes to a cDNA probe from the T cell-type IL-1R. Sequence data obtained from a cDNA clone of this mRNA indicate that the message is identical to the T cell IL-1R mRNA throughout the coding region. A smaller mRNA, also homologous to the T cell IL-1R mRNA, accumulated in induced THP-1 cells and has a shorter 3'-untranslated region than the larger. Data are presented which suggest that neither form of this message encodes the 63-kDa IL-lR, but rather that this protein is the product of a separate nonhomologous mRNA.
The IL-1R on T cells and fibroblasts are identical as determined by molecular cloning and sequencing (13). In contrast, * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Based on such data, it has been suggested that these two IL-1R are the products of separate genes.
Previous work has shown that monocytes respond to IL-l by up-regulating endogenous IL-l production (16). More recently, Uhl et al. (17) have reported an IL-lb binding protein on primary human monocytes that has a mass of -80 kDa, similar to that of the T cell IL-1R. To study the monocyte receptor in more depth, PGE, and dexamethasone were used to increase the expression of IL-1R on primary monocytes and the monocytic cell THP-1. IL-l receptors on induced cells were examined for immunoreactivity, electrophoretic mobility, and ligand binding characteristics.
Mono&c RNA also was examined by Northern blot analysis for expression of T cell-type IL-1R mRNA-related species. In this paper, we report that 1) dexamethasone and PGE2 act synergistically to greatly increase the number of cell surface IL-1R on THP-1 cells, 2) IL-la and -/3 bind to the same receptor on these cells, and 3) the binding protein has an apparent mass of -63-66 kDa and is immunologically distinct from the T cell IL-1R. However, we show that induction of the IL-1R protein on monocytic cells also results in the induction of two mRNAs which are strongly homologous to the mRNA encoding the T cell-type IL-1R. A cDNA clone of the larger of these two messages was sequenced and shown to be identical to cDNA clones derived from the T cell IL-1R mRNA, suggesting the possibility that the 63-kDa IL-1R might differ from the 80-kDa IL-1R only through post-translational modification. This explanation was ruled out by the introduction of an exogenous T cell IL-1R cDNA into THP-1 cells using a vaccinia virus vector. The vaccinia IL-1R virus (vat-IL-lR-T)-infected cells express an 80-kDa form of IL-1R immunologically indistinguishable from the T cell IL-lR, in addition to the endogenous 63-kDa form of IL-1R. This experiment indicates that monocytic cells are capable of translating and post-translationally modifying a T cell-type IL-1R protein in a manner similar to that found for T cells or fibroblasts and strongly suggests that the endogenous 63-kDa IL-1R is the product of a separate gene. Possible reasons for the lack of a detectable translation product from the endogenous T cell-type IL-1R mRNA are discussed. Lines-Previously, Akahoshi and co-workers (28) had shown that glucocorticoids such as prednisolone and dexamethasone could increase IL-1R expression on peripheral blood mononuclear cells. To determine whether dexamethasone exerts these regulatory effects on monocytes, populations of cells enriched for monocytes (>90% pure) were derived from human donors and cultured in the presence of 10m7 M dexamethasone for 12-14 h under endotoxin-free conditions. Direct binding assays using radiolabeled IL-la were performed and consistently

Induction of Monocytic
showed an approximately lo-fold increase in IL-1R expressed on the surface of dexamethasone-induced cells compared to untreated cells cultured in parallel (Table I).
Since even carefully purified primary monocytes may contain some proportion of contaminating lymphoid cells, three human monocytic cell lines were tested for their ability to respond to induction by dexamethasone in direct IL-1 binding assays. U937 cells, a monoblast line, HL-60 cells, a myeloblast line, and THP-1 cells, originally derived from a patient with acute monocytic leukemia, were treated with 5 rig/ml of the phorbol ester PMA. This treatment induces differentiation of these cell lines toward a more mature monocyte-macrophage phenotype.
Following treatment with PMA, no IL-1R were detected on any of the cell lines. However, incubation of PMA-treated cells with 10e7 M dexamethasone for lo-16 h induced the expression of IL-l binding proteins in all three cell types. THP-1 cells consistently yielded higher receptor numbers compared to the others and were selected for further study.
It has been reported that -50% of surface IL-1R on monocytes are rapidly internalized after binding IL-l (17). It has also been shown that the addition of PMA to THP-1 cells results in a rapid increase in IL-l/3 mRNA transcription (29, 30). As a result, the up-regulation of surface IL-1R number by dexamethasone may be due to down-regulation of the production of IL-l in monocytic cells. We therefore tested PGE*, another down-regulator of IL-l production in monocytes (31, 32), for its ability to induce IL-1R in THP-1 cells. The addition of 10m6 M PGEz to PMA-treated THP-1 cells resulted in binding of radiolabeled IL-lp but at a significantly lower level than that seen with dexamethasone induction of PMA-treated cells. Interestingly, when PMA-treated THP-1 cells were pretreated with 10m6 M PGE, for 10 h prior to the addition of 10M7 M dexamethasone and assayed 12 h later by direct radiolabeled ligand binding, a synergistic effect of the two drugs was observed. As shown in Table I Cross-reactivity-Primary mono&es induced with dexamethasone, and THP-1 cells induced with PGE, and dexamethasone, were washed briefly in glycine HCl buffer, pH 3.2, to remove any receptor-bound IL-l, split into duplicate samples, and chemically cross-linked to radiolabeled IL-la in the presence and absence of excess unlabeled IL-la. For comparison, human dermal fibroblasts were cross-linked in parallel. Fibroblasts have previously been shown to express an IL-1R which is identical at the molecular level to the IL-1R found in T cells, but which is usually expressed in greater numbers than that found in resting T cells. Cell lysates were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions and gels were subsequently analyzed by autoradiography. As shown in Fig. 1, the IL-1R on induced primary monocytes (lane a) has an apparent mass of -63-66 kDa (subtracting -17 kDa for the mass of the cross-linked IL-l), significantly smaller than that of the T cell-type IL-1R (lane e). The IL-1R protein crosslinked on THP-1 cells (lane c) appears as a smear of -66 kDa, similar to that seen on primary monocytes. The competition of radiolabeled ligand binding by excess unlabeled IL-la (lanes b, d, and f) confirms the specificity of the reaction. When this experiment is repeated using IL-l@ as the labeled ligand, the results are similar (data not shown).
The serologic relatedness of the monocyte and T cell-type IL-1R was examined by immunoprecipitation of radiolabeled cell extracts with a variety of antibodies directed against the T cell-type IL-1R. Primary monocytes and human dermal fibroblasts were metabolocially labeled with "'S and cleared lysates were prepared. Immunoprecipitations were performed using either a mixture of blocking and nonblocking mAbs, or a polyclonal anti-T cell-type IL-1R serum (see "Materials and Methods"). As shown in Fig. 2A, both the mAb mixture and the polyclonal antiserum recognized a 75-80-kDa protein from the dermal fibroblast lysates (lanes b and d, respectively) Fw.
A, induced primary monocytes (lanes a and c) or dermal fibroblasts (lanes b and d) were radiolabeled with Tran'?S-label as described in the text and analyzed in a standard radioimmunoprecipitation assay. Lanes a and b, lysates reacted with a mixture of anti-T cell IL-1R mAbs; lanes c and d, lysates reacted with a polyclonal rabbit antiserum raised against purified T cell-type IL-1R protein.
B, induced monocytes (lanes f, g, and i) or dermal fibroblasts (lanes hand;), were cross-linked to '""I-IL-18 as described in the text. Cell lysates were immunoprecipitated using the mixture of mAbs (lanes g and h) or polyclonal rabbit anti-T cell IL-1R serum (lanes i andj). but did not react with any polypeptides in the monocyte lysates (lanes a and c). Since it was possible that the pool of IL-1R in monocytes is too low or turns over too slowly to allow for efficient radiolabeling, the above experiment was repeated using surface '""I-iodinated monocytes and fibroblasts with identical results. In addition, immunoprecipitation of ""I-IL-lfi cross-linked lysates was performed using these antisera and is shown in Fig. 2B. Again, both the mAb mixture and the polyclonal antiserum reacted only with cross-linked lysates from dermal fibroblasts (lanes h and j, respectively) and showed no reactivity with cross-linked monocyte lysates (lanes g and i).

Direct Ligund and Inhibition
of Binding Assays-The K, values for IL-la and IL-18 on induced THP-1 cells were determined in direct binding assays. The K, for radiolabeled IL-la (3 X lo9 M-') was typically higher than that seen with labeled IL-l@ (5 x 10" M-l). Because this difference in binding affinities is significant, it was possible that the two ligands might be reacting with different IL-l binding proteins or protein complexes on the surface of THP-1 cells. To address this, inhibition of binding assays using homologous and heterologous competing ligands were performed. As shown in Table II, both radiolabeled IL-la and IL-l/3 could be successfully cross-competed by the corresponding unlabeled ligand. This indicates that despite an apparent difference in the affinity of radiolabeled IL-la and IL-l@ for the monocyte IL-lR, both ligands bind the same receptor molecule on induced cells.
The immunoreactivity of the monocyte IL-1R was further examined in inhibition of binding assays where the unlabeled  competitor was a monoclonal antibody (Ml or MlO) raised against the T cell IL-1R. These mAbs completely block binding of IL-la or IL-10 to the T cell IL-1R. In these assays, these mAbs are unable to block binding of IL-la or IL-l@ to the monocyte IL-lR, even at concentrations exceeding 7 X lo-' M (data not shown).
Induction of a T Cell-type IL-1R mRNA in Monocytic Cells-The differences in electrophoretic mobility and immunoreactivity between the monocyte and T cell IL-1R suggested that these two proteins may be separate gene products. To determine if an mRNA species with sequence similarity to the T cell IL-1R could be found in monocytic cells, THP-1 RNA was examined for its ability to hybridize to a radiolabeled probe derived from the coding region of the T cell IL-1R gene.
Northern blot analysis was performed on oligo(dT)-selected mRNA isolated from THP-1 cells before and after PMA treatment, and following induction with 10e7 M dexamethasone for 4, 8, or 16 h after PMA treatment. Blots were hybridized with a "'P-labeled antisense RNA derived from the coding region of the T cell IL-1R (see "Materials and Methods"). The results of this analysis are shown in Fig. 3. This figure was constructed from autoradiograms obtained after washing hybridized Northern blots under stringent conditions (0.1 x SSC, 0.1% SDS at 68 "C). Autoradiograms of hybridized blots after washing under less stringent conditions (2 x SSC, 1% SDS at 63 "C) were not significantly different from those shown here. Poly(A)+ RNA from non-PMA-treated, noninduced THP-1 cells (lane b) did not hybridize with the T cell IL-1R probe. After treatment with PMA (lane c) some hybridization of the probe with an mRNA species that comigrates with authentic fibroblast IL-1R mRNA (lane a) could be detected. The addition of dexamethasone to PMAtreated THP-1 cells caused a dramatic increase in the level of this mRNA within 4 h (lane d) and the message remained abundant for at least 16 h (lane f). Also, by 4-h post-dexa-methasone addition, another, smaller hybridizing mRNA species was detected which appeared to accumulate with time. This mRNA species (denoted by a dot in Fig. 3) migrates slightly slower than 18 S rRNA. When similar Northern blots were hybridized to a second RNA probe derived from nonoverlapping sequences in the coding region of the T cell IL-1R mRNA, the results were identical to those shown here. These data indicate that both of these induced mRNAs contain sequences which are highly homologous to those found in the coding region of the T cell-type IL-1R mRNA.
In previous experiments, a T cell IL-1R probe was shown to hybridize to an -18 S transcript that was present in poly(A)' RNA preparations from dermal fibroblasts, placenta, and keratinocytes." It is possible that the smaller transcripts arise as a result of the use of an apparent poly(A) addition consensus sequence which occurs 518 bases downstream of the translation termination codon in the human T cell IL-1R mRNA (13). If this alternative polyadenylation site were to be utilized, it would result in an mRNA of -2500 bases, exclusive of poly(A). To further characterize these smaller mRNAs, two identical Northern blots containing 2 fig of oligo(dT)-selected mRNA from THP-1 cells induced for 6 h with 10m7 M dexamethasone were hybridized under stringent conditions with "'P-labeled transcripts derived from T cell IL-1R sequences within the coding region (Fig. 3, lane g) or downstream of the alternative polyadenylation site (Fig. 3, lane h). The larger mRNA is recognized by both probes, confirming that it is related to authentic T cell IL-1R mRNA through its 3'-as well as its 5'-proximal sequences. The smaller mRNA species is not detectable on the blot hybridized with 3'-proximal (UTR) sequences. This latter blot was exposed for a longer period to confirm the absence of the smaller mRNA species. Thus, the smaller mRNA species differs from the larger mRNA in these cells at least in the 3' sequences and, based on electrophoretic mobility and analogy with the T cell IL-1R gene, is likely to be -2700 bases in length, inclusive of poly(A).
Since THP-1 cells respond synergistically to PGE, and dexamethasone by expressing higher numbers of IL-1R than cells treated with dexamethasone alone, mRNA was isolated from doubly treated cells and analyzed by similar Northern blot analysis. The pattern of hybridization of RNA from cells treated with lo-" M PGEn and 10-j M dexamethasone was indistinguishable from that of cells treated with lo-' M dexamethasone alone (data not shown).
To determine if the expression of these mRNAs was unique to monocytic cell lines, total RNA was extracted from primary monocytes which had been treated for 0,2,4, or 8 h with 10-j M dexamethasone. In this experiment (Fig. 3C), an abundant T cell IL-lR-related mRNA could be detected following 2 h of steroid treatment (lane j) which declined within 4 h of treatment (lane iz). Numerous repetitions of this experiment indicate that this message is induced rapidly (within 2 h) but can vary in the time of its disappearance. In all cases looked at thus far, the message begins to decline by 8 h and is undetectable by 10 h post-dexamethasone treatment. The smaller, -2700-base mRNA species is only faintly visible in the 2-h dexamethasone-treated sample (lane j). This decreased level of expression suggests that the accumulation of this smaller mRNA may be cell type-specific.

Molecular
Cloning of the T Cell-like IL-IR mRNA from THP-1 Cells-To examine more carefully the relationship between authentic T cell-type IL-1R mRNA and the homologous RNA induced in THP-1 cells, a random-primed cDNA library was generated in the vector pDC302 (33) from THP-"J. E. Sims, unpublished data.
1 poly(A)+ RNA following induction with PGE, and 8 h of dexamethasone treatment as described above. A 3657-base pair cDNA clone was isolated using a radiolabeled probe derived from the coding region of the T cell-type IL-1R. Dideoxynucleotide sequencing of this clone showed that it was identical to the T cell-derived IL-1R mRNA. The clone includes 151 nucleotides upstream of the initiating methionine and 1618 nucleotides downstream of the translation termination codon. This clone, therefore, does not contain a complete copy of the T cell-derived IL-1R mRNA but does indicate that a message whose coding region is identical to that which codes for an 80-kDa IL-1R in T cells and fibroblasts is expressed upon induction of monocytic cells. Expression of an 80-kDa IL-IR in THP-1 Cells following Infection with a Recombinant Vaccinia Virus-The correlation between time of induction of the T cell-type mRNA and expression of the 63-kDa monocyte IL-1R suggested that the differences observed between the T cell and the monocytetype IL-1R might be a result of dissimilar post-translational modifications.
It was of interest therefore, to determine if introduction of an exogenous T cell IL-1R gene into THP-1 cells would result in expression of the 63-or 80-kDa form of IL-1R.
Previously, we had constructed a recombinant vaccinia virus expressing the human T cell-type IL-1R gene." The recombinant virus used in these experiments, vat-IL-lR-T, was constructed using a restriction fragment containing only the coding sequences plus 30 nucleotides of the 5'-UTR of the T cell IL-1R gene. This construct, therefore, is missing approximately 300 nucleotides of 5'-and 2912 nucleotides of 3'untranslated sequences. This was done to ensure that the extensive UTRs on the T cell IL-1R gene could not interfere with translation of the virally introduced T cell-type IL-1R mRNA as has been reported for other translationally regulated gene products (34). In the resulting recombinant virus, the trimmed gene is under the control of the strong vaccinia ~7.5 early-late promotor.
The use of vaccinia virus as a eukaryotic expression system has been well documented (for a review see Ref. 25). The broad host range of vaccinia virus makes it a useful tool and productive infections using vat-IL-lR-T have been achieved in epithelial, lymphoid, and fibroblast cells of human and murine origins.4 In all infections examined to date, vat-IL-lR-T expressed a protein which is indistinguishable from the naturally occurring T cell-type IL-1R on the basis of electrophoretic mobility, serologic reactivity with mAbs and polyclonal antiseras, and IL-l binding.
PMA-treated, dexamethasone-induced THP-1 cells were infected with vat-IL-lR-T or mock-infected, harvested 24 h post-infection and affinity-cross-linked to radiolabeled IL-la. Equivalent amounts of protein were electrophoresed on 10% polyacrylamide gels and analyzed by fluorography. These results are shown in Fig. 4. In these experiments, the control cells used were uninfected, induced THP-1 cells. In separate experiments, control cells were infected with a recombinant vaccinia virus which contains the murine IL-1R gene in a backward, thereby nonexpressible, orientation. This was done to control for possible effects due to perturbation of the cells by viral infection. The IL-1R protein recovered from these control recombinant infected cells was indistinguishable from that seen in uninfected THP-1 cells. were subsequently immunoprecipitated using a polyclonal serum raised against the T cell-type IL-1R (lanes g and h). Molecular weights determined from prestained markers are indicated on the right. that of the monocyte IL-1R. As is evident in lane c, the amount of IL-1R protein cross-linked in infected cells is much greater than in uninfected cells (lane b) due to the efficiency of the viral promoter. The high level of expression and diffuse nature of the band made separation of the virus-expressed T cell IL-1R from the 66-kDa monocyte IL-1R impossible on this gel. To ensure that the infection was not affecting endogenous receptor expression, the experiment described above was repeated but with cells harvested 10 h post-infection, at which time accumulation of vaccinia expressed IL-1R protein would be less (panel B). Lane e shows induced cells infected with vat-IL-lR-T and cross-linked to IL-la. Both forms of IL-l binding proteins are visible under these conditions. Lane f shows induced, but uninfected, IL-la cross-linked THP-1 cells expressing only the 66-kDa form of the IL-1R. The immunoreactivity of the 80-kDa IL-1R expressed in infected THP-1 cells was examined by immunoprecipitation of these infected and uninfected cross-linked cell lysates with polyclonal serum raised against the T cell IL-1R. As expected, this antiserum immunoprecipitated the 80-kDa T cell IL-1R from infected cells (lane g), but did not immunoprecipitate the 66-kDa monocyte IL-1R from either infected or uninfected cells (lanes g and h). These experiments clearly show that the 66-kDa IL-1R expressed in monocytic cells does not derive from cell-specific post-translational modification of the T cell IL-1R protein.

DISCUSSION
Primary monocytes were shown to respond to lo-' M dexamethasone by increasing their number of IL-l surface receptors -lo-fold.
THP-1 cells, a monocytic cell line, exhibited an even more pronounced upregulation of IL-1R in response to lOA" M PGE, following differentiation with a phorbol ester. The precise mechanism involved in glucocorticoid induction of IL-1R on mononuclear cells is not fully understood.
Steroids are known to mediate some of their anti-inflammatory actions by down-regulating the production of IL-l in monocytes (35,36). IL-l in turn is known to down-regulate IL-1R expression in large granular lymphocytes (37), T cells, fibroblasts (38-40), and monocytes (17) at least in part through ligand-induced internalization. Thus, drugs which decrease endogenously produced IL-l would likely increase the measurable IL-1R protein present on the cell surface. Consistent with this, PGE, has been reported to decrease IL-l production in mononuclear cells, and it has also been reported to increase IL-1R numbers on the surface of fibroblasts (41). An alternative, though not mutually exclusive, explanation for increased surface IL-1R is stimulation of transcription of the monocyte IL-1R mRNA by the action of dexamethasone, possibly through a steroid-activated enhancer element. An induction of the T cell-type mRNA as a result of dexamethasone treatment is clearly evident in monocytic cells. Whether a similar induction of the mRNA encoding the 63-66-kDa IL-1R protein also occurs is unknown.
Affinity cross-linking of induced THP-1 cells with radiolabeled IL-la or IL-l@ yields an IL-l binding protein with an approximate mass of 63-66 kDa. This value differs from the M, of the IL-1R on uninduced monocytes reported by Uhl et al. (17). It is possible that the IL-1R which exists on monocytic cells in the absence of induction is different than that which is expressed following steroid treatment. Alternatively, the apparent difference in M, might result from differences in the polyacrylamide gel electrophoresis system used in the two laboratories.
Our data clearly show that the IL-1R induced in monocytic cells is significantly smaller than the T cell IL-1R and, currently, we have no evidence supporting the expression of a less abundant larger form. It is of some interest, also, that no additional higher molecular weight forms of IL-1R were seen following cross-linking of monocytic cells, as has been reported by several labs for the T cell 43). It has been proposed that these larger forms may represent dimerization of the IL-1R or association of the receptor with other cellular proteins. Whether this does not occur with the monocyte IL-lR, or is not detectable under these cross-linking conditions, is unclear. The monocyte IL-1R frequently appears on 10% polyacrylamide gels as a doublet (see Fig. 4). The doublet sometimes seen following IL-1 cross-linking of monocytic cells could be a result of slight differences in carbohydrate modifications. The induced monocyte IL-1R can bind either IL-la or ILlp, but with different apparent affinities.
The K, value for IL-la (3 X lo9 M-') is consistently higher than the K, values for IL-l/3 (5 X 10' M-l) in our direct binding assays. However, these apparent differences may be more a result of damage done to the ligand during radiolabeling than a quantitation of true binding affinities. It can be concluded, however, that the monocyte IL-1R distinguishes between radiolabeled IL-la and IL-l/3, with respect to binding affinity, more than does the human fibroblast, or T cell IL-1R. In addition, binding of either form can be completely cross-competed by the heterologous ligand, thus indicating that both IL-la and IL-l/3 bind to the same receptor on monocytic cells, as has been found with all other cell types examined to date.
The immunoreactivity of the monocyte and T cell-type IL-1R was compared in immunoprecipitation assays using antibodies derived from immunization of mice and rabbits with the T cell-type IL-1R. No reactivity with the 63-kDa form of the IL-1R was seen in metabolically or surface-labeled monocyte lysates, or with the receptor cross-linked to '"51-IL-l/3. In other experiments, mAbs which inhibit the binding of IL-1 to the T cell-type IL-1R showed no inhibition of IL-l binding to monocytes. Taken together, these data indicate that the two receptors are immunologically distinct. This was somewhat surprising as it might be expected that the binding sites of two receptor molecules which bind identical ligands might show some conservation of amino acids and/or conformation. It is possible that the difference in both M, and immunoreactivity of the two IL-1R is a result of differences in post-translational modification. However, the inability to detect any serologic cross-reactivity with a variety of distinct antibodies also suggests the possibility that the two IL-1R are the product of separate genes (see below).
Cross-linking of induced cells to IL-l at different times following induction show that the -63-kDa IL-1R is detectable within 8 h and accumulates for at least 15 h.* Closely correlated with the appearance of the surface receptor is the induction of two forms of mRNA which, by Northern blot analysis, are very similar to the T cell IL-1R mRNA. A 3657base pair cDNA clone from the larger of these messages which is found in both induced THP-1 cells and primary monocytes, is identical to the T cell IL-1R throughout the coding region and in the represented 5'-and 3'-untranslated flanking sequences. The smaller of these two messages, which is more pronounced in induced THP-1 cells than in primary monocytes, hybridized only with probes derived from the coding region of the T cell IL-1R mRNA, and not with a probe derived from 3'-proximal untranslated sequences. The truncation of this message likely occurs through the use of an alternative polyadenylation site which occurs in authentic T cell IL-1R mRNA 518 bases downstream of the termination codon (13).
The appearance of an abundant mRNA highly homologous to the T cell-type IL-1R mRNA seemed to support the idea that the two IL-1R differ only in their post-translational modification.
To resolve the question of whether the T cell IL-1R and the monocyte IL-1R are the product of separate genes, we introduced a copy of the T cell-type IL-1R mRNA lacking all but 30 nucleotides of 5'untranslated sequences and lacking 93% of the 3'-UTR through infection with a recombinant vaccinia virus. This virus was used to infect differentiated, induced THP-1 cells which were then crosslinked to IL-la.
Both the 63-and 80-kDa forms of IL-1R were detected. The larger of these was similar to the T cell IL-1R as determined by electrophoretic mobility. The smaller form co-migrated with the 63-kDa form of IL-1R visible in induced, uninfected cell samples, or in cells infected with a control vaccinia virus. Immunoprecipitation of the crosslinked infected lysates showed the 80-kDa IL-1R expressed in these cells reacted with T cell-type IL-1R mAbs and polyclonal antiserum, and that the 63-kDa form did not. Thus THP-1 cells are capable of post-translationally modifying a T cell-type IL-1R protein in a manner analogous to that found in T cells and fibroblasts. From these experiments, we conclude that the 63-kDa monocyte IL-1R is the product of a gene which is sufficiently different from the T cell-type IL-1R gene at the level of nucleotide sequence that it cannot be detected with any of three probes (representing a total of 1200 T cell IL-lR-specific nucleotides) under these conditions used here.
The mechanism responsible for the translational regulation of the T cell-type IL-1R mRNA in monocytes is unknown. It is unclear whether this mRNA is completely translationally inactive or if low levels of the 80-kDa IL-1R exist on the cell surface but are undetectable in our assays. Recently, there have been several interesting reports on gene regulation in mono&c cells. Beutler et al. (44) found that murine peritoneal macrophages contain low levels of untranslated TNF mRNA which can be made translationally active through treatment with lipopolysaccharide. Subsequently, it was shown that the addition of dexamethasone to lipopolysaccharide-treated peritoneal macrophages (44) or macrophage cell lines (45) has a modest effect on TNF mRNA accumulation but greatly decreases its translation.
Descoteaux and Matlashewski (46) have reported that while lipopolysaccharide in-duces TNF mRNA expression and releases the translational block in bone marrow macrophages, the addition of rTNF induces TNF mRNA expression but does not allow for its translation.
In human monocytic cells, Ch'ng and co-workers (47) found significant levels of creatinine kinase B mRNA attached to ribosomes, but blocked at the level of elongation. Overexpression of the 3'-UTR of the creatinine kinase mRNA released the block and allowed translation to proceed. Analagously, in the case of dexamethasone-induced monocytic cells, it is possible that the level of translation of the T celltype IL-1R might be altered in response to certain physiological stimuli that we have not yet defined.

17.
The relationship of the 63-kDa monocyte IL-1R to that of the non-T cell-type IL-1R found on B lymphocytes is unclear. Their ligand binding characteristics and apparent A4, similarities suggest that the receptors may be closely related. It is interesting that the IL-1R found on B lymphocytes more closely resembles the IL-1R on cells of a myeloid origin than those found on T lvmnhocvtes. This seems consistent with "-" the findings of several laboratories that suggest that monocyte and B-lymphocyte precursor cells are closely related (48), and that some B and pre-B cell lines can be induced to become macrophage like (49, 50). Unambiguous resolution of this question will require molecular cloning of at least one of these two receptor mRNAs.